extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains.

The earliest known step in the activation of the high affinity IgE receptor, FcεRI, is the tyrosine phosphorylation of its β and γ subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of FcεRI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of β and γ occurs in intact cells only for FcεRI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of FcεRI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in FcεRI activation. Participation of membrane domains accounts for previously unexplained aspects of FcεRI-mediated signaling and may be relevant to signaling by other multichain immune receptors.

The earliest known step in the activation of the high affinity IgE receptor, Fc⑀RI, is the tyrosine phosphorylation of its ␤ and ␥ subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of Fc⑀RI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of ␤ and ␥ occurs in intact cells only for Fc⑀RI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of Fc⑀RI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in Fc⑀RI activation. Participation of membrane domains accounts for previously unexplained aspects of Fc⑀RI-mediated signaling and may be relevant to signaling by other multichain immune receptors.
The plasma membrane contains specialized regions that have distinct compositions and can serve unique functions in the regulation of cell surface receptor activation. For example, caveolae have been shown to associate with certain signaling proteins (1,2) and have been implicated in receptor activation (3)(4)(5)(6), vesicular transport (7,8), and the uptake of small molecules (9). Compositionally related membrane domains, which lack the invaginated morphology of caveolae as well as the membrane protein caveolin, have also been identified and biochemically separated from caveolae (10). These membrane domains, like caveolae, are resistant to solubilization in nonionic detergents such as Triton X-100, are enriched in sphingolipids and glycosylphosphatidylinositol-linked proteins, and are associated with palmitoyl-anchored signaling molecules including Src family tyrosine kinases (10 -14). Detergent-resistant membrane domains isolated from rat basophilic leukemia (RBL) 1 cells, a mast cell line, contain at least 30% of the cellular Lyn, a Src family tyrosine kinase, and no detectable caveolin (15).
Aggregation of Fc⑀RI on mast cells and basophils by multivalent antigens leads to phosphorylation of immunoreceptor tyrosine-based activation motifs within the ␤ and ␥ receptor subunits by Lyn (16 -19). This initiates a signaling cascade culminating in secretion of inflammatory mediators and cytokines that play an important role in the allergic response (20,21). The molecular mechanism by which aggregation of Fc⑀RI initiates its phosphorylation by Lyn is incompletely understood. Selective binding of Lyn directly to unphosphorylated Fc⑀RI ␤ (22) has been proposed to mediate an initial transphosphorylation of aggregated Fc⑀RI (23), but this does not account for the capacity of Fc⑀RI lacking the ␤ subunit (24,25) or chimeric receptors containing only the ␥ cytoplasmic tail (26 -28) to become tyrosine-phosphorylated upon aggregation. The involvement of detergent-resistant membrane domains in Fc⑀RI signaling was recently suggested by the observation that aggregation of Fc⑀RI on RBL cells significantly increased the amount of active Lyn associated with these structures (15). Furthermore, fluorescence microscopy studies showed that aggregation of Fc⑀RI at the surface of intact cells co-redistributes ganglioside-enriched membrane patches that are related to the isolated membrane domains (29,30). The aggregation-dependent association of Fc⑀RI with these less fluid regions of the membrane (30,31) is also consistent with decreased lateral and rotational mobility of aggregated Fc⑀RI (reviewed in Ref. 32). In the present study, we establish conditions for preserving the interaction of aggregated Fc⑀RI with these membrane domains following cell lysis, and we demonstrate the importance of this interaction to the initial step in signaling, the tyrosine phosphorylation of Fc⑀RI.
Immunoblotting-Electrophoresis of samples was carried out on 12.5% acrylamide SDS gels under nonreducing conditions, and semidry transfer to Immobilon P (Millipore, Bedford, MA) was performed as described (15). Anti-phosphotyrosine immunoblots were performed using 0.1 g/ml monoclonal antibody 4G10 conjugated to horseradish peroxidase (UBI, Lake Placid, NY) and Supersignal ECL substrate (Pierce, Rockford, IL). For the results in Fig. 2, tyrosine phosphorylation of the Fc⑀RI ␤ subunit was quantified from anti-phosphotyrosine immunoblots of post-nuclear supernatants of 10 6 RBL cells lysed in 0.2% Triton X-100. The prominent 34-kDa band detected in these blots after Fc⑀RI stimulation was identified as ␤ based on selective immunodepletion by IgE-specific agarose beads. 2 This band was quantified with a 256 gray-scale scanner (Umax Vista-S6E) and NIH Image software.
Immunoprecipitations-After adjusting the sucrose fractions to 0.2% Triton X-100 to extract Fc⑀RI from the membrane domains, Fc⑀RI was immunoprecipitated for 90 min with trinitrophenyl-conjugated Sepharose 4B (which efficiently binds anti-DNP IgE). The immunoprecipitates were washed twice with 0.2% Triton X-100 and once in lysis buffer lacking detergent prior to elution by boiling in nonreducing SDS sample buffer.
In Vitro Kinase Assays-Kinase assays were performed by adding kinase buffer (20 mM Tris, pH 7.6, 10 mM MgCl 2 , 1 mM ATP, and 1 mM Na 3 VO 4 ) to the sucrose fractions and incubating at 37°C for 15 min. The reaction was quenched either with 5 ϫ nonreducing SDS sample buffer or by adding 50 mM EDTA followed by immunoprecipitating Fc⑀RI.

RESULTS AND DISCUSSION
In order to determine if the interaction of Fc⑀RI with membrane domains is involved in the activation of this immunoreceptor, we developed conditions that preserve this association during the isolation of these complexes by equilibrium sucrose density ultracentrifugation. As shown in Fig. 1A, limiting amounts of Triton X-100 used for cell lysis preserve the association of aggregated Fc⑀RI (q, f) with the detergent-resistant membrane domains which migrate as low density vesicles (fractions 3-7). In 29 separate experiments, 54 Ϯ 7% of biotin-IgE Fc⑀RI complexes aggregated with streptavidin associate with the membrane domains (q). Significant but lesser amounts of antigen-aggregated receptors associate (f; 11 Ϯ 1%, n ϭ 6), most likely reduced by the partial reversal of IgE-antigen binding during the overnight ultracentrifugation. In contrast, monomeric Fc⑀RI (E) is nearly absent from the membrane domains (3 Ϯ 1%, n ϭ 26) and found almost entirely in the 40% sucrose fractions containing solubilized proteins (fractions 10 -16). The association of Fc⑀RI with isolated membrane domains depends on its aggregation at the cell surface, as less than 5% association is seen for Fc⑀RI aggregated after cell lysis or for Fc⑀RI aggregated with antigen on cells and then dissociated with monovalent hapten after lysis. 2 The interaction between aggregated Fc⑀RI and the membrane domains is very sensitive to the detergent:cell lipid ratio during solubilization and ultracentrifugation, as indicated by its disruption when concentrations of Triton X-100 greater than 0.05% are used (15). This sensitivity is similar to that observed by Pribluda et al. (23) for Fc⑀RI coupling to Lyn in cell lysates, and it contrasts with cytoskeleton-mediated detergent insolubility of aggregated Fc⑀RI (33)(34)(35), which is not disrupted by high Triton X-100 concentrations.
Although this reduction in Triton X-100 used for cell lysis dramatically increases the amount of aggregated Fc⑀RI that remains associated with detergent-resistant membrane domains, these domains are otherwise very similar to those isolated after lysis in high Triton X-100 (Ն0.2%). When directly compared, domains from low and high detergent lysis conditions contain the same fraction of cellular Lyn, and neither has detectable amounts of Src. 2 In addition, both preparations contain a similar spectrum of tyrosine kinase substrates as revealed in in vitro tyrosine kinase assays (Ref. 15 and as described below), and both contain similar amounts of cellular protein (Ͻ2% of the total). 2 By these criteria, the domains obtained using 0.05% Triton X-100 for cell lysis appear to be identical to other membrane domains previously described that do not contain caveolin (10,(13)(14)(15). Furthermore, the aggregation-dependent association of Fc⑀RI with membrane domains shows selectivity among transmembrane cell surface receptors, as Fc⑀RI but not Type I interleukin-1 receptors, both expressed on Chinese hamster ovary cells, associate with membrane domains following aggregation. 3 Association of Fc⑀RI with membrane domains does not require tyrosine phosphorylation of the receptor subunits. As shown in Fig. 1B, RBL cells permeabilized with Streptolysin O in the presence of excess EDTA to inhibit kinase activity show a similar amount of aggregation-dependent association of Fc⑀RI with domains as intact cells (Fig. 1A). As previously shown with broken cells (36), stimulated tyrosine phosphorylation of Fc⑀RI ␤ and other substrates is prevented by EDTA in these permeabilized cells. 2 The presence of Lyn and aggregated Fc⑀RI within the same subregions of the plasma membrane suggests that domainassociated Lyn could be responsible for the initial phosphorylation of the immunoreceptor tyrosine-based activation motifs. Fc⑀RI associates with membrane domains very rapidly at 37°C (E, Fig. 2A) and is more than 50% complete within 30 s, whereas substantially less than 50% of the maximal tyrosine phosphorylation of Fc⑀RI ␤ occurs during this time (q, Fig. 2A). The amount of ␤ tyrosine phosphorylation declines after 2 min at 37°C, and domain-associated receptor also decreases between 5 and 30 min in parallel with its internalization. 2 At 4°C, the association of Fc⑀RI with domains occurs more slowly (E, Fig. 2B), but is clearly more rapid than the ␤ tyrosine phosphorylation during the first 5 min (q, Fig. 2B). Fc⑀RI internalization and downstream signaling such as Ca 2ϩ mobilization and phosphatidylinositol hydrolysis do not occur at 4°C (37), indicating that they are not required for domain association. These results demonstrate that association of Fc⑀RI with membrane domains on cells is an early, aggregationdependent event that is sufficiently rapid to mediate receptor tyrosine phosphorylation.
Evidence for Fc⑀RI tyrosine phosphorylation occurring within membrane domains of intact cells is shown in Fig. 3. Stimulation of biotin-IgE-sensitized RBL cells with streptavidin dramatically increases the tyrosine phosphorylation of many proteins. When lysates of these cells are analyzed by sucrose gradient ultracentrifugation, most of the proteins with 3 K. A. Field, D. Holowka, and B. Baird, manuscript in preparation.  (15) were stimulated for 5 min at 37°C with 500 ng/ml antigen (f, DNP-bovine serum albumin), 10 nM streptavidin (q), or left unstimulated (E) prior to lysis in 0.025% Triton X-100 at 4 ϫ 10 6 cells/ml. Lysates were then diluted 1:1 with 80% sucrose containing 0.025% Triton X-100 and loaded into sucrose step gradients (right axis) followed by ultracentrifugation at 250,000 ϫ g overnight at 4°C. After fractionating the gradients in 0.2-ml aliquots, the distribution of 125 I-IgE-Fc⑀RI was determined and is expressed as the fraction of total 125 I present in the gradient, including the pellet. Error bars show the range of duplicate gradients run on the same day. B, RBL cells sensitized as above and suspended in 20 mM phosphate, pH 7.5, 150 mM NaCl, and 5 mM EDTA were permeabilized with 0.4 units/ml of Streptolysin O (Burroughs-Wellcome, Research Triangle Park, NC) for 15 min at 37°C. The permeabilized cells were then stimulated with streptavidin (q) or not (E) followed by lysis and ultracentrifugation as in A. Staining with Trypan blue confirmed that nearly 100% of the cells were permeabilized by Streptolysin O, and immunoblotting showed no stimulated phosphorylation of Fc⑀RI ␤ under these conditions. 2 enhanced tyrosine phosphorylation are found with the solubilized proteins at 40% sucrose (fractions 11-16), as expected (15). Associated with the membrane domains (fractions 3-8) after stimulation are tyrosine-phosphorylated proteins of approximately 90, 53/56, 45, 34, and 25-30 kDa. The 53/56-kDa doublet was identified as Lyn by reprobing the blot with rabbit anti-Lyn (UBI). 2 Significantly, the 45-, 34-, and 25-30-kDa bands appear only with stimulation and are markedly enriched in membrane domains relative to the other fractions. The domain-associated proteins of 34 and 25-30 kDa correspond to phosphorylated ␤ and ␥ 2 Fc⑀RI subunits, respectively, as identified by immunoprecipitating Fc⑀RI from the sucrose gradient fractions (Fig. 3B). Fig. 3 clearly shows that the tyrosine-phosphorylated ␤ and ␥ subunits are almost entirely associated with membrane domains. The majority of other tyrosine kinase substrates phosphorylated as the result of Fc⑀RI aggregation are located in the solubilized protein fractions, presumably because they are cytosolic or associated with membranes that are solubilized in 0.05% Triton X-100. Syk, the ZAP-70-related tyrosine kinase responsible for phosphorylating the majority of substrates downstream of Fc⑀RI (19,38,39), is also found exclusively in these soluble fractions, 2 as expected because activated Syk is localized primarily in the cytosol after receptor stimulation (40,41). 2 Thus, following Fc⑀RI aggregation on cells, Lyn phosphorylates the ␤ and ␥ subunits of domainassociated receptors. This apparently leads to a transient association and the activation of Syk, followed by Syk-mediated phosphorylation of downstream substrates, most of which are not stably associated with membrane domains.
Additional support for the involvement of these domains in initiating Fc⑀RI activation comes from in vitro tyrosine kinase assays performed on sucrose fractions, followed by immunoprecipitation of Fc⑀RI in the presence of 0.2% Triton X-100 (which releases Fc⑀RI from membrane domains). Fig. 4A shows that aggregated Fc⑀RI associated with membrane domains isolated after cell lysis in 0.05% Triton X-100 (MDϩ) are efficiently tyrosine-phosphorylated in vitro, whereas receptors in the sucrose fractions containing solubilized proteins (40ϩ and 40Ϫ) are not phosphorylated, and membrane domains from unstimu-lated cells (MDϪ) also show no phosphorylated Fc⑀RI. Streptavidin-aggregated Fc⑀RI from cells lysed in 0.2% Triton X-100 migrate at a high density (50 -70% sucrose) in these gradients (15). When in vitro tyrosine kinase assays are performed on these high density sucrose fractions (HDϩ), a relatively small amount of ␤ subunit phosphorylation is seen. This fraction does contain a small amount of Lyn that may be responsible for the phosphorylation detected, 2 but it is not known whether this represents Lyn directly associated with Fc⑀RI, Lyn contaminating this fraction from the 40% sucrose fraction, or fragments of membrane domains which remain receptor-associated in 0.2% Triton X-100. Consistent with the last possibility, the in vitro phosphorylation in the HDϩ fraction shows a Triton X-100 sensitivity similar to that of the MDϩ fraction (see below).
When in vitro tyrosine kinase assays are performed on membrane domains, stimulated Fc⑀RI phosphorylation is highly sensitive to the concentration of Triton X-100 present. As shown in Fig. 4B, addition of submicellar (0.01%) Triton X-100 to the membrane domains causes a slight enhancement of Fc⑀RI ␤ and ␥ tyrosine phosphorylation, but higher Triton X-100 concentrations dramatically reduce this phosphorylation. The association of Lyn with membrane domains, as well as its activity toward the exogenous substrate, enolase, is not significantly affected by Triton X-100, 2 and neither is phosphorylation of Lyn itself or the 45-kDa substrate (Fig. 4B). The exquisite sensitivity of Fc⑀RI in vitro phosphorylation to Triton X-100 indicates that lipid-mediated association of these receptors with the membrane domains is required for this activation step. Fig. 4C supports this conclusion, demonstrating that treatment of isolated membrane domains with 0.05% Triton X-100 (f) dissociates the aggregated receptors from these membrane domains as indicated by reanalysis on a second sucrose gradient. Treatment of the same domains with 0.01% Triton X-100 (q) does not dissociate the receptor, although it does cause a slight change in the distribution of the receptor within the gradient relative to untreated membrane domains (E). Thus, Fc⑀RI associated with membrane domains is functionally coupled to Lyn, and this association is easily disrupted by detergent.
The involvement of membrane domains in this early step of Fc⑀RI activation provides a new model in which the initial phosphorylation of the ␤ and ␥ subunits by Lyn is mediated by lipid-protein interactions. Although previous results have explained how the phosphorylation of Fc⑀RI ␤ and ␥ and subsequent events proceed after the association of active Lyn with a receptor cluster (23,25,38,39), the structural basis for the initial interaction between Lyn and Fc⑀RI in the activation process has remained poorly defined. Several studies that detected association of Lyn with unstimulated Fc⑀RI utilized methods that could stabilize the association of these receptors with membrane domains, including chemical cross-linking (18) or low detergent:cell lipid ratios (23). We find that unstimulated receptors do not co-isolate with the Lyn-containing domains to a large extent (Fig. 1), although weak and/or transient interactions could occur on intact cells. The size and stability of the domains on the surface of intact, unstimulated cells are unknown. These domains are likely to be small and dynamic in composition, but appear to coalesce together with aggregated Fc⑀RI (29,30). Thus, localization of Lyn within membrane domains could serve to sequester this kinase away from Fc⑀RI prior to receptor aggregation and, in turn, provide a pool of active or readily stimulated Lyn for aggregated Fc⑀RI that stably associate with the domains. Support for this aspect of the model comes from our observation that isolated membrane domains contain abundant tyrosine kinase activity even in the absence of Fc⑀RI activation (15), as well as from other studies on Src family members that associate with detergent-resistant membrane domains. These other investigations have found that Fyn, Lck, and Fgr associated with isolated membrane domains show higher specific activity in vitro than soluble forms of these kinases (42,43), possibly because of the capability for kinases concentrated within domains to trans-autophosphorylate readily. 4 Our results demonstrate that aggregation of Fc⑀RI causes its rapid and efficient association with specialized domains in the plasma membrane that are enriched in the tyrosine kinase, Lyn. Fc⑀RI associated with membrane domains are rapidly tyrosine-phosphorylated in intact cells, and this phosphorylation is also observed in vitro preferentially for receptors associated with membrane domains. The interaction of Fc⑀RI with these specialized membrane domains does not depend on the ␤ subunit, 3 and thus can account for the initiation of receptor signaling independent of specific protein-protein interactions between the ␤ subunit and Lyn (24 -28). In addition, this receptor-membrane domain association may facilitate coupling to processes such as Ca 2ϩ mobilization, lipid metabolism, and exocytic vesicle fusion. Recent evidence indicates that specialized membrane domains, including caveolae, are involved in the signaling of other cell surface receptors such as certain growth factor receptors (3,5) and glycosylphosphatidylinositollinked mitogenic receptors (12,44,45). Receptor-domain interactions also may be important for other multichain immune recognition receptors that utilize Src family kinases during their initial signaling steps (46,47). FIG. 4. In vitro tyrosine phosphorylation of Fc⑀RI associated with membrane domains. A, preferential phosphorylation of Fc⑀RI associated with membrane domains from stimulated cells. Kinase assays were performed on fractions from sucrose gradients without Na 3 VO 4 containing either detergent-resistant membrane domains (MD), the 40% sucrose fraction (40), or immune complexes in high density sucrose (HD), from either unstimulated RBL cells (Ϫ) or cells stimulated with 10 nM streptavidin for 5 min at 37°C (ϩ). Fc⑀RI was then immunoprecipitated and subjected to anti-phosphotyrosine immunoblotting as in Fig. 3B. The relative amount of 125 I-IgE loaded in each lane is (left to right) 0.04, 1.00, 0.76, 0.34, and 0.42. Experiments where ATP and Mg 2ϩ were omitted from the kinase assay, or where cells without IgE were used, showed no detectable phosphorylation of ␤ and ␥ under these conditions. 2 B, detergent sensitivity of in vitro Fc⑀RI phosphorylation. Membrane domains isolated as in Fig. 1 from streptavidin-stimulated cells were incubated with the indicated concentration of Triton X-100 prior to performing kinase assays on the sucrose fractions. The samples were then boiled with SDS, electrophoresed, and immunoblotted with anti-phosphotyrosine. C, extraction of Fc⑀RI from membrane domains with Triton X-100. Membrane domain fractions isolated from streptavidin-stimulated cells as for B were treated with no Triton X-100 (E), with 0.01% Triton X-100 (q), or with 0.05% Triton X-100 (f), readjusted to 40% sucrose, and ultracentrifuged overnight within a sucrose gradient (right axis) as in Fig. 1.


Reproductive toxicology. Tris(2-chloroethyl)phosphate.

Bcl-3 as an oncoprotein is overexpressed in nasopharyngeal carcinoma (NPC). Nedd4 binding protein 2 (N4BP2), which is located in the NPC susceptibility locus, is a Bcl-3 binding protein. This study is aimed to explore the association between N4BP2 genetic polymorphism and the risk of NPC. We performed a hospital-based case-control study, including 531 sporadic NPC and 480 cancer-free control subjects from southern China. PCR-sequencing was carried out on Exons, promoter region and nearby introns of the N4BP2 gene. The expression pattern of N4BP2 and Bcl-3 was also analyzed. We observed a statistically significant difference in haplotype blocks ATTA and GTTG between cases and controls. In addition, three novel SNPs were identified, two of which were in exons (loc123-e3l-snp2, position 39868005, A/G, Met171Val; RS17511668-SNP2, position 39926432, G/A, Glu118Lys), and one was in the intron6 (RS794001-SNP1, position 39944127, T/G). Moreover, N4BP2 was at higher levels in a majority of tumor tissues examined, relative to paired normal tissues. These data suggest that haplotype blocks ATTA and GTTG of N4BP2 is correlation with the risk of sporadic nasopharyngeal carcinoma in the Southern Chinese population and N4BP2 has a potential role in the development of NPC.

Background
Bcl-3 was originally identified as expressed in chronic B cell lymphocytic leukemia [1]. Many cell growth and survival promoters can induce Bcl-3 expression, and Bcl-3 overexpression has been detected in other cancers such as nasopharyngeal carcinoma (NPC) [2,3]. Nedd4 binding protein 2 (N4BP2, GenBank:AY267013) is a Bcl-3 binding protein, N4BP2 protein contains a polynucleotide kinase domain (PNK) at the N-terminus and a Small MutS Related (Smr) domain with nicking endonuclease activity near C-terminus [3]. MutS is central to the DNA mismatch repair (MMR) systems that are responsible for maintain-ing genome stability and protecting against mutation, Mutations in these genes are linked to the development of certain types of cancer [4,5]. Since N4BP2 contains a MutS-related domain, N4BP2 may play a role in MMR.
NPC is an epithelial tumor with an exceptionally high incidence in southern China, particularly in the Guangdong province [6,7]. Etiological and epidemiological studies have suggested that susceptibility genes may determine the predisposition to developing NPC [8,9]. Previously, we reported the use of 382 polymorphic microsatellite markers to identify a candidate susceptibility locus that mapped to chromosome 4p15.1-q12 (D4S2950-D4S2916) in a subset of NPC families [10]. Further analysis identified SNPs within or near this region, strongly suggesting the presence of an NPC susceptibility locus adjacent to the LOC344967 [11], very close to the N4BP2 gene.
We thus hypothesized that SNPs or other variation in the N4BP2 gene lead to a predisposition to developing NPC. We further hypothesized that the N4BP2 gene plays a role in tumorigenesis. To address these hypotheses, we examined N4BP2 haplotypes among NPC patients from southern China. We also examined mRNA levels of Bcl-3 and N4BP2 in NPC cell lines and tissues.

Subjects
A total of 531 sporadic NPC patients and 480 unrelated age, sex-, and geographically-matched healthy individuals from southern China were used for our case-control study. NPC patients were recruited from Sun Yat-sen University Cancer Center and the presence of differentiated nonkeratinizing NPC or undifferentiated NPC was confirmed by histological analysis. Control subjects were recruited from the People's Hospital of Guangdong Province. The characteristics of cases and controls are shown in Table 1. Although the incidence of NPC is generally higher among males than females, no significant difference in sex distribution existed between case and control groups in this study. The average age in the control group was 37 ± 10, and in the case group was 40 ± 10; thus the age distribution here is a good representation of the broader NPC patient population given that NPC incidence peaks at the relatively young age of 45.

DNA preparation
Genomic DNA was extracted from 5-10 ml peripheral blood using the QIAamp DNA Blood Midi kit (Qiagen, German).

Primer design
Between 250 and 400 bp of sequence surrounding SNPs sites were submitted to the primer design program [12]. The primers used for SNPs are shown in Table 2.

PCR Amplification
Long-distance PCR was performed in a total volume of 15 μl containing 200 ng of genomic DNA, 1.5 μl 10× Buffer, 50 μM dNTPs, 0.3 μM each primer, and 1U Taq DNA polymerase. Samples were amplified with each pair of

Tissue collection and RT-PCR
A total of 21 tissues were collected from Sun Yat-sen University Cancer Center. Six paired matched tissues from different organs included esophagus, stomach, liver, lung, cervix and breast; nine nasopharyngeal tissues contained

SNP analysis
We identified a total of twelve SNP associated with the N4BP2 locus, four of which were upstream of the N4BP2 gene and eight which were within N4BP2 gene (Table 3).

Haplotype analysis
Haplotype frequencies and distributions were estimated using a standard EM algorithm. Interestingly, Four SNPs (SNP6-7-9-10) combined haplotype Block 2 ATTA and GTTG exhibited notable difference between case and control groups (Table 5). Permutation tests for allelic association confirmed that block ATTA and GTTG are closely linked ( Table 6) and confirmed the difference of Block 2 ATTA and GTTG in cases and controls.

N4BP2 and Bcl-3 expressed in cells and tissues
Bcl-3 and N4BP2 were detected in all cell lines examined ( Figure 1A). Interestingly, gene expression levels appeared to vary among the cell lines, with the lowest levels being detected in the Sune line. N4BP2 and Bcl-3 mRNA levels appeared to be higher in tumor than in matched normal tissue ( Figure 1B). N4BP2 and Bcl-3 were also detected in nasopharyngeal tissues ( Figure 1C). These observations suggest that N4BP2 expression levels correlate with the progression of cancer including NPC.

Discussion
We previously showed, by linkage analysis that an NPC susceptibility locus maps to chromosome 4 near the LOC344967. Here, we extend this analysis in an effort to identify a bona fide NPC susceptibility gene. N4BP2 is a candidate gene in this region, and we thus sought to examine the correlation between genetic polymorphisms in N4BP2, a mismatch repair gene, and the incidence of NPC. SNPs have been shown to be extremely useful for studying the association between genomic regions and disease [14]. Several studies have demonstrated that polymorphic variation in mismatch repair genes contributes to susceptibility to certain cancers [15,16]. In addition, several pieces of evidence suggest that nasopharyngeal carcinogenesis is associated with individual susceptibility caused by SNPs. Our results show that, although there is no significant difference in SNPs between cases and controls, there are two haplotypes, ATTA and GTTG, the distribution of which differed between case and control groups. Just as the report that almost not any difference in the allele frequencies of five SNPs within the TNFSF4 gene individuals suffered from coronary artery disease versus the controls while there were significantly more frequent of the possible haplotypes from this five TNFSF4 SNPs in individuals with coronary artery disease than controls [17]. Insight into this paradox has been provided in a recent review by Schaid [18]. Haplotypes, the grouping of closely linked alleles on a chromosome, make an impor-tant contribution to the study of the genetic basis of disease. Schaid [18] explained that for case-control studies, methodological approach based on haplotype has more advantage than single-locus analysis as the SNPs are in LD with a causative diallelic locus; in particular, haplotypebased methods have greater power when the marker alleles are in strong LD with causative alleles. Haplotype methods are more useful for variants which are more recently evolved, rarer and more causative than for variants which are older and more common. This may help explain why the haplotypes ATTA and GTTG exhibit differences in frequency between case and control groups while individual SNPs do not.
N4BP2 is a Bcl-3 binding protein, and Bcl-3 is an oncoprotein that is overexpressed in certain cancers, including NPC. Our analysis of N4BP2 and Bcl-3 expression levels suggest that expression of these genes is correlated, suggesting they may be co-regulated. We also found that N4BP2 and Bcl-3 are expressed in all NPC cell lines examined and were higher for certain cancers. These observations are consistent with previous reports.

Conclusion
Above all, we found that two N4BP2 haplotypes, ATTA and GTTG, are correlated with NPC. This will be useful for predict the development of NPC. In addition, N4BP2 and Bcl-3 mRNA levels were elevated in tumors, including NPC tumors, which suggest new therapeutic targets for fighting NPC.


text:
A Peptidyl-prolyl cis/trans-Isomerase (Cyclophilin G) in Regulated Secretory Granules*

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a β-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolylcis/trans-isomerase. p27 exhibits isomerase activity with ak cat /K m of 0.18 μm −1 s−1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolylcis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.

The horseshoe crab (or limulus) is an arthropod with a unique innate humoral immune system that differs substantially from the immunoglobulin-based acquired immune system of vertebrates (1,2). A single type of hemocyte, which comprises approximately 99% of the total, plays a major role in the host defense system of this animal (3,4). These hemocytes contain two types of granules, larger (L-) 1 and smaller (S-) granules, both of which are exocytosed by a trace amount of bacterial endotoxin, lipopolysaccharide (LPS), when exposed to Gram-negative bacteria (3)(4)(5)(6). These granules contain LPSand ␤-glucan-sensitive coagulation factors, lectins, and antimicrobial proteins, which after exocytosis participate in the establishment of proteolytic cascades that ultimately immobilize and kill invading pathogens (1,7).
Recently, we purified a serine protease zymogen factor G from hemocytes (8). The autocatalytic activation of factor G by a trace amount of (1,3)-␤-D-glucan on the surface of fungi triggers a protease cascade that culminates in hemolymph coagulation. Factor G is a heterodimeric protein composed of subunits ␣ and ␤. Subunit ␣ is a novel mosaic protein involved in ␤-glucan recognition, whereas subunit ␤ is a 37-kDa serine protease zymogen (9). During immunoblotting analyses of horseshoe crab hemocyte lysate with subunit ␤-specific antisera, we found a 27-kDa protein (p27) that cross-reacts with subunit ␤ antiserum. Thus, anticipating a related serine protease, we initiated a study of p27.
In this study, we purified p27 and cloned its cDNA. The deduced amino acid sequence of p27 unexpectedly revealed that it was a cyclophilin (CyP) homologue with peptidyl-prolyl cis/trans-isomerase (PPIase) activity. A PPIase, which catalyzes the cis/trans-isomerization of Xaa-Pro bonds, was first isolated from porcine kidney cortex (10), and sequence analysis revealed that it was identical to cyclophilin A (CyPA), a cytosolic protein isolated from bovine thymocytes which binds to the immunosuppressant cyclosporin A (CsA) (11)(12)(13). CyPA, by making a complex with CsA, suppresses immune reactions by inhibiting a protein phosphatase, calcineurin (14). Although CsA inhibits the PPIase activity of CyPA, its calcineurin inhibitory activity is independent of its PPIase activity.
Other non-cytosolic CyP homologues with PPIase activity have been found to participate in the processing of secretory proteins (15). Cyclophilin B (CyPB) is mainly found in the endoplasmic reticulum (ER) and is thought to have a role in the folding of nascent peptide chains (16,17). The ninaA gene product, a CyP homologue in Drosophila photoreceptor cells, co-localizes with rhodopsin in secretory transport vesicles, suggesting a role in protein trafficking and macromolecular assembly (18).
This study characterizes a new 27-kDa CyP homologue, cyclophilin G, which is located in the L-granule, one of two types of regulated secretory granules in horseshoe crab hemocytes. This is the first report demonstrating the presence of a PPIase in regulated secretory granules, and suggests an expanded role for CyPs in maintaining the integrity of stored secretory proteins.

EXPERIMENTAL PROCEDURES
Materials-Hemocyte lysate of the Japanese horseshoe crabs (Tachypleus tridentatus) was prepared as described previously (19). Land S-granules were purified from the hemocytes by the method described in Ref. 20. Sephadex G-150, CM-Sepharose CL-6B, Superdex 75 HR 10/30, and glutathione-Sepharose 4B were products of Pharmacia Fine Chemicals (Uppsala, Sweden). ␣-Chymotrypsin was obtained from Worthington and succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4methylcoumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) was from Peptide Institute Inc. (Osaka, Japan). Oligonucleotides were synthesized by Sawady Technology (Tokyo, Japan). Cyclosporin A and FK506 were kindly provided by Sandoz AG (Basel, Switzerland) and Fujisawa Pharmaceutical Co., Ltd. (Ibaraki, Japan). gt22A horseshoe crab hemocyte cDNA library was prepared by using a Superscript™ Lambda system (Life Technologies, Inc.). The rabbit antiserum against factor G subunits and p27 was prepared by using glutathione S-transferase (GST)fusion proteins of a factor G fragment and recombinant p27 as antigens, respectively. All other chemicals were of analytical grade or the highest quality commercially available.
Purification of p27-Dextran sulfate-Sepharose CL-6B column (inner diameter, 5.0 ϫ 14 cm) chromatography of the lysate (590 ml) derived from 97.5 g (wet weight) of the horseshoe crab hemocytes was performed as described (8). The 2.0 M NaCl fractions containing p27 were lyophilized, and applied to a Sephadex G-150 column (inner diameter, 3.2 ϫ 98 cm) in 20 mM sodium acetate (pH 5.5) containing 0.5 M NaCl. Fractions containing p27 were pooled, diluted 10-fold with 20 mM sodium acetate (pH 5.5), and then applied to a CM-Sepharose CL-6B (inner diameter, 1.0 ϫ 5.0 cm) equilibrated with the same buffer. After washing with 20 mM sodium acetate (pH 5.5) containing 0.05 M NaCl, bound proteins were eluted by a linear salt gradient from 0.05 M to 0.4 M NaCl in 20 mM sodium acetate (pH 5.5). Further purification of p27 was performed by a Superdex 75 HR 10/30 chromatography in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl.
SDS-PAGE and Immunoblotting-SDS-PAGE was performed according to the method of Laemmli (21). Immunoblotting was carried out by the standard method with antiserum against factor G subunit ␤ or p27 (22). The immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Nippon Bio-Rad Laboratories) and an ECL™ Western blotting detection system (Amersham Japan, Tokyo).
Partial Amino Acid Sequence of p27-p27 was further purified by reversed-phase high performance liquid chromatography on a Phenyl-5PW RP column (inner diameter, 4.6 ϫ 75 mm) (Tosoh, Tokyo, Japan) for sequence analysis. Purified p27 was reduced, S-alkylated by iodoacetamide, and digested with lysyl endopeptidase (Wako Pure Chemical Industries Ltd., Tokyo, Japan). The generated peptides were separated on a Chemcosorb 5-ODS-H column (inner diameter, 2.1 ϫ 150 mm) (Chemco Scientific Co., Ltd., Osaka, Japan). The amino-terminal sequence analyses of each peptide and intact p27 were carried out with an Applied Biosystems 477A or 473A gas-phase protein sequencer. Amino acid analysis was performed on a PICO⅐TAG system (Waters).
cDNA Cloning of p27-Two degenerated oligonucleotide primers (5Ј-CTGAATTCGTNTAYTTYGAYATHAC-3Ј and 5Ј-GCGAATTCTTRAA-RTTYTCRTCNGG-3Ј), each of which contained an EcoRI site (underlined), were synthesized based on the partial amino acid sequences of p27. A partial cDNA fragment for p27 was amplified by polymerase chain reaction (PCR) with the first strand cDNA derived from the horseshoe crab hemocyte as a template, 100 pmol each of the oligonucleotide primers, and 2.5 units of Pfu polymerase (Stratagene). The generated DNA fragment containing a partial p27 cDNA fragment was used as a probe for the screening of gt22A horseshoe crab hemocytes cDNA library (23). A positive clone was isolated and its cDNA fragment was subcloned into pBluescript II SK(ϩ) (Stratagene). The nucleotide sequence analysis was carried out on ABI PRISM™ 373A or 377 DNA sequencer using ABI PRISM™ dye terminator cycle sequencing ready reaction kit. All sequences were confirmed by sequencing overlapping fragments.
Homology Search-A computer-assisted homology search of p27 was performed in the DDBJ homology search system using the programs FASTA version 3.0 (24) and BLAST version 1.4.9 (25).
PPIase Assay-PPIase assay was performed by the chymotrypsincoupled assay (12,26). The sample was preincubated at 10°C for 2 min with 6.7 M Suc-Ala-Ala-Pro-Phe-MCA in 0.2 M Tris-HCl (pH 8.0), in the presence and absence of CsA or FK506, in a total volume of 1.485 ml. The reaction was started by adding 15 l of 0.1 mM ␣-chymotrypsin, and the fluorescence was monitored for 5 min with excitation at 380 nm and emission at 440 nm.
Under these assay conditions, the rate constant for the cis to trans isomerization can be obtained by measuring the rate constant of the substrate hydrolysis at the slow phase. The apparent first order rate constant (k app ϭ k H 2 O ϩ k enz ) was determined from the slope of the Guggenheim plot (27). k H 2 O is the rate constant in the absence of PPIase, and k enz is that catalyzed by PPIase. One unit of PPIase activity was defined as k enz (s Ϫ1 ). The k cat /K m was calculated from the equation, Preparation of Recombinant p27-A cDNA fragment encoding the mature p27 was created by a PCR and inserted into a BamHI site of pGEX-2T expression vector (Pharmacia Biotech Inc.). The expression and purification of the GST-p27 fusion protein was carried out with glutathione-Sepharose 4B according to the manufacturer's protocol. The purified GST-p27 was digested by trypsin (N-tosyl-L-phenylalanyl chloromethyl ketone-treated, Worthington), and then released p27 was purified by passing it through benzamidine-Sepharose (28) and glutathione-Sepharose 4B columns. The amino acid composition and aminoterminal sequence of the prepared recombinant p27 was confirmed.
Expression of p27 in COS Cells-COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. A plasmid containing the full-length cDNA for p27 in pcDNAI/Amp (Invitrogen) was transfected by the calcium phosphate method (29). Forty-eight hours after the transfection, the medium was changed to a serum-free medium and the culture was continued for another 24 h. The cell lysate and the culture supernatants were analyzed by immunoblotting using anti-p27 antiserum.
Immunofluorescence Microscopy-The horseshoe crab hemolymph were fixed, immediately after bleeding, in 2% paraformaldehyde EM (Taab Laboratories Equipment Ltd., Berkshire, UK) for 10 min at room temperature. The washed hemocytes were mounted on a glass coverslip. The cells, permeabilized with ice-cold methanol for 10 min, were incubated with primary antibody for 2 h at room temperature. After the washing with phosphate-buffered saline containing 0.1% saponin, the cells were incubated with rhodamine-conjugated swine anti-rabbit IgG secondary antibody (1:50 dilution, DAKO) for 1 h, followed by washing with phosphate-buffered saline. The specimen was mounted in Entellan neu (Merck) and viewed with a Carl Zeiss LSM 410 confocal laser scanning microscope. Anti-p27 antiserum, preimmune serum, or antihorseshoe crab tachylectin 1 (L6) (30) was used as primary antibody at a dilution of 1:100.

RESULTS
Purification of p27-Western blotting analysis with antiserum against factor G revealed two bands of 37 and 27 kDa in the horseshoe crab hemocyte lysate, which reacted specifically with anti-subunit ␤ but not with anti-subunit ␣ antisera (Fig.  1). The 37-kDa band was indistinguishable from factor G subunit ␤, whereas the 27-kDa band did not correspond with any fragments of factor G or activated factor G (8). We started the purification of this protein (p27) by using anti-subunit ␤ antiserum as a probe.
FIG. 1. Western blotting analysis of the horseshoe crab hemocyte lysate with anti-factor G subunit-specific antibody. The horseshoe crab hemocyte lysate (10 l) was subjected to 12.5% SDS-PAGE followed by Western blotting by using anti-factor G subunit ␣ and ␤ antiserum. Molecular masses of the reacted protein bands are shown.
The hemocyte lysate was initially fractionated with dextran sulfate-Sepharose CL-6B chromatography ( Fig. 2A). Whereas the 37-kDa subunit ␤ of factor G eluted from the column with 0.25 M NaCl corresponding to factor G activity (8), p27 was found to elute in the 2.0 M NaCl fractions. The fractions containing p27 were pooled and subjected to gel filtration on Sephadex G-150 at pH 5.5 in the presence of 0.5 M NaCl (Fig. 2B). The p27-containing fractions were pooled, diluted to reduce NaCl concentration, and then applied to a CM-Sepharose CL-6B column at pH 5.5 (Fig. 2C). p27 bound to the column was eluted with a NaCl gradient and was further purified by gel filtration on a Superdex 75 HR column (Fig. 2D). The purified p27 gave a single band (mass ϭ 27 kDa) on SDS-PAGE under reducing conditions (Fig. 2D, inset, fraction 15), and its mobility did not change under non-reducing conditions (data not shown). Forty g of p27 was obtained from 97.5 g (wet weight) of hemocytes (Table I).
Isolation of p27 cDNA-To obtain a partial amino acid sequence, amino-terminal sequences of the purified p27 and its proteolytic fragments were determined as described under "Experimental Procedures" (Fig. 3, underlined). Two degenerated primers were synthesized based on the partial sequences (Fig.  3, boxed) and were used in reverse transcriptase-PCR with hemocyte poly(A) ϩ RNA as a template. A PCR fragment of 280 bp was obtained, and sequencing confirmed that it encoded p27. Using this fragment as a probe, one clone was isolated from 500,000 clones of the hemocyte cDNA library.
Sequence analysis of this clone showed that the cDNA contained an open reading frame of 660 bp followed by a TAA termination codon, and that it was flanked by a 104-bp 5Јnoncoding sequence containing in-frame termination codons and 914 bp of 3Ј-noncoding sequence. A poly(A) ϩ signal was missing. The open reading frame encoded a p27 precursor of 220 amino acid residues. All the partial amino acid sequences were consistent with the deduced sequence, confirming that it encoded p27 (Fig. 3, underlined). The amino-terminal sequence of the purified p27 was located at the 24th amino acid of the deduced sequence, consistent with a 23-residue pro-sequence, which exhibited the characteristics of a typical hydrophobic ER signal sequence. Thus, mature p27 was composed of 197 amino acids with a calculated molecular mass of 21,614 Da. The amino acid compositions calculated from the mature protein of 197 amino acids agreed well with the analytical values obtained from the purified protein (Table II). Although one potential glycosylation site (Asn 148 ) for an N-linked carbohydrate chain (Asn-Xaa-Ser/Thr) was present in the sequence, no amino sugar was detected in the purified p27 (Table II).
A sequence homology search indicated that p27 had extensive similarity with proteins belonging to CyPB family (Fig. 4). p27 showed the highest similarity with human and mouse CyPBs (70% identity). p27 also showed more than 60% sequence identity with other vertebrate CyPBs and CyP homologues in Caenorhabditis elegans (CyP-5 and 6). A CyP homologue in yeast and an eye-specific CyP homologue in Drosophila, NinaA, had 50% and 36% identity, respectively. The 15 residues conserved in all CyPs, which are involved in close contact with substrates or CsA, were also found in the p27 sequence (Fig. 3, boldface).
Two distinct differences were found between p27 and other members of the CyPB family. All the members of CyPB family contain a conserved 10-residue ER retention signal at their carboxyl termini, which differs from a typical motif, K/HDEL. This signal was missing in p27. In addition, p27 had an insertion of 8 residues in the middle of the molecule, which has not been observed in any other CyPs.
PPIase Activity of p27-Since p27 showed high sequence similarity with CyPs having PPIase activity, the isomerase activity of p27 was assessed at each purification step (Fig. 2). At each step, the elution profile of p27 antigen correlated well with PPIase activity. However, p27 represented only a minor part of total PPIase activity in the hemocytes, since most of the activity was eluted in the flow-through fractions of the dextran sulfate-Sepharose CL-6B column chromatography, the first step of the purification procedure ( Fig. 2A).
The chymotrypsin-coupled assay for PPIase activity showed that p27 increased the hydrolysis of a peptide substrate by ␣-chymotrypsin by catalyzing cis to trans isomerization of Pro at the P2 site (Fig. 5A). The k cat /K m of purified p27 was estimated to be 0.18 M Ϫ1 s Ϫ1 , which was 5-50-fold lower than the other CyPBs. The PPIase activity of p27 was inhibited by CsA, an inhibitor for CyP family (Fig. 5B). The CsA concentration (IC 50 ) required for 50% inhibition of the p27 (1.3 nM) activity was 8.3 nM, similar to that reported for other eukaryotic CyPs (31). On the other hand, another PPIase inhibitor, FK506, did not show any inhibitory activity on p27 (data not shown).
Recombinant p27 was prepared by excising an expressed insert from a GST-p27 fusion protein. Its amino-terminal sequence and amino acid composition showed that recombinant p27 started with the 4th Lys residue, indicating that the first 4 residues were removed after trypsin digestion (data not shown). This recombinant p27 showed a similar mobility with the purified protein on SDS-PAGE and reacted with the anti-subunit ␤ antiserum (Fig. 6A). The recombinant p27 also exhibited a PPIase activity as the purified p27 (Fig. 6B), ruling out the possibility that p27 was co-purified with minor contaminant that has isomerase activity.
Localization of p27-CyPBs have been reported to localize in the ER through their carboxyl-terminal ER retention signal (32). We tested whether p27, which lacks this retention signal, would also localize to the ER when expressed in COS cells. COS7 cells were transfected with the full-length cDNA for p27, and cell lysate and culture supernatant of the transfected cells were analyzed by using anti-p27 antiserum. p27 was detected in the culture supernatant, but not in the cells (Fig. 7).
We next analyzed the distribution of p27 in the horseshoe crab hemocytes. The fixed and permeabilized hemocytes were stained with anti-p27 antiserum, followed by rhodaminelabeled secondary antibody. Confocal laser scanning microscopic analyses indicated that intracellular granular structures of the hemocyte were stained with anti-p27 (Fig. 8A) but not with preimmune serum (Fig. 8B). The staining pattern with anti-p27 antiserum was essentially same as that of antiserum against tachylectin-1 (L6), a lectin abundantly present in the L-granule (20,30) (Fig. 8C). We further examined subcellular localization of p27 in the horseshoe crab hemocytes, which contain two types of secretory granules (L-and S-granules) (4). The L-and S-granules were purified from the cells (20) and were analyzed by immunoblotting (Fig. 8D). The anti-p27 an-  3. Nucleotide (upper) and deduced amino acid sequence (lower) of p27. The amino acid sequence is numbered beginning at the amino terminus of the mature protein. In-frame termination codons are dotted underlined. The amino acid residues confirmed by sequencing purified peptides are underlined. The amino acid sequences used to design degenerate oligonucleotide primers for the PCR are boxed. The potential carbohydrate attachment sites for Asn-Xaa-Ser/Thr, which was confirmed not to carry the carbohydrate, is shown by an open diamond. Residues involved in the contact to CsA in human CyPB are indicated in boldface type. tiserum identified p27 protein in the L-granules in addition to the hemocyte lysate, but not in the S-granules. p27 was not detected in the hemolymph plasma. Similar results were obtained with anti-factor G subunit ␤ antiserum. 2

DISCUSSION
Although the cross-reactivity of p27 with antiserum against subunit ␤ of the horseshoe crab ␤-glucan-sensitive serine protease zymogen factor G (Fig. 1) led us to initially speculate that it might be a new serine protease, p27 instead exhibits an amino acid sequence most similar to that of CyPs and PPIases (Figs. 3 and 4). The amino acid sequence of p27 showed little similarity with that of factor G. p27 and factor G subunit ␤ should share a conformation-specific epitope(s), although its biological significance is unknown.
Representing only a small portion of total hemocyte isomerase activity, this protein was found to localize to the hemocyte L-granules and was purified approximately 8600-fold with an overall yield of 1.8% (Table I). Comparative sequence analysis revealed that p27 was most homologous with members of the CyPB family (Fig. 4), and functional data supported this conclusion; in a chymotrypsin-coupled assay, p27 exhibited isomerase activity that was inhibited by CsA, but not by FK506 (Fig. 5). These results indicate that p27 is a CyP-type enzyme, distinct from the FK506/rapamycin-binding protein (FKBP)type enzyme (15). But unlike other cyclophilins, which are proteins functioning in cytosol or ER, p27 is instead found only in the L-granules of horseshoe crab hemocytes. Thus, p27 is a new type of CyPB, hereby designated as cyclophilin G (CyPG).
A sequence comparison between CyPG and other CyPs offers insight into the mechanisms underlying its unique biological activities. Similar to other CyPBs, the CyPG precursor contains an amino-terminal hydrophobic pro-peptide, but unlike them it lacks a well conserved 10-amino acid carboxyl-terminal sequence, VEKPFAIAKE, which functions as an ER retention signal (Fig. 4) (32). When transiently expressed, CyPG is not retained in the cells but instead is secreted into the medium (Fig. 7), indicating that the amino-terminal pro-peptide functions as a signal peptide targeting the polypeptide to the ER, but because CyPG lacks the ER retention signal, it is not retained.
The specific signal(s) which target proteins to the regulated secretory granules in hemocytes are not yet known. The signal(s) for targeting CyPG to the L-granule might be present in the unique central 8-amino acid insertion, which is not found in any known CyPBs. Alternatively, it may be present in the amino-terminal pro-peptide, or CyPG might instead localize through a conformation-specific signal patch(es). It might be noted that the amino-terminal signal sequences of CyPG and 2 Y. Takaki and T. Muta, unpublished data. factor G subunit ␤ (8,9), both of which are localized in the L-granules, are similar: FLVFVTTLS and FLVFITLS, respectively, although the mature proteins exhibit little sequence similarity. In addition, both CyPG and C. elegans CyP-5 share similar carboxyl-terminal and amino-terminal signal sequences; an analysis of the subcellular localization of CyP-5, which is not currently known, may provide a key insight into this question (33).
The specific function of CyPG in the hemocyte granule is not known. The L-granule, one of two regulated secretory granules in the hemocytes, is known to contain many proteins involved in the defense system, such as coagulation factors, lectins, and antimicrobial proteins (1,20). These components are stored in the granules until they are released from the cells after being stimulated by bacterial endotoxin. Since CyPs generally function as "molecular chaperones," CyPG might similarly be involved in the quality control of stored protein components, i.e. in the refolding or repair of partially denatured proteins.
For instance, a serine protease zymogen called factor C triggers an LPS-sensitive coagulation cascade, and it contains autocatalytic cleavage sites: -Lys(P3)-Pro(P2)-Arg(P1)-Ser(P1Ј)-and -Ser(P3)-Pro(P2)-Phe(P1)-Ile(P1Ј)- (34,35). The cleavage of the latter site can be catalyzed by ␣-chymotrypsin (36) and is essential for the activation (37). In each cleavage site, a proline is located at the P2 site, and an appropriate cis/trans conformation of these X-Pro bonds may be critical for the autocatalytic activation of the zymogen. CyPG might function to maintain the correct conformation of such X-Pro bonds. Similarly, 7 out of 8 well defined proline residues in the crystal structure of coagulogen, a clottable protein that is the final target of the hemolymph clotting cascade, have the trans-configuration, implying that the cis/trans conformation of prolines is strictly regulated in these granules (38).
Moreover, CyPG was not detected in the S-granules (Fig.  8D). The protein/peptide components in the S-granule are much smaller and rich in cystines, compared with those in the L-granule (20). Those S-granule components could be stabilized by disulfide bond(s), thus the PPIase activity to repair peptide folding might not be required. Maintaining the conformational integrity of the proline-rich L-granule proteins may be essential to sustain the integrity of the organism's host defense, and CyPG may thereby provide a key immunological function.
In summary, this report demonstrates the purification, cloning, expression, and characterization of CyPG (p27), a unique PPIase that is present in the regulated secretory granules of horseshoe crab hemocytes. This is the first report to establish the existence of such "quality control" molecules in regulated secretory granules, and although the exact function of CyPG is not known, the recognized function of cyclophilins as molecular chaperones suggests that it may play a role in maintaining the conformational integrity of stored granular proteins. Indeed, the presence of at least 11 isoforms of CyPs in C. elegans (33) has suggested that CyPs may exhibit more varied biological activities than previously recognized. Further elucidation of the function of CyPG in the horseshoe crab hemocyte granule may therefore offer interesting insights into the diverse functions of this interesting group of molecules.
Acknowledgments-We thank Dr. N. Takahashi (Tonen Corp.) and Dr. K. Tawada (Kyushu University) for helpful advice in the PPIase assay, H. Iwanari (Institute of Immunology Inc.) for preparing anti-FIG. 6. Comparison of the purified and recombinant p27 proteins. A, the purified p27 and the recombinant p27 excised from a GST-p27 fusion protein were subjected to 15% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-factor G subunit ␤ antibody. See "Experimental Procedures" for details. B, the PPIase activities of purified p27 and recombinant p27 were measured as in factor G subunit ␤ antiserum, Dr. H. Nishimura (Hokkaido University) for providing COS7 cells, Dr. M. Sakaguchi (Kyushu University) for comments on immunofluorescence staining, and Dr. D. L. Sylvestre (HAART, Inc.) for critical comments on the manuscript. We are also grateful to C. Yano for technical assistance with the amino acid analyses.