extfieldsofstudy: Medicine Biology

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Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase

The BBSome, a regulator of ciliary membrane protein composition, is required only for the export phase of a process that continuously cycles phospholipase D through cilia.

Introduction
Bardet-Biedl syndrome (BBS; OMIM accession no. 209900) is a rare inherited disorder characterized by retinal degeneration, anosmia, kidney anomalies, polydactyly, hypogonadism, and obesity (Beales, 2005;Zaghloul and Katsanis, 2009;Sheffield, 2010). The phenotype of BBS is indicative of defects in the function of cilia, and specifically in cilia-mediated signaling. Mutations in at least 12 genes (BBS1-BBS12) cause the syndrome (Jin and Nachury, 2009). Seven of the BBS gene products (BBS1,2,4,5,7,8,and 9) form a biochemically stable complex, the BBSome (Nachury et al., 2007). The BBSome subunits are well conserved in organisms with cilia, indicating that the BBSome fulfills an important ciliary function. Recently, we identified Chlamydomonas reinhardtii mutants for BBS1, BBS4, and BBS7. Because large quantities of cilia can be isolated from C. reinhardtii, it was possible to carry out a direct biochemical comparison of wild-type and bbs mutant cilia. (Because cilia and flagella are essentially identical organelles, here, we refer to the two flagella of C. reinhardtii as cilia.) Remarkably, loss of the BBSome has little effect on the overall composition of cilia or the ciliary axoneme; rather, a small subset of membrane-associated proteins, several of which are predicted to have signaling function, are present at abnormal levels in bbs cilia . A redistribution of ciliary membrane proteins is also characteristic for Bbs knockout mice (Berbari et al., 2008b;Domire et al., 2011;Seo et al., 2011;Zhang et al., 2011). However, the mechanism by which BBSome deficiency causes changes in ciliary protein composition remains unclear.
Data from Caenorhabditis elegans, C. reinhardtii, and mammalian cells have revealed that only intact BBSomes enter cilia Lechtreck et al., 2009;Seo et al., 2010Seo et al., , 2011. Within cilia, BBSomes are moved by intraflagellar T he BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched 150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency. corrected when wild-type BBSomes are introduced into the cytoplasm and cilia, that retrograde IFT acts upstream of the BBSome in the PLD export pathway, that PLD can enter the cilium independently of the BBSome and IFT, and, finally, that BBSome disruption causes secondary defects such as changes in the lipid composition of the ciliary membrane. We conclude that the C. reinhardtii BBSome functions downstream of IFT in the export phase of a process that cycles membrane signaling proteins through the cilium. The results further suggest that the absence of a protein from BBSome-deficient cilia is not necessarily due directly to failure of the BBSome to import the protein into the cilium.

PLD redistributes from the cell body into cilia in bbs mutants
We previously reported, based on silver-stained gels, that PLD, AMPK, and THB1 are enriched in C. reinhardtii bbs1, bbs4, and bbs7 cilia . We have now generated an antibody that is specific for C. reinhardtii PLD (Fig. 1 A) and, using that antibody, are able to confirm that PLD is enriched in cilia of bbs1, bbs4, and bbs7 cells ( Fig. 1 B). In addition, we have extended the findings to a newly identified mutant for BBS8 (Fig. 1 B). This mutant has an insertion in the first exon of BBS8 that results in a STOP codon predicted to terminate the normally 530-aa protein at aa 38 ( Fig. S1 A). Therefore, this mutant is likely to be null for BBS8. Its isolated cilia lack BBS4, indicating that the BBSome without BBS8 is either unstable as in bbs1-1 or is unable to enter the cilium as in bbs7-1 . To distinguish between these two possibilities, we assessed the level of BBS4 in the cytoplasm of the bbs8-1 mutant; BBS4 was strongly reduced, indicating that the BBSome is largely unstable in the absence of BBS8 (Fig. S2 B). All four bbs mutants have normal length cilia (Fig. S2 A). Anterograde and retrograde IFT velocities as determined by differential interference contrast (DIC) microscopy are similar to that of wild type (Fig. S2 C); anterograde IFT frequency is also similar to wild type but retrograde IFT frequency appears to be slightly reduced (Fig. S2 D), although this was more difficult to measure because the retrograde particles are near the limit of detection for DIC. The levels of IFT particle proteins in the bbs cilia are similar to wild type ( ; this slight increase, if real, may be an attempt by the mutant cells to compensate for loss of the BBSome. These results are consistent with our previous observation using immunofluorescence microscopy that the ciliary distribution of IFT complex A protein IFT139 and complex B protein IFT46 is unaffected in bbs4-1 . Importantly, PLD can also be detected in wild-type cilia, particularly in the ciliary membrane, by means of Western blots probed with the anti-PLD antibody, indicating that PLD is a bona fide ciliary protein (Fig. 1, A and C). In wild type and bbs4-1, ciliary PLD is largely associated with the ciliary membrane, defined as the detergent phase of Triton X-114 phase partitioning (Fig. 1 C). To estimate just how great the transport (IFT), a process by which large membrane-associated protein particles are transported bidirectionally along the axonemal microtubules (Kozminski et al., 1993;Rosenbaum and Witman, 2002;Blacque et al., 2004;Lechtreck et al., 2009). These particles consist of over 20 different proteins organized into two sub-complexes, termed IFT complexes A and B (Cole and Snell, 2009). Kinesin motors move these particles toward the ciliary tip (anterograde IFT); cytoplasmic dynein 1b (cytoplasmic dynein 2 in mammals) returns the particles to the cell body (retrograde IFT). Ciliary assembly is largely unaffected in C. reinhardtii bbs mutants and in Bbs knockout mice, and organization of IFT complexes A and B appears unaffected in the C. reinhardtii mutants Lechtreck et al., 2009). BBS proteins are significantly less abundant than IFT proteins in cilia of C. reinhardtii, and only a subset of IFT particles carries BBSomes . This suggests that the BBSome is an IFT cargo or cargo adapter dispensable for ciliary assembly and basic IFT in most cell types.
In mammalian cells, BBSome deficiencies affect the presence of ciliary transmembrane proteins, specifically of G protein-coupled receptors (GPCRs), in cilia. Melanin-concentrating hormone receptor 1 (MCHR1) and somatostatin receptor type 3 (SSTR3) fail to localize to neuronal cilia in Bbs2 / and Bbs4 / mice (Berbari et al., 2008b). The BBSome interacts with the ciliary targeting motif in the IP3 loop of SSTR3, and IP3 SSTR3 -GFP fusion proteins translocate into cilia in a BBSome-dependent manner (Berbari et al., 2008a;Jin et al., 2010;Domire et al., 2011). The BBSome could facilitate the transport of proteins from the plasma membrane through the barrier of the ciliary transition zone into the ciliary membrane proper . However, recent data show that the localization of some ciliary GPCRs is unaffected by a BBSome deficiency, whereas still others, e.g., dopamine receptor 1 in Bbs4 / mice and MCHR1 and the hedgehog effector Smoothened in Bbs3 / mice, accumulate in BBSome-deficient cilia (Domire et al., 2011;Seo et al., 2011;Zhang et al., 2011). This suggests that both import and export of ciliary GPCRs are affected in Bbs mutants.
We previously identified several putative signaling proteins (phospholipase D [PLD], an AMP-regulated protein kinase [AMPK], and a single domain globin [THB1]) that accumulate excessively in the ciliary membranes of C. reinhardtii bbs1, bbs4, and bbs7 mutants . Although lacking receptor functions, these membrane-associated proteins could modulate ciliary signaling, e.g., by protein phosphorylation and the synthesis of signaling lipids, providing a potential explanation for the disruption of phototactic behavior that is a hallmark of bbs mutants in C. reinhardtii.
To investigate the mechanism by which these signaling proteins are accumulated in the bbs cilia, we performed a detailed analysis of one of the proteins, PLD, in wild type versus the bbs4-1 mutant that is null for BBS4 . We chose to focus on PLD, both because it has a mammalian orthologue, PLD6, and because of its likely involvement in phospholipid signaling (Munnik et al., 2000). We have found that BBSome deficiency causes a massive redistribution of PLD from the cell body to the ciliary membrane, that the biochemical defects in bbs cilia increase with time but can be rapidly wild type, 0.2% of the total PLD is in the cilia, whereas in the mutant, 60% of the total PLD is in the cilia.
Immunofluorescence microscopy confirmed our biochemical data: PLD was readily detected in a spotted distribution along the length of bbs4-1 mutant cilia but was barely detectable in wild-type cilia ( Fig. 1 F and see Fig. 4 B). Within mutant cilia the signal strength was more pronounced in the proximal region of the cilium. Labeling by the anti-PLD antibody was similar in wild-type and bbs4-1 cell bodies, albeit signal strength was reduced in the mutant. Label was distributed in a spotted pattern at the cell periphery ( Fig. 1 G for bbs4-1); this could represent vesicles underlying the plasma membrane. In summary, the bbs4-1 mutation causes a dramatic redistribution of PLD from the cell body into cilia (Fig. 1 H).
increase is in the mutant cilia, we used Western blotting to quantitate the relative amounts of PLD in isolated membranes of wild-type and bbs4 mutant cilia. We determined that PLD is 150-200× more abundant in the mutant versus wild-type ciliary membrane (Fig. 1 D). A similar enrichment was observed in cilia of mutant versus wild-type gametes (unpublished data).
To distinguish whether the observed enrichment of PLD in the cilia of bbs mutants is a result of a general increase in PLD levels in the mutant cells or to defects in ciliary trafficking of PLD, we determined the distribution of PLD in wild-type and bbs mutant cells and cilia. Western blotting revealed that the total amount of PLD in bbs4-1 cells actually was reduced by 40% in comparison to wild-type cells (Fig. 1, E and H). Moreover, in Figure 1. BBSome deficiency causes PLD redistribution from the cell body into cilia. (A) Western blot of isolated wild-type and bbs4-1 cilia probed with antibodies to PLD and, as loading controls, antibodies to the membrane-associated protein FAP12, the axonemal dynein subunit IC2, and the IFT complex B protein IFT172. (B) Western blot of isolated cilia from wild type (wt) and bbs1-1, bbs4-1, bbs7-1, and bbs8-1 probed with antibodies to BBS4 and PLD. IC2 was used as loading control. (C) Western blots of wildtype and bbs4-1 cilia (FL) and ciliary fractions (Ax, axonemes; AP and DP, aqueous and detergent phase from Triton X-114 phase partitioning, respectively) were probed with antibodies to PLD and BBS4, and with antibodies to the axonemal protein IC2 and the matrix protein IFT139 as loading controls. (D) Western blot of isolated ciliary membrane from wild type and bbs4-1 probed with anti-PLD. The bbs4-1 sample was diluted as indicated. IFT139, small amounts of which can be detected in the ciliary membrane, was used as a loading control. The signal strength obtained from the undiluted wild-type sample is between that of the 1:128 and 1:256 dilution of the bbs4-1 sample. (E) Western blots of whole cells (WC), deciliated cell bodies (CB), and isolated cilia (cilia) from wild type and bbs4-1 were probed with anti-PLD. Equivalent amounts of cells and cilia were loaded (i.e., one whole cell, one cell body, and two cilia). The IFT particle protein IFT139 was used as a loading control. (F) Wild-type and bbs4-1 gametes were stained with anti-IFT139 and anti-PLD. PLD was readily detected in cilia from bbs4-1 but not detected in wild-type cilia. PLD were consistently detected in isolated wild-type ciliary membranes, where the PLD was maintained at a relatively constant low level during the time period analyzed (Fig. 3 B, bottom). In contrast, PLD was detectable in the newly assembled mutant whole cilia at least as early as 80 min after deciliation; it then accumulated steadily over the course of several hours as the cilia aged (Fig. 3, B [top] and C). The data indicate that PLD accumulation in mutant cilia is a dynamic process that continues in full-length steady-state cilia. The amounts and kinetics of accumulation of several ciliary transmembrane proteins, specifically PKD2, FMG1, and the mastigoneme protein, were similar in wild-type and mutant cilia, suggesting that the transport of these proteins is unaffected by BBS4 deficiency.
In the course of the cell cycle, cilia are normally formed after division, at least 6 h before our synchronously grown cells were used for biochemical analyses. We here term such 6-hour-old cilia "standard cilia." Comparison of regenerated and standard cilia from wild type and bbs4-1 using silver-stained gels revealed a protein of 24 kD that was present in wild-type cilia and in newly regenerated bbs4 cilia but was significantly reduced in standard bbs4 cilia ( Fig. 3 D). The protein was tentatively identified as carbonic anhydrase 6 (CAH6) based on recovery of a single peptide by mass spectrometry (Fig. S1 B); the identification was confirmed using an antibody to C. reinhardtii CAH6 (Fig. 3 B). Quantitative analysis of age-sorted cilia showed that at 80 min after deciliation the mutant cilia contained 50% of the CAH6 present in comparable wild-type samples (Fig. 3, B and E). Over the next five hours CAH6 was progressively lost from mutant cilia, whereas little reduction of CAH6 was observed in wild-type cilia (Fig. 3, B, D, and E). Because a substantial amount of CAH6 is present in newly formed mutant cilia, it is unlikely that BBSome defects inhibit CAH6 ciliary import. The data rather suggest that bbs cilia increasingly lose the ability to retain CAH6. Thus, CAH6 loss from bbs cilia appears to be caused indirectly by the BBSome defect.

Excess PLD is rapidly removed from bbs cilia when BBSomes are reintroduced
The progressive redistribution of PLD from the cell body into cilia of bbs mutants could be caused by a failure of PLD ciliary export. Therefore, to test directly whether BBSomes

The lipid composition of bbs4 cilia is altered
To assess whether the accumulation of PLD in bbs cilia affects the lipid composition of the ciliary membrane, we performed a metabolomic and lipidomic analysis of purified wild-type and mutant cilia (Table 1 and Fig. 2). Phosphatidylethanolamine (PE; Fig. 2, A and B), the substrate of PLD in C. reinhardtii (Munnik et al., 2000), was reduced by 30-50% in bbs4-1 cilia. Phosphatidic acid (PA) and ethanolamine, the products of PLD activity, were increased by 50% and 4,000%, respectively (Table 1 and Fig. 2 C). DAG and stearic acid, putative downstream products of PA enzymatic degradation, were each increased by 500%; a two-to threefold increase was also observed for glycerol and glycerol phosphate ( Fig. 2 D and not depicted). The changes in PE, PA, and DAG occur predominately in species carrying 18:3/18:0 fatty acids, indicating that they are linked, i.e., that DAG 18:3/18:0 is formed by dephosphorylation of PA 18:3/18:0 ( Table 1). The amounts of other membrane lipids, including that of the major ciliary membrane lipids ergosterol and stigmasterol, which account for 32% and 20% of the total ciliary lipid, respectively, were similar in both strains (unpublished data). In conclusion, the lipid composition of the ciliary membrane of the bbs4-1 mutant is altered and the observed changes are as predicted for increased PLD activity. The data also reveal that only a limited number of phospholipid species are present in the ciliary membrane of C. reinhardtii (Table 1).

The defects in protein content of bbs4 cilia increase with time
To distinguish whether the abnormal amounts of PLD in bbs cilia are deposited there during initial ciliary assembly or accumulate there with time after ciliary formation, we took advantage of the ability of C. reinhardtii to fully assemble cilia de novo within 60 min after amputation of the cilia by pH shock (Fig. 3 and Fig. S3 E). We simultaneously deciliated large populations of wild-type and bbs4-1 cells and allowed them to synchronously regenerate their cilia; we then isolated the recently formed cilia at time points ranging from 80 to 380 min after deciliation and compared them by SDS-PAGE and Western blotting ( Fig. 3 A). At the loadings used, PLD was not detected in wild-type whole cilia (Fig. 3 B, top), but small amounts of The distribution of phospholipids in cilia of wild type and bbs4-1 as a percentage of total phospholipid. PG, phosphatidylglycerol; PI, phosphatidylinositol.

PLD export from cilia requires retrograde IFT
We previously showed that the BBSome cycles in association with IFT through cilia . To determine if this association is important for BBSome function and normal export of PLD from the cilium, we analyzed the mutant strain dhc1b-2 (previously referred to as dhc1b ts ). This strain contains a hypomorphic allele of the gene encoding the heavy chain of dynein 1b, the motor for retrograde IFT Witman, 2012). At the permissive temperature (21°C) used for all experiments involving dhc1b-2 here, 60% of the mutant cells assembled two cilia that were slightly shorter than those of wild type (Fig. S3, A and B). The velocity and frequency of retrograde IFT and the amounts of the retrograde dynein subunits DHC1b and D1bLIC were reduced in the dhc1b-2 cilia ( Fig. 5 A and Fig. S2, C and D). The amount of BBS4 was elevated in dhc1b-2 cilia, as expected for a protein cycled through the cilium by IFT. However, despite the presence of BBS4/BBSomes, PLD was highly enriched in the mutant cilia ( Fig. 5 A), indicating that the BBSome-dependent export of PLD from cilia requires the retrograde motor for IFT.
As an independent test of the involvement of IFT in export of PLD, we analyzed the mutant ift74-1, which expresses a truncated version of the IFT complex B protein IFT74 (Fig. 5 B; unpublished data). The truncated IFT74 is assembled into complex B and transported into the cilium as part of the IFT particle; the ciliary levels of the other complex B proteins are near normal, but the amount of IFT complex A is greatly reduced. Compared with wild-type cilia, BBS4 was reduced by 65% and are required to remove PLD from cilia, bbs4 and wild-type gametes were mixed and the distribution of PLD in cilia of the resulting zygotes was analyzed at various time points after mating. The zygotes possess four cilia, two derived from the mutant gametes and two from the wild-type gametes (Fig. 4 A). After cell fusion, BBSomes contributed by the wild-type cytoplasm become immediately available to bbs4 mutant cilia. With immunofluorescence microscopy, PLD is readily detectable in cilia of mutant but not wild-type gametes (Fig. 4 B, a-c). Ten minutes after mixing of the gametes, some zygotes were observed that possessed two cilia with PLD derived from the bbs4 parent and two cilia without detectable PLD derived from the wild-type parent (Fig. 4, B [d-f] and C). However, even at this early time point, PLD was reduced or not detectable in cilia of the majority of zygotes (Fig. 4, B [g-l] and C). The share of zygotes without detectable ciliary PLD increased progressively from 18% at 10 min to 96% at 50 min after mixing of the gametes (Fig. 4 C). The time span required for gametes to complete fusion after mixing is variablesome zygotes may form as early as 15 s after mixing, with typically 50% of the gametes having completed fusion after 5 min in good mating reactions (Hunnicutt and Snell, 1991). The presence of a few zygotes with cilia still containing PLD at later time points can be explained by delayed fusion of some gametes. In conclusion, PLD is removed in <10 min from the formerly mutant cilia once BBSomes become available. Therefore, an efficient pathway for PLD export from cilia is present in C. reinhardtii and this pathway requires the BBSome. (B) Schematic presentation of the enzymatic breakdown of PE into PA, ethanolamine, DAG, and downstream products. (C and D) Gas chromatography-mass spectrometry profiles showing the elution times for small metabolites from wild-type and bbs4-1 cilia. Ethanolamine (peaks marked by red arrows in C) is enriched 40× and free stearic acid (peaks marked by red arrows in D) is enriched 6× in bbs4-1 cilia. Accuracy of quantitation is ±10%.
The fla17-1 strain has an in-frame deletion of three exons of the IFT139 gene and produces a smaller-than-normal version of IFT139 (Fig. 5 C;Iomini et al., 2009). These fla mutants fail to assemble cilia at elevated temperatures. At the permissive temperature used here IFT is sufficient for ciliary assembly but the velocity and frequency of retrograde IFT is reduced and the cilia develop small bulges containing complex B components, indicative of a defect in returning cargo to the cell body (Iomini et al., 2009). Compared with wild type, BBS4 was highly enriched in cilia of fla17-1 (Fig. 5 C), suggesting that the BBSome also is not returned normally to the mutant cell body. Despite the abundance of BBSomes, PLD also was highly enriched in fla17-1 cilia (Fig. 5 C), indicating that PLD export from cilia is dependent on IFT complex A. In contrast, the amounts of BBS4 and PLD were near normal in PLD was strongly accumulated in ift74-1 cilia (Fig. 5 B). The data suggest that entry of BBSomes into cilia requires IFT74 and/or IFT complex A and that the residual IFT and BBSomes in ift74-1 cilia are insufficient to remove PLD from cilia. The result is consistent with a recent observation in C. elegans that a mutation in complex A protein IFT144 abrogates entry of BBSomes into cilia (Wei et al., 2012).
IFT complex A has an important role in retrograde IFT (Pedersen and Rosenbaum, 2008;Pedersen and Christensen, 2012). To more directly investigate if complex A has a role in BBSome transport and PLD export, we analyzed strains fla15-1 and fla17-1, which carry mutations in the complex A proteins IFT144 and IFT139, respectively (Piperno et al., 1998;Iomini et al., 2009). The fla15-1 strain has a point mutation that converts a highly conserved cysteine in IFT144 to an arginine. to PLD levels in standard bbs4-1 cilia, i.e., cilia harvested without prior deciliation at least 6 h after the beginning of the light phase. (D) Silver-stained gel comparing matrix and membrane isolated from standard and 2-h-old regenerated wild-type and bbs4-1 cilia. Solid arrows, proteins altered in bbs4-1 ciliary membrane; open arrow, THB1. Note the differences in composition in the membrane of young and old cilia; no major differences were observed between young and old axonemal (not depicted) and matrix fractions, indicating that age-induced changes in ciliary protein composition are largely restricted to the ciliary membrane. (E) Graph showing the loss of CAH6 in bbs4-1 cilia as a percentage of that in wild-type cilia over time. The data shown in C and E are from a single representative experiment out of two repeats.
for entry of PLD into cilia. We took advantage of the fla10-1 mutant, which carries a temperature-sensitive mutation in one of the heavy chains of the anterograde IFT motor kinesin-2, so that IFT can be rapidly turned off by a temperature shift (Kozminski et al., 1995). At the permissive temperature (22°C), fla10-1 cilia contained slightly elevated amounts of PLD, which can be explained by the somewhat reduced amounts of IFT complex A and BBSomes (as indicated by reduced amounts of IFT139 and BBS4, respectively) in the mutant cilia (Fig. 6 A). Cilia isolated from fla10-1 that had been incubated for 2.5 h at the nonpermissive temperature (32°C) were largely depleted of IFT81 (complex B) and IFT139 (complex A). This loss of IFT proteins at the restrictive temperature was accompanied by an increase in ciliary PLD; in contrast, the amounts of PLD in wild-type and bbs4-1 cilia were essentially unaffected by the temperature shift. To determine the temporal sequence of IFT depletion and PLD accumulation in fla10-1 cilia, we analyzed cilia isolated at various time points (1-6 h) after shifting fla10-1 cells to 32°C (Fig. 6 B). During the incubation time the number of mutant cells carrying one or two cilia decreased from >90% to 70% and the mean length of the cilia that they did have decreased from 11 to 6 µm; the number and length of cilia of wild type was unaffected at 32°C (Fig. S3, C and D). Starting at 2 h at 32°C, by which time the level of IFT proteins was considerably reduced, PLD began to accumulate in fla10-1 cilia. Importantly, PLD continued to accumulate in fla10-1 cilia after 4 h at 32°C when IFT proteins were nearly undetectable; after 6 h at 32°C it reached a level approaching that of bbs mutants (>30× above the level before temperature shift; Fig. 6 C). The data indicate that PLD entry into cilia does not require IFT. Note that despite the depletion of IFT proteins, BBS4 remained present in fla10-1 cilia. The accumulation of PLD in the presence of BBS4 provides additional evidence that BBSomes alone are insufficient to control PLD levels in cilia in the absence of IFT.

Discussion
BBSome deficiency has been shown to alter the membrane protein composition of cilia in C. reinhardtii and mammals (Berbari et al., 2008b;Lechtreck et al., 2009;Domire et al., 2011;Seo et al., 2011;Zhang et al., 2011). We showed previously that BBSomes travel through cilia in association with a subset of IFT particles . These observations raise the possibility that BBSomes contribute to IFT-mediated transport of ciliary membrane proteins. Here we analyzed the role of the BBSome and IFT in the ciliary transport of PLD, a membraneassociated protein that accumulates in cilia of C. reinhardtii bbs mutants. Our data reveal that PLD continuously enters the cilium in a BBSome-and IFT-independent manner but that export of PLD from the cilium is dependent on the BBSome, probably functioning as a cargo adaptor for IFT.

PLD cycles through the cilium and redistributes from the cell body to the cilium in the absence of the BBSome or IFT
PLD is abundant in wild-type cell bodies and 0.2% of the PLD is present in wild-type cilia. However, in bbs4-1 mutants cilia of fla15-1 (Fig. 5 C). The data suggest that IFT139 is involved in retrograde transport of BBSomes and removal of PLD from cilia.

PLD can enter cilia independently of IFT
Our data suggest that retrograde IFT particles transport PLD in a BBSome-dependent manner. Because PLD enters cilia through a BBSome-independent process, we investigated if IFT is required . PLD is removed from cilia of bbs4-1 cells when BBSomes are introduced by fusion with wild-type cells. (A) Schematic presentation of the PLD ciliary export assay. In wild-type gametes BBSomes are present in both cytoplasm and cilia, and nearly all of the cell's PLD is in the cell body; bbs4-1 gametes lack BBSomes and have undergone a massive redistribution of PLD from the cell body to the cilia. Wild-type (BBS4, mt) and bbs4-1 (bbs4, mt+) gametes were mixed and allowed to fuse. The resulting zygotes have four cilia (two each from the wild-type and mutant gametes); BBSomes are present in the shared cytoplasm. (B) Gametes (a-c) and quadriciliated zygotes (d-l) were stained with antibodies to PLD and to IFT139 to visualize the cilia. Arrow in c, bbs4 gamete; arrowhead in c, wild-type gamete; arrows in f and h, cilia containing PLD derived from mutant gametes; arrowheads in f, cilia lacking PLD derived from a wild-type gamete; arrowheads in j, four cilia of a zygote without detectable PLD. Bar, 5 µm. (C) Quadriciliated zygotes were scored by eye for the amount of PLD in their cilia. Black bars, quadriciliated cells with two wild-type cilia and two cilia with levels of PLD characteristic of the bbs4-1 gamete; gray bars, quadriciliated cells with two wild-type cilia and two cilia with detectable but reduced amounts of PLD; white bars, quadriciliated cells without detectable PLD in their cilia.
A similar role for the BBSome in mammalian primary cilia is suggested by recent observations on dopamine receptor 1, a GPCR that is rapidly transported to and from cilia in response to environmental cues (Domire et al., 2011). Import of dopamine receptor 1 into cilia is BBSome independent whereas translocation of the protein from cilia requires the BBSome (Domire et al., 2011;Zhang et al., 2011). In addition, very recent results indicate that accumulation of the hedgehog signaling proteins Smoothened and Patched 1 occur in cilia of cells from Bbs / mice . Like PLD in C. reinhardtii, Smoothened is believed to continuously shuttle through the cilium (Ocbina and Anderson, 2008). Thus, BBSome-mediated export may be a feature of many membrane-associated proteins that undergo such cycling.

BBSome function depends on retrograde IFT
To investigate the role of IFT in the BBSome-dependent export of PLD from the cilium, we analyzed the distribution of BBS4 and PLD in cilia isolated from the dynein 1b mutant dhc1b-2, which is characterized by reduced retrograde movement of IFT particles. Using silver-stained 1D and 2D gels, we previously determined that THB1 is enriched in dhc1b-2 cilia, but we could not ascertain the levels of PLD in these cilia ). Now, using our anti-PLD antibody and Western blotting, we found that PLD also is significantly accumulated in dhc1b-2 cilia despite the presence of BBSomes. Likewise, BBSomes are present albeit reduced during PLD accumulation in fla10-1 cilia at restrictive temperature. Therefore, the presence of BBSomes in cilia is insufficient to remove PLD when IFT is defective. Because the primary effect of the dhc1b-2 mutation is on retrograde IFT, these results indicate that retrograde IFT is critical there is a massive redistribution of PLD from the cell body into the cilia, such that about half of the PLD is now contained in the cilia. Taking into account the relative volumes of the cell body (>500 µm 3 ) and the cilium (<0.5 µm 3 ; and ignoring possible differences in the fraction of each that is accessible to PLD), PLD in bbs4-1 cells is enriched 1,000× in the two cilia relative to the cell body. Because this concentration occurs in bbs mutants, PLD must be able to move into the cilia in the absence of BBSomes. A similar great enrichment was observed in cilia of the conditional mutant fla10-1 after IFT proteins (but not BBS4) were depleted by incubation at the nonpermissive temperature. This strongly suggests that entry of PLD into cilia can occur even in the absence of IFT. Recently, Kee et al. (2012) reported that small molecules and proteins ≤40 kD can freely pass from the cytoplasm into cilia. Thus, PLD, which is only 25 kD, may enter the cilia slowly by diffusion and then become trapped there in the absence of an active export mechanism.
Because PLD accumulates progressively over time in BBSome-and IFT-deficient cilia, it is likely that the protein is continuously cycled through the cilium in wild-type cells, with IFT-dependent export mediated by the BBSome. The high efficiency of this PLD export pathway, as demonstrated by the rapid clearing of accumulated PLD when BBSomes are reintroduced into bbs mutant cilia, suggests that PLD entering wild-type cilia will be rapidly picked up and removed from the organelle. A system in which the movement of a protein into the cilium is BBSome independent but its export is BBSome dependent would allow fine tuning of the amount of the protein in the cilium by regulating the number of BBSomes that enter the cilium or by regulating the affinity of the BBSome for the protein. . Ciliary PLD is elevated in retrograde IFT mutants. (A) Western blot of isolated wild-type and dhc1b-2 cilia probed with antibodies to the proteins indicated. The amounts of the dynein 1b heavy chain, DHC1b, and light intermediate chain, D1bLIC, are reduced in the mutant cilia because of the instability of the dynein motor complex. BBS4 and in particular PLD are enriched in mutant cilia. IC2 was used as a loading control. For quantification of relative protein levels, values for band intensities were first normalized to the IC2 band intensity and the ratios (left, parentheses) were then calculated. (B) Western blot of cilia isolated from wild type and ift74-1 probed with antibodies to the proteins indicated. In ift74-1 the IFT complex B protein IFT74 is truncated. Note reduced amounts of IFT complex A proteins and BBS4 and increased levels of PLD in the mutant cilia. Relative protein levels (in parentheses) were determined as in A. (C) Western blot probing isolated cilia from wild type, bbs4-1, fla17-1 (expressing a truncated IFT139; arrowheads), and fla15-1. Note increase of BBS4 and PLD in fla17-1. (D) Summary diagram showing the IFT motors, IFT complexes A and B, the BBSome, and PLD. The BBSome functions as an IFT cargo adapter linking PLD and other cargoes to retrograde IFT particles. PLD is predicted to be myristoylated and palmitoylated (Fig. S4 C), offering a possible explanation for its membrane association. Dynein is shown linking the IFT particle to a doublet microtubule, which provides a track for IFT. BBSome-dependent ciliary protein export • Lechtreck et al. and Anderson, 2008) or in which the same protein was knocked down by shRNA (Kim et al., 2009) or inhibited pharmacologically (Firestone et al., 2012). Thus, IFT-dependent BBSomemediated export of signaling molecules from the cilium may be widespread. However, another hedgehog signaling protein, Gli2, which also is believed to cycle though the cilium (Kim et al., 2009), does not accumulate in Bbs / mice , suggesting the existence of distinct export (and import) mechanisms for different ciliary proteins.

IFT complex A is critical for BBSome export
The aforementioned observations suggest that coupling of PLD to retrograde IFT particles requires the BBSome. To explore how the BBSome interacts with the IFT machinery, we used mutants defective in IFT particle proteins. IFT complex A proteins and BBS4 are similarly decreased in cilia of ift74-1, which expresses a truncated complex B IFT74 protein. PLD is highly enriched in ift74-1 cilia, indicating that the residual BBSomes are insufficient for its export. This suggests that IFT74 is critical for the entry of IFT complex A into cilia and that complex A could mediate binding of the BBSome to IFT complex B and the associated dynein motors during retrograde IFT. To test for a possible role of IFT complex A as a docking site for the BBSome, we analyzed fla15 and fla17-1, which are defective in the IFT complex A proteins IFT144 and IFT139, respectively, and have reduced retrograde IFT (Piperno et al., 1998;Iomini et al., 2009). The distributions of BBS4 and PLD were nearly normal in fla15, whereas PLD and in particular BBS4 were enriched in cilia of fla17-1. Therefore, IFT139 could be involved in binding the BBSome to IFT complex A during retrograde IFT, rendering it critical for the export of the BBSome and PLD from cilia (Fig. 5 D). It may be relevant that the Tubby family protein TULP3 similarly interacts with complex A in mammals to promote ciliary trafficking of a subset of GPCRs, including MCHR1, and that retrograde transport of TULP3 specifically requires IFT139 (Mukhopadhyay et al., 2010).
In summary, PLD accumulates in cilia of mutants with defective BBSomes (bbs4-1, bbs1-1, bbs7-1, and bbs8-1; Lechtreck et al., 2009), with reduced amounts of BBSomes in cilia (ift74-1), and with reduced retrograde IFT of the BBSome (dhc1b-2 and fla17-1). We propose that the BBSome functions as a cargo adapter linking PLD to retrograde IFT particles to ensure export of PLD from cilia. Further work will be necessary to understand how PLD is delivered to the cilium, how PLD interacts with the BBSome during the export phase of its cycle, and how the BBSome interacts with IFT complexes A and B.

BBSome function in IFT assembly and turnaround
In C. elegans mutants null for bbs-7 and bbs-8, IFT complex A separates from complex B and the two complexes are transported anterogradely by kinesin-2 and OSM-3, respectively (Ou et al., 2005). These observations led to the hypothesis that the BBSome couples complex A to complex B and coordinates the activity of the two anterograde IFT motors. However, more recently it has been reported that a hypomorphic mutation at a for PLD export from the cilium. Inasmuch as loss of the BBSome has little if any effect on IFT (Figs. 1 C, 3 B, 6 A, and S2; Lechtreck et al., 2009) but defects in retrograde IFT affect export of both the BBSome and PLD, IFT must function upstream of the BBSome in the PLD export pathway; i.e., retrograde IFT moves the BBSome out of the cilium, and the BBSome in turn carries PLD from the cilium (Fig. 5 D). Export of Smoothened from mammalian primary cilia similarly is dependent on the retrograde IFT motor, as indicated by the accumulation of Smoothened in cilia of mouse cells having a mutation in the dynein 2 heavy chain Dync2h1 (Ocbina Figure 6. PLD accumulates in cilia after depletion of IFT proteins. (A) Western blot of cilia isolated from wild type, bbs4-1, and fla10-1 at 22°C and after 2.5 h at 32°C. The blot was probed with antibodies to the proteins indicated. Depletion of IFT proteins and accumulation of PLD in response to the temperature shift was observed only in cilia from fla10-1. (B) Western blot comparing fla10-1 cilia isolated at various time points from 0 to 6 h (T0-T6) after shifting the cells to 32°C. The blot was probed with antibodies to the proteins indicated. Note progressive depletion of IFT proteins and concomitant accumulation of PLD. (C) Relative amounts of PLD, BBS4, and IFT172 in the Western blot analysis shown in B. Maximum band intensity for each protein was set to 1. At later time points the mean length of fla10-1 cilia was reduced (Fig. S3 D). Because similar amounts of protein were loaded for each sample, the slight increase observed for BBS4 during the second half of the incubation time is likely to reflect the higher number of cilia loaded.

C. reinhardtii PLD has homologues in mammals and many other ciliated eukaryotes
Our finding that C. reinhardtii PLD is apparently a bona fide ciliary protein that cycles through the cilium raises the question of whether a homologue occurs in the cilia of other organisms. Close homologues of C. reinhardtii PLD are encoded in the genomes of many vertebrates, nonvertebrate metazoans (with C. elegans being a notable exception), and some ciliated protists; they are absent from Arabidopsis thaliana, yeasts, and other nonciliated organisms (Fig. S4, A and B). Such a phylogenetic distribution is often indicative of a centriolar or ciliary function of the gene product. The mammalian homologue of PLD is PLD6 (C. reinhardtii to H. sapiens BLAST E = 8e37; 40% identity); in a phylogenetic analysis PLD6 groups more closely with C. reinhardtii PLD than with other murine PLDs (Fig. S4 B). In mice, overexpressed PLD6 localizes to mitochondria and is anchored to the mitochondrial surface by a transmembrane domain (Huang et al., 2011). This transmembrane domain is not predicted in C. reinhardtii PLD but, like C. reinhardtii AMPK and CAH6, the protein is predicted to be myristoylated and palmitoylated (Fig. S4 C), likely accounting for its association with the ciliary membrane. PLD6 is widely expressed in human tissues; in mice, expression is especially high in lung and testis, tissues that are rich in cilia, and Pld6 / mice are infertile (Huang et al., 2011;Watanabe et al., 2011). It will be of interest to determine if mammalian PLD6 functions also in cilia, and whether BBSome deficiency promotes ciliary accumulation of PLD6. It may be relevant that cilia of Bbs / mice accumulate unusual intraciliary vesicles at the ciliary tip (Shah et al., 2008); this might be brought about by an excess of ciliary DAG caused by ciliary accumulation of PLD6. Phospholipid signaling also has been implicated in the functioning of olfactory cilia (Klasen et al., 2010), and malfunction of olfactory cilia has been reported in Bbs8 / mice (Tadenev et al., 2011).

BBSome deficiency results in secondary defects in ciliary composition
Quantitative analysis of age-sorted cilia revealed that the ciliary protein CAH6 is progressively lost from bbs4-1 cilia. Because CAH6 is initially present in mutant cilia, it is unlikely that BBSome defects inhibit CAH6 ciliary import. Rather, the progressive loss of CAH6 from bbs4-1 cilia is likely to be caused indirectly by BBSome deficiency. Loss of a peripheral membrane protein such as CAH6 could follow from the severe changes in membrane lipid content that occur in bbs4-1 cilia, presumably as a result of increased ciliary PLD activity; the ability of proteins, such as CAH6, which is predicted to be fatty acid modified (Fig. S4 C), to associate with membranes is influenced by the lipid composition of the membrane (Levental et al., 2010). An altered lipid composition of bbs cilia also could contribute in other ways to changes in the pattern of ciliary membrane proteins. Loss of certain GPCRs from cilia has been reported in murine Bbs / mutants (Berbari et al., 2008b). In light of our data on CAH6 in bbs mutant cilia of C. reinhardtii, it seems possible that absence of GPCRs from mammalian cilia also could be an indirect consequence of the loss of the BBSome. conserved site in the complex A protein DYF-2 (the C. elegans homologue of IFT144) causes complete dissociation between the BBSome and moving IFT particles, yet complexes A and B remain associated in anterograde IFT (Wei et al., 2012). These results indicate that in C. elegans the BBSome is not essential for stabilizing the binding of complex A to complex B, in good agreement with our previous observation that the colocalization of complexes A and B is not affected in cilia of C. reinhardtii bbs4-1 mutants . In the C. elegans IFT144 mutant, the BBSome is still formed but remains at the base of the cilium. Moreover, complex B but not complex A accumulates at the tip of the cilium, suggesting that complex B fails to be coupled to complex A for retrograde IFT. Based on these observations, Wei et al. (2012) proposed that in wild-type C. elegans the BBSome regulates assembly of complexes A and B into intact IFT particles at the base of the cilium, and is then carried as cargo by IFT to the tip of the cilium, where it has a similar role in reorganizing the IFT particle for retrograde transport. In the mutant, the BBSome still functions to assemble the IFT particle at the base of the cilium, but because it is no longer transported to the ciliary tip, IFT particle reorganization is defective and complex B accumulates there.
We previously reported that in C. reinhardtii the BBSome is not an integral part of the IFT machinery but rather appears to be a cargo or cargo adapter, a conclusion now supported by the observations of Wei et al. (2012). However, in C. reinhardtii loss of the BBSome does not seem to affect the assembly of complexes A and B , does not lead to an accumulation of complex B at the tip of the cilium , and does not obviously alter the balance of complex A to complex B within the cilium as would be expected if complex B were being left behind during retrograde transport (Figs. 5 C,6 A,and S2 B). Therefore, in contrast to the situation in C. elegans, it is unlikely that the BBSome has a key role in regulating IFT particle assembly at either the base or the tip of the C. reinhardtii cilium.

The function of PLD in C. reinhardtii
The function of PLD in C. reinhardtii is likely to involve phospholipid signaling (Meijer et al., 2002). Consistent with this, we found that the accumulation of PLD in cilia of C. reinhardtii mutants is correlated with major changes in the phospholipid content of the ciliary membrane. It is possible that PLD cycles through the wild-type cilium as part of a system that monitors and responds to the cell's environment (e.g., to light or to osmotic conditions) via a phospholipid-based signaling pathway. We previously reported that C. reinhardtii bbs mutants are nonphototactic. Phototaxis is dependent on membrane depolarization and Ca 2+ influx (Witman, 1993), both of which activate PLD in C. reinhardtii (Munnik et al., 2000). Changes in membrane lipid composition could affect the functioning of membrane channels and hence phototaxis. Consistent with this, we previously observed that bbs4-1 cells with newly formed cilia were able to phototax for over an hour, but lost that ability as the cilia aged and presumably accumulated PLD and acquired an altered membrane lipid composition ). P-40 in HMEK; and allowed to settle onto poly-l-lysine-coated multi-well slides for 3-6 min. Slides were then submerged into 20°C methanol for 6 min and air dried. All subsequent steps were performed as described in the following paragraph.
For immunofluorescence microscopy of vegetative cells, cells in HMEK were mixed with an equal volume of 6% formaldehyde/0.5% Nonidet P-40 in HMEK, allowed to settle onto coated slides for 8-15 min, washed with PBS, and air dried. The following primary antibodies and dilutions were used: anti-PLD (1:100), anti-IFT139 (1:1), and anti-acetylated -tubulin (1:2,000, mouse; Sigma-Aldrich). Specimens were incubated overnight at 4°C with primary antibodies in PBS/2% BSA/0.05% Tween20. Secondary antibodies linked to Alexa Fluor 488, 568, or 594 (1:1,000; Invitrogen) were applied for 90-120 min at room temperature. Specimens were mounted with ProlongGold (Invitrogen). Images were acquired using Axiovision software (Carl Zeiss) and an AxioCam MRm camera (Carl Zeiss) on a microscope (Axioskop 2 plus; Carl Zeiss) equipped with a 100× NA 1.4 oil DIC Plan-Apochromat objective (Carl Zeiss) and epifluorescence. Image brightness and contrast were adjusted using Photoshop (CS5.1; Adobe), and figures were assembled using Illustrator (CS5.1; Adobe). Capture times and adjustments were similar for images mounted together. An Axio Imager.Z1 equipped with a 100× NA 1.3 EC Plan-Neofluar objective, an Apotome, an axiocam MRm, and Axiovision software (all from Carl Zeiss) was used to obtain optical sections of the immunostained cell shown in Fig. 1  Isolation of cilia Flagellar isolation using the dibucaine method and flagellar amputation by pH shock were performed as previously described by Witman (1986) and Lefebvre (1995), respectively. In brief, cells were concentrated and repeatedly washed in 10 mM Hepes, pH 7.4, resuspended in HMS (10 mM Hepes, pH 7.4, 5 mM MgSO 4 , and 4% wt/vol sucrose), and placed on ice; all subsequent steps were performed at 4°C. 2 ml of dibucaine (25 mM in H 2 O) were added per 10 ml of sample; cells were diciliated by rapidly drawing them up and down in a pipette. 20 ml of 0.7 mM EGTA in HMS was then added to the suspension. Cell bodies were removed by differential centrifugation (3 min at 1,150 g); the supernatant was collected and the remaining cell bodies were removed by centrifugation (10 min at 1,700 g) through a sucrose cushion (10 ml 25% sucrose in HSM). The supernatant was harvested and cilia were pelleted at 27,000 g for 20 min. For deciliation by pH shock, 0.5 M acetic acid was added to cells in M medium with rapid stirring until the pH reached 4.2; after 50 s the solution was neutralized using 0.25 N KOH. Cells were immediately placed into fresh culture medium and allowed to regenerate cilia in bright light at RT with shaking (60 rpm). Isolated flagella were extracted with 1% NP-40 in HMDEKP (30 mM Hepes, 5 mM MgSO 4 , 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, and 1% protease inhibitor cocktail [Sigma-Aldrich]) for 20 min on ice, and axonemes and membrane-plus-matrix were separated by centrifugation (27,000 g for 10 min). Alternatively, cilia were extracted with 1% Triton X-114, which allows subsequent separation of the membrane and matrix fractions by phase partitioning. After removing the axonemes, the supernatant was incubated briefly at 37°C and phases were separated by centrifugation (3,300 g for 10 min at RT). The aqueous phase was treated with 1% Triton X-114, the detergent phase was diluted with HMDEKP, and the phase separation was repeated to yield the final aqueous and detergent phases.
For temperature-shift experiments with fla10-1, cells were concentrated and incubated in Modified Medium I (Witman, 1986) with gentle shaking (60 rpm) either at room temperature (22°C) or at 32°C in an incubator. For time course experiments, equal aliquots of concentrated fla10-1 cells were transferred at various time points from 22 to 32°C and incubated for the time period indicated. Then, cilia from all samples were isolated simultaneously using warm (32°C) 10 mM Hepes to wash cells maintained at the restrictive temperature. All experiments using the dhc1b-2 and other IFT mutants were performed at or below 22°C.
Lipidomic and metabolomic analyses of cilia We used two distinct approaches for the lipidomic analysis of cilia, liquid chromatography coupled to mass spectrometry and an approach based solely on mass spectrometry. In brief, cilia were extracted with methanol/ chloroform and lipid extracts were separated by gradient elution normal phase HPLC and the analytes detected by positive atmospheric pressure chemical ionization. For metabolomic analysis by gas chromatography coupled to mass spectrometry, samples were derivatized to their trimethylsilyl derivatives. Chromatography was on a 30-M DB-5MS column. Full scan

BBS likely involves progressive deterioration of ciliary function
The observation that ciliary defects resulting from BBSome deficiency increase with time potentially has implications for the pathomechanism of BBS in mammals. Several features of BBS in murine Bbs / models or human patients with BBS also increase with time or manifest themselves only years or decades after birth (Beales, 2005). The outer segments of photoreceptor cells in the eye, for example, initially develop and function normally in Bbs2 and Bbs4 knockout mice. About 6 wk postnatally, rhodopsin mislocalization becomes apparent and retinal degeneration begins (Nishimura et al., 2004). Therefore, BBSome defects appear to initiate a cascade of events that increasingly deteriorate ciliary protein composition and function. The time scales in which ciliary defects develop in different organisms and different tissues within an organism may be different. Nevertheless, the mechanism by which cells use the BBSome to control ciliary protein transport and to maintain the balance of ciliary signaling proteins might very well be conserved.

Materials and methods
Strains and culture conditions Wild-type and mutant strains of C. reinhardtii were mostly maintained as described in Lechtreck et al. (2009). The bbs8-1 strain was generated as described in the legend of Fig. S1. The retrograde IFT mutants fla15 and fla17-1 were aerated without CO 2 supplement and maintained at 22°C.

Mating experiments and fluorescence microscopy
To generate gametes, cells were grown for 10 d on Tris-acetate-phosphate medium plates (Gorman and Levine, 1965) and resuspended in modified medium I without nitrogen (8 ml/plate; M-N; http://www.chlamy.org/ SG.html), and the suspension was incubated overnight in constant light with gentle agitation. The next morning, cells were incubated in 20% M-N supplemented with 10 mM Hepes for 3 h. To obtain a series of zygotes of distinct age, gametes were mixed at various consecutive time points. Next, the cells were harvested (3 min at 2,000 rpm, at room temperature); resuspended in 10 mM Hepes, 5 mM MgSO 4 , 5 mM EGTA, and 25 mM KCl (HMEK); mixed with an equal volume of 6% formaldehyde/0.1% Nonidet


extfieldsofstudy: Chemistry Medicine

text:
Consequences of glycan truncation on Fc structural integrity

Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of CH2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual CH2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, CH2 domains collapsed onto CH3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability.

Introduction
Proteins-based therapeutics are an important class of biopharmaceuticals that have substantially increased the number of treatable diseases. 1,2 Among the biopharmaceuticals currently in development, monoclonal antibodies (mAbs) make up the largest portion. Antibodies perform multiple functions-each antibody is composed of two Fab regions capable of binding antigen and one Fc region, which modulates antibody clearance and activation of antigen destruction through effector function pathways such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). 3 The Fc region consists of four structural domains, two C H 2 and two C H 3 domains (Fig. 1). In antibodies of the IgG class, each C H 2 domain in the Fc region has a complex biantennary oligosaccharide, termed a glycan (Fig. 2), attached at Asn297 (in Kabat numbering scheme 4 ). Due to the presence of different terminal sugars, the composition of these glycans can be Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of C H 2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual C H 2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, C H 2 domains collapsed onto C H 3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability. Abbreviations: mAb, monoclonal antibody; RMSD, root mean square deviation; RMSF, root mean square fluctuation; Rg, radius of gyration; HpASA, hydrophobic accessible surface area; GAL, galactose; FUC, fucose, MAN, mannose; NAG, N-acetyl glucosamine one of several forms G2F, G0F, and G2FS2 (Fig. 2). Microheterogeneity in Fc glycan composition varies with species and expression systems used to produce candidate mAbs during research and commercial manufacturing. 5,6 Glycan composition is also known to influence the activation of specific effector function pathways differentially. 7,8 For example, truncating the G2F glycoform by degalactosylation to G0F can decrease CDC activation without affecting ADCC activation. 9,10 Effector function activity can also be modulated by defucosylation, which has been shown to increase ADCC activity. 11 These discoveries have inspired the development of different engineering approaches to enhance/optimize antibody effector function activity by altering the Fc-bound glycan profile or by exchanging amino acids in the protein backbone. 12,13 Glycan modification techniques such as these have led to the first market-approved glycoengineered monoclonal antibody, 14 and may be useful in optimizing effector function activities of therapeutic Fc-fusion proteins as well. 15 www.landesbioscience.com mAbs 905 REPORT

REPORT
In a separate study by Zheng et al., full deglycosylation was shown to expand the Fc hydrodynamic radius, increasing its susceptibility to proteolytic cleavage by papain. 20 Other studies have shown that partial and full deglycosylation can also increase the propensity of IgG1 Fc fragments to aggregate. 21 Therefore, understanding the mechanism that underlies the stability differences of fully and partially deglycosylated IgG Fc fragments is important to the development of therapeutic mAbs. 22 Effective characterization of protein-based therapeutics such as mAbs is important to facilitate their translation from discovery to commercially-available drugs. Achieving this goal partly depends on the sensitivity of analytical tools to provide chemical/physical information. Due to the size and complexity of fulllength mAbs, however, characterization of their higher order structure with currently available experimental methodologies is challenging. The tools available for characterization, including differential scanning calorimetry, analytical ultracentrifugation, circular dichroism, isothermal calorimetry, can provide a global view of mAb structure/stability, but cannot determine specific protein sequence or structural sites that can affect experimental observations. On the other hand, X-ray crystallography can provide detailed structural information, but structures are relatively static and do not provide dynamic information beyond crystallographic B-factors. 23 In principle, protein solution dynamics can be determined using nuclear magnetic resonance (NMR), but intact and IgG fragments are too large for routine NMR 3-D structural analyses. Moreover, NMR experiments require samples with highly concentrated protein, which can distort antibody structure and lead to aggregation. 24 Therefore, it is important for biopharmaceutical development to seek new analytical tools to detect protein structural changes with a high degree of sensitivity.
Atomistic and coarse-grained molecular dynamics (MD) simulations can play an important role in fulfilling this need. [25][26][27][28][29] MD can assess both the global and local conformational behavior of mAbs in solution under a variety of different conditions. Our recent analyses of a murine IgG2a using molecular dynamics provided new insights into mAb dynamics by comparing structural change differences upon thermal stress and deglycosylation. 30 Studies such as these and others illustrate how MD can be an important addition to the set of tools available for characterization of protein structural changes that may occur during the shipping and storage of biopharmaceuticals. 31,32 In this work, explicit water simulations were performed on the following Fc glycoform variants DEGLYCO, M3N2, and G2F, which were modeled using the crystal structure of a human mAb, PDB entry 1HZH (Table 1; Fig. 2). Each system was simulated at 300 K and 375 K for 200 ns in two independent production runs of 100 ns each. Total simulation time was 400 ns per Fc glycoform variant, and the overall total simulated time for this study was 1.2 μs. Simulations were evaluated for differences in domain ensemble averages, FcR (I-III) binding site closure, local structure perturbations, and sugar dynamics/ conformational preferences. Clustering glycan-glycan conformations in simulation trajectories revealed a preferred glycan-glycan conformation that is significantly different than the one in the crystal structure (1HZH). Moreover, the preferred glycan-glycan From a biopharmaceutical development perspective, truncating or removing glycans attached to therapeutic mAb candidates can also have undesirable consequences such as reduced Fc thermostability and increased aggregation. Consequently, it is important to ensure a consistent glycan profile across manufacturing batches or to match the glycan profile of an innovator product when developing a biosimilar version. 16 Therefore, much effort has been devoted to the development of rapid high through-put methods for glycan profile determination. 17,18 In a set of experiments demonstrating the effect glycan composition can have on Fc effector function activity, the thermostability of C H 2 domains decreased as more carbohydrates were removed from the glycan. 19 Figure 1. Fc structure (A) indicating the locations of structural domains, two attached oligosaccharides colored cyan, and the position of Gly237 residues used to calculate the C H 2-C H 2 distance. For chain H, the C H 2 domain is red and the C H 3 domain is orange. For chain K, the C H 2 domain is green and the C H 3 domain is blue. The hinge, which was present in all glycoform variant simulations, has been left out of this representation for the sake of clarity. C H 2 domain schematic (B) with β-strands labeled and residue indices for strands in Kabat numbering scheme. Two phenylalanine residues 241 and 243, important for C H 2 domain stability, are located on β-strand A. The oligosaccharide attachment residue, Asn297, is located in loop C'E.  19 Desialylation and degalactosylation, however, did not affect C H 2 domain thermostability to the same degree (C H 2 Tm for G0 is 71.5 °C). 19 Because NAG9 does not form interactions with C H 2 domains, the loss of C H 2 domain thermostability upon the removal of NAG6 (see above for M3N2) can be attributed to key C H 2 domain stabilizing interactions formed between NAG6 and Phe243 (Kabat) of β-strand A. 34,35 No further destabilization of C H 2 domains was observed upon removal of MAN5 or MAN8 from M3N2 to produce MN2 (NAG1-NAG2-MAN4), (C H 2 Tm for MN2 is 67.3 °C). 19 However, full deglycosylation did lower the C H 2 melting temperature below that of MN2 (C H 2 Tm for DEGLCYO is 65.9 °C). 19 This provides supporting evidence for the importance of interactions between MAN4 and Phe241. 34,35 The significance of the MAN4-Phe241 and NAG6-Phe243 interactions in stabilizing C H 2 domains was also supported by the work of Voynov et al. who showed that mutations, F241S and F243S, significantly lowered C H 2 domain melting temperatures for fully glycosylated glycoforms (G0, G1, and G2). 28 Here, individual domain ensemble averages are reported to understand the stabilizing effect that full length glycans have on C H 2 and C H 3 thermostability. Simulation evaluations began by measuring the degree of structural change using root mean square deviation (RMSD), radius of gyration (Rg), and hydrophobic accessible surface area (HpASA) for glycoform variants during equilibrated simulations. For each of these global measures, the magnitudes of structural change were then compared with the thermodynamic melting temperature differences among glycoform variants or other available experimental data. To calculate domain ensemble averages for all glycoform conformation has several novel inter-glycan contacts that are not formed in the crystal structure (1HZH). Consistent with the above observations, structural differences among the glycoform variants during simulations suggest that glycan truncation/ removal can cause quaternary structural deformation as a result of the loss or disruption of a significant number of these inter-glycan contacts. Glycan truncation can also increase the structural deformation of C H 2 domains, demonstrating the importance of specific carbohydrates in stabilizing C H 2 domains. In elevated temperature simulations, glycan truncation appears to affect structure deformation differentially in locations (Helix-1 residues 246-251 and Helix-2 residues 310-315) that are far from the oligosaccharide attachment point (Asn 297). These helices form part of the FcRn binding site and their preservation is important in maintaining pharmacokinetic and pharmacodynamic properties of therapeutic mAb candidates. Observations from these glycan truncation simulations have improved our understanding on how glycan composition can impact pharmaceutical stability of protein therapeutics and demonstrated the usefulness of molecular dynamics simulations in biopharmaceutical development studies.

Results
Individual domain ensemble averages. The Fc structure is described in Figure 1, and Figure 2 presents the location of carbohydrates in glycan structure and glycan composition Figure 2. Structure, composition, and branches are labeled for oligosaccharides of G2FS2. The glycan has three terminal carbohydrates: FUC-2, and two N-acetyl neuraminic acids or sialylic acids, labeled S. Desialylating G2FS2 produces G2F. Alternate glycan compositions and structures are shown using a simplified notation. Note that MN2, not shown, can be formed by removing MAN5 and MAN8 from M3N2. ) across all glycoforms. This was expected because the average HpASA was calculated in the absence of the oligosaccharide and there are two hydrophobic residues (Phe241, Phe243) that become solvent-exposed when ignoring the glycan. In comparing the average HpASA for C H 2 domains across glycoform variants at 300 K, values were approximately the same given that the range of HpASAs for C H 2 domains among glycoform variant was ~125 Å 2 , which is a small percentage of the total average C H 2 domain HpASA (~3500 Å 2 ). HpASA standard deviations for C H 2 domains of glycoform variants were even smaller than the range ( Table 2). At 375 K, the average HpASA for C H 2 domain (chain K) of DEGLYCO is less than it was at 300 K (3509.5 Å 2 , 300 K; 3170.6 Å 2 , 375 K). This was due to the collapse of Fc quaternary structure at elevated temperature (Fc ensemble averages are described in the next section). All other domains of both chains (H and K) for glycoforms variants had similar average HpASA values between 300 K and 375 K simulations (± 100 Å 2 ). The lack of differences in average HpASA values among glycoform variants suggests that glycan truncation/removal does not impact the average HpASA of individual structural domains.
Overall, analyses of Fc individual structural domains during simulations indicate that tertiary structure changes in C H 2 and C H 3 domains were relatively small among different glycoform variants at both 300 K and 375 K ( Table 2). Furthermore, C H 3 domain tertiary structure changes upon glycan truncation/removal were smaller than those in C H 2 domains. This is expected because glycosylation sites are located within C H 2 domains. Moreover, the destabilization of C H 2 domains upon glycan truncation (G2F vs. M3N2) and deglycosylation (G2F variants, the last 90 ns of both trajectories at the same temperature were used. Salient observations are described below. At  For example, the C H 2-C H 2 distance in the crystal structure of MN2 is 4.7 Å shorter than it is in the structure for G2F. 35 Full deglycosylation shortened the C H 2-C H 2 distance even further. Based on these observations, it was concluded that progressive glycan truncation reduces Fc activity by shortening the distance between C H 2 domains, appearing to close the FcR (I-III) binding site and decrease FcR binding affinity. To investigate the effect glycan truncation has on overall Fc quaternary structure/activity during simulations, Fc average quaternary structure changes were determined at both temperatures. FcR binding site closure was monitored by computing the distance between Gly237 residues of β-strand A (see Materials and Methods). At 300 K, the C H 2-C H 2 distance did not change appreciably in G2F and M3N2 simulations (Fig. 3A). For DEGLYCO, C H 2 domains moved closer together such that the average C H 2-C H 2 distance was ~4 Å less than the average distance for G2F ( Table 2). The mutual approach of C H 2 domains in deglycosylated Fc simulations is consistent with observations from crystallographic studies (described above). At an elevated temperature (375 K), the C H 2-C H 2 distance actually increased for DEGLYCO over time. The increase was not due to domain separation as disulfide bonds in the hinge prevent this from happening. Instead, C H 2 domains collapsed onto C H 3 domains by twisting relative to one another, extending the distance between Gly237 residues (Fig. 3B). For DEGLCYO at 375 K, the extent of C H 2 domain collapse onto C H 3 domains can also be seen by comparing average HpASA values for both chains. At 300 K, the average HpASAs for C H 2 and C H 3 domains of chain K were 3509.5 Å 2 and 2367.2 Å 2 , respectively. Collapse of C H 2 domains onto C H 3 domains of chain K decreased the average HpASA for both domains in 375 K simulations (C H 2, 3170.6 Å 2 ; C H 3, 23041.1 Å 2 ). In M3N2 simulations at 375 K, C H 2 domains partially collapsed onto C H 3 domains, but the C H 2-C H 2 distance was restored to the initial simulation distance of 15 Å after 40 ns. C H 2 domains began collapsing again after 70 ns suggesting that the collapse of C H 2 domains in M3N2 simulations at 375 K could be a transient event. In G2F simulations at 375 K, the C H 2-C H 2 distance remained relatively constant and similar to the initial simulation distance (15 Å).
RMSD ensemble averages for the complete Fc fragment ( Table 2) were similar to the changes in C H 2-C H 2 distance during simulations (Fig. 3A and B). At 300 K, the structure of DEGLYCO deformed by 4.65 Å compared with the crystal structure, and M3N2 and G2F deformed by 4.25Å and 3.74 Å, respectively. The same increasing trend of average RMSD among glycoform variants was even more apparent at 375 K (DEGLYCO, 8.09 Å; M3N2, 6.97 Å; G2F, 5.60 Å). The Fc ensemble average RMSD for DEGLYCO was 2.5 Å greater than the average RMSD for G2F in elevated temperature simulations ( Table 2). The greater Fc quaternary structural deformations in elevated temperature simulations compared with 300 K simulations was likely due to the collapse of C H 2 domains onto C H 3 domains. By comparing Fc average RMSD differences among glycoform variants to their C H 2-C H 2 distance changes during simulations, it appears that these two descriptors of structural change are interrelated, i.e., the movements of the individual C H 2 domains with vs. DEGLYCO) was consistent with thermodynamic measurements summarized at the beginning of this section. Qualitative recapitulation of experiment results provides confidence in subsequent simulation analyses.
The Fc average radius of gyration (Rg) during DEGLYCO simulations was slightly larger than it was during M3N2 and G2F simulations at 300 K (DEGLYCO, 26.68 Å; M3N2, 26.48 Å; G2F, 26.30 Å), ( Table 2). This observation is unexpected because C H 2-C H 2 approach would seemingly decrease the overall volume of the quaternary structure (see above), but it is in agreement with experimental observations of a slight increase in hydrodynamic radius and susceptibility to papain cleavage upon deglycosylation. 20 In simulations at 375 K, the Fc average Rg for DEGLYCO was less than it was for G2F and M3N2 due to the collapse of C H 2 domains onto C H 3 domains (DEGLYCO, 24.12 Å; M3N2, 25.75 Å, G2F, 25.55 Å), ( Table 2). The collapse of C H 2 domains onto C H 3 domains in DEGLCYO simulations also affected the Fc average HpASA, which was calculated in the absence of the glycan for all glycoform variants ( Table 2). While the Fc average HpASA was approximately the same for all glycoform variants at 300 K (DEGLYCO, 14034.9 Å 2 ; M3N2, 14125.8 Å 2 ; G2F, 14015.6 Å 2 ), at 375 K, the average HpASA decreased due to the reduction in overall accessible surface area, resulting from C H 2 domain collapse (DELGYCO, 13185.5 Å 2 ; M3N2, 14081.0 Å 2 ; G2F, 14126.8 Å 2 , see Table 2). Overall, the observations from Fc and structural domain ensemble averages indicate that glycan truncation and deglycosylation can affect both C H 2 domain tertiary structure and Fc quaternary structure. The differences in quaternary structure deformation among glycoform variants were, however, significantly larger than differences in tertiary structure deformation. This suggests that individual C H 2 domains move with respect of each other and with respect to C H 3 domains. Similar observations have also been made in earlier simulations of a full length murine IgG2a mAb. 30 The consequence of these inter-domain movements is that the effector function activity can become impaired.
Local structure deformations upon glycan truncation/ removal. Glycan truncation/removal can also perturb residues in loops that constitute the FcR binding site such as BC, FG, and C'E loops (Fig. 1B). Crystal structures of G2F, G0F, and M3N2F suggest that removal of NAG6 perturbs the C'E loop. 35 Variants with an attached NAG6 carbohydrate (e.g., G0F) do not show differences in C'E loop perturbations when compared with the crystal structure of G2F. NMR studies have supported these findings and added that glycan truncation can also perturb the lower hinge region as well as the FG loop after full deglycosylation. 33 To monitor the local structure changes upon glycan truncation, average residue-wise RMSD values were computed. Since C H 3 domains were mostly unaffected by glycan truncation ( Table 2), this section focuses on the local structure changes in C H 2 domains only. The average residue-wise RMSD values are reported for β-strands and connecting loops by averaging between C H 2 domains of heavy chains, H and K (see Materials and Methods). The average residue-wise RMSD values were compared for similarities and differences at 300 K and 375 K among glycoform variants to identify local structure that is susceptible to glycan truncation and removal. temperature (300 K vs. 375 K) and the extent of glycan truncation. Helix-1 (residues 246-251) and Helix-2 (residues 310-315), which form part of the FcRn binding site, are located far from the glycan attachment site (Asn 297). Deformation of these helices could affect FcRn binding if these regions are unable to refold after temperature normalization. FcRn binding is an important determinant of pharmacokinetic and pharmacodynamic properties of therapeutic mAb candidates. 36 Glycan dynamics. Enzymatic sialylation of galactosylated glycan termini was shown to be anti-inflammatory in mice. [37][38][39] From a glycoengineering standpoint, enzymatic sialylation of normally galactosylated IgGs has the potential to improve therapy for patients suffering from inflammatory diseases such as rheumatoid arthritis. While a recent report suggests that sialylation produces an anti-inflammatory effect by biasing the FcR binding site toward the "closed" conformation, 40 it remains unclear how galactosylated termini are accessible for sialylation given their location in the cavity formed by C H 2 domains. Crystal structures of galactosylated Fc fragments show that glycan termini are normally buried between C H 2 domains. In early solution NMR measurements of Fc glycoforms, the α1-6 branch was completely immobilized to the surface of C H 2 domains while the α1-3 branch appeared to be more dynamic (Fig. 2). 41,42 Immobilization would seemingly prevent the α1-6 branch from enzymatic sialylation or interactions with receptors. A more recent NMR study indicates that the termini of both glycan branches are highly dynamic. 43,44 Here, we investigated the dynamics of both glycan branches at 300 K and 375 K using Root Mean Square Fluctuation (RMSF), ( Table 3). RMSF is a measure of how much a carbohydrate fluctuates about its average position. It is, therefore, an appropriate measure of glycan dynamic behavior. RMSF values for each carbohydrate were averaged between H and K glycans and across trajectories (see Methods).
At 300 K, the most mobile carbohydrates in G2F as measured by average RMSF were the branch termini GAL7 (1.0 Å), GAL10 (2.0 Å), and FUC2 (1.0 Å), (Fig. 2 and 5A). In comparing carbohydrates of the α1-6 and α1-3 branches, NAG9 and MAN8 (α1-3) fluctuated more than NAG6 and MAN5 (α1-6). Therefore, the α1-6 branch was less mobile than the α1-3 branch in 300 K simulations, agreeing with earlier NMR studies. Moreover, upon visual inspection of simulation trajectories, it was found that the α1-6 branch never lost all protein-glycan interactions as was proposed to explain α1-6 sialylation. 43,44 The retention of contacts between α1-6 carbohydrates and C H 2 domains can also be seen in Table 3. The NAG6-F243 contact and all GAL7 contacts were retained during simulations ~100% of the time (NAG6, GAL7 are located on the α1-6). It is possible, however, that 100 ns of simulation time may not be enough to see GAL7 lose its contacts, despite a reported α1-6 branch correlation time of 37 ns. 44 Average RMSF values between M3N2 and G2F were similar except at MAN5 (α1-6), (Fig. 5). The truncated glycan, M3N2, had similar carbohydrate mobilities to G2F in the more mobile α1-3, but the α1-6 branch was more mobile in M3N2 than in G2F due to the loss of glycan-protein interactions. Thus, GAL7 and NAG6 glycan-protein interactions at an elevated temperature (375 K), the perturbation differences in loop FG among variants did not exist (see below). Therefore, local structure deformation differences among glycoform variants were mostly localized to loop C'E loop. Furthermore, the localization of local structure perturbation differences to loop C'E suggests that differences in C H 2 domain ensemble average RMSDs among glycoform variants ( Table 2) could be a result of loop C'E deformation differences.
At 375 K, local structure perturbations in C H 2 domains were greater than at 300 K, and glycan truncation generally increased the extent of perturbation for local structure regions (Fig. 4A  and B). Comparing local structure perturbations between 300 K and 375 K, the local regions most affected by an increase in temperature were Helix-1 and Helix-2. At 375 K, both these helices deformed 2.0 Å more than they did at 300 K, indicating that these regions were highly susceptible to deformation at elevated temperatures. For DEGLCYO, the local regions most perturbed from the crystal structure were in Helix-1 (4.5 Å), loop BC (3.5 Å), β-strand C (3.0 Å), β-strand C' (3.0 Å), loop C'E (3.5 Å), Helix-2 (4.0 Å), and loop FG (2.5 Å). Comparing average residue-wise RMSDs among glycoform variants at 375 K, the regions of local structure with similar perturbations were in loops BC and FG loop, and all β-strands (Fig. 4A). Only β-strand C' differed among glycoform variants as it was heavily perturbed in one of the M3N2 simulations at 375 K. Local regions with differences in perturbation among glycoform variants were in Helix-1 (DEGLYCO, 4.0 Å; M3N2, 3.0 Å; G2F, 2.5 Å), loop AB (DEGLYCO, 4.5 Å; M3N2 and G2F, 3.0 Å), Helix-2 (DEGLYCO, 4.0 Å; M3N2, 3.0 Å; G2F, 2.5 Å), and loop C'E (DEGLYCO and M3N2, 3.5 Å; G2F, 2.5Å). Based on these differences, the extent of perturbation for these local structure regions generally increased due to glycan truncation/ removal. Taken together, observations from 300 K and 375 K simulations suggest that the amount of local structure perturbation within C H 2 domains depended both on the simulation NAG9, GAL10) facing in an outward direction. Overall, the inter-glycan contacts in the preferred conformation were shifted compared with those in the crystal (Fig. 6), and crystal contacts MAN4(H)-MAN8(K) and MAN5(H)-MAN8(K) were not well maintained ( Table 3). The critical stabilizing C H 2 domain contacts NAG6-F243, and MAN4-F241 were ~100% retained for both glycans. Comparatively, fucose-C'E loop contacts (FUC2-Y296 [H], FUC2-Q295 [K]) were partially lost during simulations, indicating much weaker interactions. Taken together, the preferred oligosaccharide conformation had significantly more inter-glycan contacts than observed in crystal structure (1HZH) and inter-glycan contacts were crucial to maintain Fc quaternary structure and/or activity. These observations may are important in restricting α1-6 branch carbohydrate dynamics. As in 300 K simulations, the most mobile carbohydrates at 375 K were the branch termini GAL7 (3.0 Å), GAL10 (3.0 Å), and FUC2 (2.5 Å). A comparison G2F carbohydrate mobilities in 300 K and 375 K simulations shows that there was an average 1.5 Å increase in average RMSF among carbohydrate moieties. At 375 K, MAN5 and NAG6 (α1-6) were more mobile than MAN8 and NAG9 (α1-3), (Fig. 5). Upon visual inspection of G2F simulation trajectories at 375 K, GAL7 did not leave the protein surface, but the greater mobility of the α1-6 branch than that of the α1-3 branch, at an elevated temperature, does suggest that this branch could expose its termini for sialylation over time.
Carbohydrate conformation/contacts. The crystal structure complex of FcRIII bound to the Fc fragment has revealed the existence of a single contact between oligosaccharides and FcRIII. 45 Because FcRIII activation is dependent on Fc glycan composition 19 (see section Fc quaternary structure ensemble averages), oligosaccharide moieties exert their influence indirectly through modification of the Fc conformation. As previously stated, glycan truncation can reduce FcR (I-III) binding by decreasing the distance between C H 2 domain, and thereby causing the FcR binding site to close. Therefore, contacts between oligosaccharides from glycans attached to different heavy chains (inter-glycan contacts) are crucial to maintain the Fc in an active state. Fc fragment crystal structures with intact glycans, however, show very few inter-glycan contacts with MAN8 mediating most of the inter-glycan contacts. 34,46,47 Moreover, Fc crystal structures obtained under different crystallization conditions have different inter-glycan interactions. 35 Thus, there is a need to investigate inter-glycan interactions and their dynamics to understand their effect on Fc conformation.
Here, the inter-glycan contacts from the crystal structure (1HZH) are compared with the preferred glycan-glycan conformation found during G2F simulations at 300 K ( Fig. 6A and B). The preferred glycan (chain H) -glycan (chain K) conformation, labeled 8240, was identified by clustering all G2F trajectory samples from both 100 ns simulations (see Methods). In total, 85% of all structures submitted for clustering had the preferred glycan-glycan conformation. The crystal structure (1HZH) with modeled GAL10(K) and FUC(H) showed only three inter-glycan contacts MAN4(H)-MAN8(K), MAN5(H)-MAN8(K), MAN8(H)-NAG3(K), (Fig. 6A; Table 3). The glycan-glycan conformation has both α1-3 branches facing down and away from the FcR binding site, and both α1-6 branch termini have GAL7 bound to the protein surface. 48 In the preferred glycanglycan conformation from simulations, both α1-3 branches were extended and faced outwards (Fig. 6B). The outward position of the α1-3 branch may allow sialylation. Compared with the crystal structure, several new inter-glycan contacts were observed in the preferred glycan-glycan structure. These are   57,58 Gray outlines show the surface of carbohydrates. Yellow dotted lines indicate inter-glycan contacts (identified at ≤ 4.5 Å for non-hydrogen atoms) between chains H and K in the crystal structure. Red dotted lines indicate inter-glycan contacts in the preferred glycanglycan conformation that were formed during simulations at 300 K and are not formed in the crystal structure for 1HZH.
www.landesbioscience.com mAbs 913 to mimic the thermal stress experienced by biopharmaceuticals as a result of occasional temperature incursions that may happen during manufacture, shipping and storage. These simulations are analogous to accelerated stability studies, which are routinely performed in pharmaceutical development. Importantly, the temperature values in molecular dynamics simulations should be carefully interpreted. In simulations, temperature is simply a measure of the amount of kinetic energy in the system. As such, elevating the temperature is a commonly used strategy to allow molecular systems to escape from deep energy minima. 49 However, elevating the simulation temperature is not a reliable strategy to reproduce experimental temperature-dependent phenomena such as changes in protein-protein binding affinity. On the other hand, the molecular origins that underpin protein conformational changes upon thermal stress can be reliably accelerated by arbitrarily increasing the temperature. [50][51][52] The molecular origins of these changes remain similar when simulations are performed at different elevated temperatures, but the time interval have important implications not only for basic understanding of Fc structure-function relationships, but also toward rational design of glycoengineered antibodies.

Discussion
In addition to simulations performed at 300 K, this study reports findings for molecular dynamics performed at an elevated temperature (375 K) used to enhance conformational sampling and perturb glycoform variants during simulations. In these elevated temperature simulations, the goal was not to unfold the IgG Fc molecular system. Protein folding/unfolding transitions occur over the millisecond to second timescales. One hundred nanoseconds of all-atom explicit solvent simulation time cannot achieve this for a molecular system as large as the Fc fragment, even when such studies are performed at an elevated temperature. Instead, the elevated temperature simulations in this study were performed to perturb the Fc molecular system as a means scheme. The inter-heavy chain disulfide bond formed by Cys229 residues, which is not formed in the 1HZH crystal structure, was modeled in using MOE. 53 Glycans for each glycoform variant were identical (M3N2 and G2F). GAL10 of chain K and FUC2 of chain H (Fig. 2), which are absent in the 1HZH crystal structure, were modeled for the G2F variant using MOE, with identical glycosidic dihedral angles to their corresponding carbohydrate, which is present in the opposite glycan. Histidines were doubly protonated, simulating a pH near or below 6, which is commonly used in formulation development. Each Fc system was solvated in a TIP3P water box with an initial box size of ~786 348 Å 3 for all glycoform variants. Chloride ions were added to neutralize all systems, resulting in average Clion concentration of ~16 mM per bath of water molecules. Energy minimizations and molecular dynamics simulations were performed using the NAMD package 54 with the AMBER99 all-atom forcefield 55 and the GLYCAM06 forcefield for carbohydrates. 56 Molecular systems were first subjected to 20 000 energy minimization steps with protein heavy atoms fixed, followed by 5000 steps of energy minimization without any restraints. Production runs were performed at 300 K and 375 K and each system was simulated for 100 ns. Trajectory samples were taken every 10 ps. For all glycoform variants, DEGLYCO, M3N2, G2F, two production simulations were performed at each temperature (Table 1). Overall, 12 production runs were performed during this study for a total simulation time of 1.2 μs.
Ensemble averages were calculated using the last 90 ns of each production run. The first 10 ns were used as an equilibration phase. System equilibration was assessed by monitoring energy, temperature, and pressure profiles. Domain ensemble averages were computed without the hinge or linker between C H 2-C H 3 domains. Reported ensemble averages are values averaged over simulation time (trajectory samples taken 10 ps apart) and between the two production runs at each temperature. RMSD and Rg values were calculated using all heavy atoms. HpASA was calculated using all carbon atoms and carbon bonded hydrogens in the absence of the glycan. The C H 2-C H 2 distance was computed between Gly237 (β-strand A) pairs, which is near the FcR (I-III) binding site (Fig. 1A).
For all trajectory samples, C H 2 structural domains were superimposed separately onto their corresponding crystal structure coordinates (chain H onto H, chain K onto K) and residue-wise RMSD values were computed using all atoms. Superimposing individual structural domains limits residue-wise RMSD values to the tertiary structure changes and excludes the effect of quaternary structural changes. Residue-wise RMSDs were then averaged across all trajectory structure samples (10 ps apart). Residue-wise RMSDs were also averaged between C H 2 domains (H and K) and across the two production runs at the same temperature (300 K and 375 K). RMSF values for glycans were computed by first superimposing glycans individually (chain H onto H, chain K onto K) onto the glycan coordinates found in the crystal structure (1HZH). RMSF values for glycan carbohydrates were also averaged between chains and production runs. necessary to observe these conformational changes is different due to varying the kinetic energy.
In the presented simulation analyses, glycoform variants were evaluated for differences in domain ensemble averages, FcR (I-III) binding site closure, local structure perturbations, and sugar dynamics/conformational preferences. Upon glycan truncation/removal, we find that simulations can recapitulate the thermodynamic trends for C H 2 and C H 3 domains. The closure of the FcR binding site was dependent on the degree of glycan truncation. During simulations at both 300 K and 375 K, most local structure deformation differences among glycoform variants were confined to the C'E loop, which contains the oligosaccharide attachment point, Asn297. In simulations at elevated temperature, Helix-1 and Helix-2 were the most perturbed local structures and were more perturbed for the deglycosylated glycoform variant than for the other variants. Within the glycan, the oligosaccharides of the α1-3 branch were more mobile than those of the α1-6 at room temperature (300K); however, at an elevated temperature (375K), the α1-6 branch was more mobile than the α1-3 branch. Furthermore, the preferred glycan-glycan conformation, identified by clustering glycan-glycan conformations in simulation trajectories, had significantly more inter-glycan contacts than those found in the crystal structure.
Observations of structural differences among glycoform variants suggest that glycan truncation and removal can cause quaternary structural deformation of the Fc region by C H 2 domain closure which results from the loss (DEGLYCO) or disruption (M3N2) of a significant number of inter-glycan contacts revealed by the preferred glycan-glycan conformation observed in simulations. These inter-glycan contacts are not formed in the crystal structure, PDB entry 1HZH. Glycan truncation also destabilized C H 2 domains by deforming the C'E loop at 300 K, suggesting that the absence of glycans can increase protein entropy, leading to domain destabilization. Glycan truncation also appears to affect structure deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point in simulations at elevated temperature. Deformation of these local structures could affect FcRn binding if these regions are unable to refold after temperature normalization. Observations from these glycan truncation simulations have improved our understanding of how glycan composition can affect mAb stability and demonstrated the usefulness of molecular dynamics simulations in biopharmaceutical development studies.

Materials and Methods
Fc coordinates were taken from Protein Data Bank (PDB) entry 1HZH (heavy chains labeled H and K) that contains atomic coordinates for the full length human IgG1 b12 antibody. 48 The N and C termini were acetylated and amidated, respectively, to neutralize their charges. All inter-chain and intra-domain cysteine (Cys) pairs were disulfide bonded. Coordinates for the hinge (225-TCPPCPAPELLGG-237, Kabat numbering scheme 4 ) were included in all Fc glycoform variant simulations. All residues noted here are numbered according to the Kabat www.landesbioscience.com mAbs 915

Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.