extfieldsofstudy: Medicine Chemistry

text:
Structural Changes Measured by X-ray Scattering from Human Flap Endonuclease-1 Complexed with Mg2+ and Flap DNA Substrate*

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5′-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1·34-mer DNA flap complex, and the D181A·34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173–9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of ∼26 Å, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5′-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolatedI o scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1·34-mer DNA flap complex has anR g value of ∼34 Å, whereas the D181A·34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1·34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.

The 5Ј-flap structure is a common DNA structural intermediate occurring during DNA replication, recombination, and repair (1). In eukaryotic DNA replication, displacement of an upstream primer by an incoming polymerase can result in the formation of a 5Ј-flap structure (2,3). The 5Ј-flap intermediates are also formed during double-stranded break repair (4,5), homologous recombination (6), and excision repair (7)(8)(9)(10). In DNA repair and replication activities, structural recognition of the 5Ј-flap by specific DNA repair nucleases is essential. The importance of the DNA metabolic reactions, involving the structure-specific nucleases, is best illustrated by the human genetic disorder xeroderma pigmentosum (11)(12)(13). This disease, characterized by severe sensitivity to sunlight and a predisposition to skin cancer, results directly from defects in the nucleotide excision pathway. Mutation defects in the repair nucleases may be a point of breakdown in this DNA repair pathway.
The design of a model flap DNA structure, similar to those conjectured to occur in the nucleotide excision pathway, has led to the discovery of human flap endonuclease-1 (FEN-1), 1 which structurally recognizes and cleaves the flap DNA structure (4, 14 -17). FEN-1, an ϳ43-kDa Mg 2ϩ -or Mn 2ϩ -dependent enzyme, demonstrates both 5Ј-flap structure-specific endonuclease activity (1,4,7) and nick-specific 5Ј 3 3Ј exonuclease activity (4,14,17,18). The exonuclease activity of FEN-1 is similar to the function of the 5Ј 3 3Ј exonuclease domain of Escherichia coli DNA polymerase I (16) and identical to the activity in RNA primer removal that is necessary for in vitro mammalian DNA replication. In its endonuclease role, FEN-1 recognizes the phosphodiester backbone of a 5Ј-flap single strand and tracks down this arm to the cleavage site, the junction where the two strands of duplex DNA adjoin a singlestranded arm (1,3). FEN-1 does not cleave bubble substrates, single-stranded 3Ј-flaps, heterologous loops, or Holliday junctions, but acting as an exonuclease, FEN-1 will hydrolyze double-stranded DNA substrates containing a gap or 3Ј-overhang. FEN-1 endonuclease activity is independent of 5Ј-flap length, and endonuclease and exonuclease activities cleave both DNA and RNA without the need for accessory proteins (19). FEN-1 does, however, interact with other proteins at the replication fork, including a DNA helicase (20), the proliferating cell nuclear antigen (21)(22)(23)(24), and possibly replication protein A (RPA) (25).
The biological significance of the FEN-1 gene (RAD27 in Saccharomyces cerevisiae and rad2 in Schizosaccharomyces pombe) is emphasized by genetic analysis in yeast. The yeast FEN-1 mutants display severely impaired phenotypes such as UV sensitivity, deficient chromosome segregation, conditional lethality, and accumulation in S phase (15, 26 -29). The yeast rad27 null mutant is a strong mutator, and the majority of mutations found are duplications. This is probably because unexcised flap strands in Okazaki fragments displaced by upstream DNA polymerization are subsequently annealed to the downstream complementary sequence. This part of the sequence will be duplicated in the next generation of DNA replication (30). FEN-1 activity requires a free 5Ј-end of the flap DNA strand. For instance, secondary structure formation of the single-stranded DNA into a hairpin structure is known to prevent the enzyme's function (31). At risk motif sequences such as trinucleotide repeats have a higher probability to form these structures. Indeed, the same FEN-1 null mutant displays length-dependent CTG tract destabilization and a marked increase in expansion frequency (32,33). Thus, FEN-1 is a key enzyme for maintaining genome integrity, and mutations in FEN-1 may give rise to a number of genetic diseases such as myotonic dystrophy, Huntington's disease, several ataxias, fragile X syndrome, and cancer (30,32).
As the role of FEN-1 in DNA replication and repair is becoming more clear, it is important to structurally characterize this enzyme to better understand how it functions either as an exo-or endonuclease. To examine the structure-function relationship of FEN-1 in its nuclease capacity, we have studied the effect of magnesium on its conformation in aqueous solution, as observed by small-angle x-ray scattering (SAXS). Experiments were also done with a D181A mutant of FEN-1, which still selectively binds to the 5Ј-flap DNA structure, but has lost its catalytic ability (34,35). We show that no measurable structural change was evident in either FEN-1 or the D181A mutant due to the presence of Mg 2ϩ . A DNA fragment was constructed so that it readily adopts a 5Ј-flap conformation. When measurements of FEN-1 and the D181A mutant were performed in the presence of the 34-mer DNA flap fragment, the FEN-1⅐34mer DNA flap complex was seen to be more compact than the D181A⅐34-mer DNA flap complex, which indicates that the wild-type FEN-1⅐34-mer DNA flap complex is in a cleavageready conformation. A time course scattering measurement showed that magnesium was able to activate the cleavage of the FEN-1⅐34-mer DNA flap complex, and the protein was found to completely release the remaining single-and doublestranded portions of the DNA products.

EXPERIMENTAL PROCEDURES
Protein Purification and Sample Preparation-Protein expression and purification were essentially carried out according to Nolan et al. (36). After FEN-1 was eluted from the column using elution buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, and 300 mM histidine), it was further dialyzed in Tris-HCl buffer, pH 7.9, containing 100 mM NaCl, 10 mM 2-mercaptoethanol, and 10% glycerol and then concentrated in a Centriprep-10 concentrator (Amicon, Inc.). Final protein concentrations used were ϳ6 mg/ml in the SAXS study for both the wild-type and D181A mutant proteins, in which 10 mM Mg 2ϩ was either present or absent. Wild-type and D181A mutant protein concentrations were ϳ4.7 mg/ml for the protein⅐DNA flap complex studies, and the protein and DNA had a 1:1 stoichiometric ratio. Final concentrations were determined by the Bradford assay (46) using bovine serum albumin ␥-globulin as a standard. Binding of the two proteins to the flap DNA substrate was confirmed by gel shift assay after labeling the 5Ј-end of the substrate with 32 P. The purity of the concentrated protein samples was checked by SDS-polyacrylamide gel electrophoresis and gave single bands as illustrated in Fig. 1a. The flap endonuclease activity of the proteins was assayed via a flow cytometry-based nuclease assay system (36) before and after each SAXS measurement. Activity was also observed by time-resolved SAXS measurements by activating the endonuclease with Mg 2ϩ in the presence of the flap DNA substrate.
Preparation of the 34-mer DNA Flap Substrate-An oligonucleotide (5Ј-CCCCCCATGCTACGTTTTCGTATACGTTTTCGTA-3Ј) was synthesized by the solid-phase phosphoramidite method using an Applied Biosystems synthesizer. The oligonucleotide was designed to form a 5Ј-flap substrate, which contains two Watson-Crick duplex arms folded by TTTT loops (37) and a 10-base-long 5Ј-flap single strand (see Fig. 1b). It was purified by eluting the material through a POROS R2/H reversephase chromatography column, followed by eluting through a POROS HQ/M anion-exchange chromatography column if necessary, equipped with a Bio-Cad Workstation 700E (PerSeptive Biosystems). The purity was analyzed using a Tris borate/EDTA-urea gel (Novex) and found to be Ͼ98% pure. NMR spectroscopic analysis was used to establish the formation of the flap structure as shown in Fig. 1b. Electrophoretic mobility shift and flap endonuclease assays were used to determine the suitability of the newly designed substrate for structural analysis.
SAXS Data Collection, Reduction, and Analysis-SAXS data were reduced as described elsewhere (38,39) to give I(Q) versus Q, where I(Q) is the scattered x-ray intensity and Q is the amplitude of the scattering vector. For elastic scattering processes, Q is equal to 4sin/, where is half the scattering angle and is the wavelength of the incident and scattered x-rays. Guinier (40) and the indirect Fourier transform (41) analyses were used to calculate R g , forward scatter (I o ), and the vector distribution function (P(r)). Aggregation was ascertained from I o , which was expected to be proportional to the molecular mass (39). Lysozyme was used as a standard for scaling I o with the implicit assumption that lysozyme has the same mean scattering density as FEN-1 and the D181A mutant. Interparticle interference contribution to the scattering at the concentrations used was assumed to be negligible since preliminary measurements from the samples at concentrations between 0.5 and 6 mg/ml scaled linearly and gave the same R g values. P(r) is the frequency of all interatomic vectors within the scattering particle weighted by the product of their scattering powers. The zeroth and second moments of P(r) are equal to I o and R g , respectively, and the maximum linear dimension of the scattering particle (d max ) was determined from the corresponding value at which the P(r) function goes to zero.
SAXS measurements were done using the instrument described elsewhere (38) at the Los Alamos National Laboratory. The instrument configuration used nickel-filtered 1.542 x-rays produced from a 1.5kilowatt sealed tube copper target source and was line-focused by a single mirror giving a full width at a half-maximum of 0.74 mm and a full height of 26 mm as measured at the detector. A 4-inch-long positionsensitive linear detector (TEC Model 210Q) was placed 64 cm from the sample. Measurements spanned a Q range of 0.015-0.27 Å Ϫ1 . The net scattering from the samples was calculated by subtracting a normalized buffer spectrum measured in the same sample cell. Time course SAXS measurements, whereby data were recorded for 30 min at subsequent intervals, was used to track endonuclease activation after addition of magnesium to protein/DNA substrate samples. Typically, measurements at these protein concentrations require ϳ6 h for sufficient statistics; however, 30-min scans were good enough for observing changes in I o . SAXS from at least two separate sample preparations were measured for all measurements. Measurements were made at 10°C. P(r) analysis of the data collected included a deconvolution of the slit geometric contribution to the scattering. Omission of this correction results in a systematic ϳ0.3-Å smaller R g for FEN-1, well within the statistics of the data measured in this report (0.5-1.0 Å; 1 S.D.). Guinier analysis of the data collected was not deconvoluted for instrument geometry.

RESULTS AND DISCUSSION
Magnesium is an essential cofactor for many enzymes involved in DNA metabolism such as DNA polymerases, nucleases, and ligases. This divalent metal, commonly ligated by acidic amino acid residues in nucleases, attacks water molecules to produce a nucleophile that can break phosphodiester bonds (34). Chelating of the metal ions out of human FEN-1 can inactivate the enzyme completely. It has been hypothesized that activation of the enzyme by adding Mg 2ϩ to the reaction requires conformational changes in the enzyme before it can cleave the DNA substrate (36). To test this hypothesis, we performed small-angle x-ray scattering from wild-type FEN-1, the D181A mutant, and their complex with DNA substrate to determine their global structure and possible conformational changes upon addition of the Mg 2ϩ cofactor.
To perform SAXS experiments, it was necessary to develop an experimental approach to produce a large quantity of flap DNA substrate. Unfortunately, the conventional approach, which utilizes annealing of three independent oligonucleotides, was inadequate for this purpose due to its low yield. To overcome this problem, we designed a single oligonucleotide that has a high propensity to form the flap DNA structure, as shown in Fig. 1b (see "Experimental Procedures").
We used NMR spectroscopy and gel mobility shift and flap endonuclease assays to determine that the newly designed oligonucleotide forms a predicted flap DNA substrate. NMR spectroscopy showed the presence of a double-stranded region registered by AϭT and GϵC base pairs and showed the presence of a TTTT loop, and wild-type FEN-1 was able to bind to DNA and yielded a correct cleavage product with an expected size of the released flap strand (10 bases) (data not shown). Based upon all of these results, we concluded that the new flap DNA substrate could be used for our further study described. Fig. 2 shows Guinier plots calculated for FEN-1 and the D181A mutant as well as for FEN-1⅐34-mer DNA flap and D181A⅐34-mer DNA flap complexes. Each sample gives a Guinier region that can be fit with a straight line with reduced 2 below 1 (Table I). Molecular masses calculated for the protein samples from I o and using the lysozyme standard were within 10% of the expected value of 43,416 Da. In addition, there was no significant upturn at low Q in the scattering profiles, except for the D181A⅐34-mer DNA flap complex. The D181A⅐34-mer DNA flap complex sample had the same concentration as the FEN-1⅐34-mer DNA flap complex and gave an extrapolated I o value consistently ϳ10% larger than that found for the FEN-1⅐34-mer DNA flap complex. This increase in I o indicates slight aggregation of the D181A⅐34-mer DNA flap complex samples. The R g and d max parameters in Table I were calculated from combinations of two to four scattering measurements using different sample preparations. Guinier plots of log(I⅐Q) versus Q 2 showed a linear Q region (0.03-0.08 Å Ϫ1 ) with a negative slope, from which the radius of gyration of cross-section (R c ) could be calculated. A linear region in such a plot suggests that the proteins have an elongated shape, at least in one dimension. The R c values are also tabulated in Table I. Fig. 3 shows the R g values calculated from the P(r) analysis of the individual measurements for the proteins alone in the presence and absence of magnesium. Also, a comparison of the P(r) functions is given in Fig. 4. The P(r) functions have a single peak at ϳ27 Å and are fairly symmetric, suggesting that the proteins are globular (ellipsoidal). Modeling the data with one ellipsoid using a Monte Carlo modeling method described elsewhere (42) gave dimensions for FEN-1 of a ϭ 13.6 Ϯ 0.2 Å, b ϭ 32.4 Ϯ 1.0 Å, and c ϭ 45.1 Ϯ 1.0 Å for the best fit. The scattering profile generated from this model also gave an R c value consistent with the R c value determined from the Guinier plots. Within the statistics of R g measurements, it is evident that magnesium has no effect on FEN-1 or the D181A mutant in the absence of DNA. The fact that Mg 2ϩ -induced conformational changes was not observed is probably because the Mg 2ϩ binding causes a localized instead of global conformational effect or the induced global conformational changes are quite small.
Next, we examine the effect of Mg 2ϩ on the DNA substrate by both scattering and modeling. Scattering profiles and P(r) functions from the free 34-mer DNA fragment in the absence and presence of Mg 2ϩ are shown in Fig. 5. In the absence of Mg 2ϩ , the P(r) function showed a peak at ϳ16 Å and decreased approximately linearly out to ϳ60 Å. A simple two-cylinder model was constructed based on the 5Ј-flap DNA structure shown in Fig. 1b. The intent of this modeling was to add further support for the self-annealed structure by comparing the P(r) function calculated from the model with the P(r) function calculated from the scattering data. In this model, one cylinder represents the double-stranded portion of the DNA with dimensions of a B-type DNA structure. The second cylinder represents the single-stranded portion and was assumed to have a diameter half that of a B-type DNA structure with a 4-Å rise/base pair. One end of the second (smaller) cylinder was positioned on the surface of the first cylinder and located at the middle of the first cylinder along its axis so that the total DNA structure basically forms a T shape. The cylinder axis of the second cylinder was then given an angle of 45 o relative to the cylinder axis of the first cylinder. A schematic of the two-cylinder model is shown in Fig. 5b (inset). The P(r) function for this model is plotted in Fig. 5b and shows the same basic features as the measurements, namely, a peak at ϳ17 Å and linearly decreasing long vectors. The scattering data are sensitive to electron density pair distribution of vectors defining the scattering object; however, phase information is lost in this type of measurement, so a unique model cannot be determined based solely on a single scattering curve. Therefore, the model having the same peak position at ϳ17 Å taken alone does not conclusively show that the 34-mer DNA fragment adopts the 34-mer DNA flap, but it has not been ruled wrong. On the other hand, this simple model, in combination with the binding affinity measurements, the nuclease activity measurements, and the NMR analysis, strongly supports that the 34-mer DNA fragment adopts the flap structure. Addition of Mg 2ϩ caused change in the scattering profile and, more important, an increase by 16.6% in the extrapolated I o scattering. This intensity increase suggests that Mg 2ϩ causes the DNA to aggregate. Aggregation of the DNA substrate in the presence of Mg 2ϩ is not surprising since ionic interaction of the cation with the negatively charged phosphate groups in the DNA backbone would decrease electrostatic repulsion between DNA molecules in solution and allow weaker attractive interactions to dominate.
A comparison of the scattering from the two proteins bound to the 34-mer DNA substrate is shown in Fig. 6. Lysozyme could not be used as a standard for molecular mass determination for the protein⅐DNA complexes as done for the proteins alone since the mean scattering density for DNA is different  4. P(r) functions for FEN-1 (a)   I X-ray scattering data from FEN-1 and D181A R g and R c were calculated from the scattering data using Guinier analysis. R g and d max were calculated using P(r) analysis. FEN-1 and the D181A mutant alone give approximately the same R g , R c , and d max values in both the absence and presence of Mg 2ϩ , suggesting that Mg 2ϩ either induces no conformational change or induces a localized or global conformational change that is not measurable within the precision of these measurements. The 34-mer DNA in the presence of Mg 2ϩ aggregates as shown by a 16.6% increase in I p , therefore, the larger R g and d max values observed relative to the case without Mg 2ϩ are not necessarily attributable only to a Mg 2ϩ -induced conformational change in the DNA substrate. The D181A ⅐ 34-mer DNA flap complex has a consistently ϳ10% larger I o relative to the FEN-1 ⅐ 34-mer DNA flap complex, suggesting that slight aggregation occurs in the mutant ⅐ DNA substrate complex. Thus, the scattering data show that the D181A mutant possibly binds and interacts differently with the DNA fragment compared with wild-type FEN-1. ϭ I protein ϩ I protein⅐DNA ϩ I DNA where n is the particle number density, ⌬ x is the scattering density of component x, and dV x is the integration volume element over component x. Assuming specific volumes of 0.73 ml/g for the protein and 0.56 ml/g for the nucleic acid, we expect, for our measurements, that the contribution of the DNA would increase I o by ϳ60% above the protein-alone measurement. This calculated increase in I o takes into account the concentration differences of the samples (ϳ4.7 instead of ϳ6 mg/ml). We observed this increase for the FEN-1⅐34-mer DNA flap complex samples, suggesting that these samples are monodisperse. In addition, as expected for monodisperse solutions, we obtained good agreement between the R g values obtained using the Guinier and P(r) analyses, 34.4 Å (see Table I). The major peak in the P(r) function for the wild-type complex is ϳ28 Å, and d max is ϳ104 Å. However, a difference was observed between the R g values for the D181A⅐34-mer DNA flap complex samples obtained from the Guinier and P(r) analyses, 40.6 and 43.9 Å, respectively, suggesting that the D181A⅐34-mer DNA flap complex samples may be aggregated. The I o value is more meaningful in evaluating the degree of aggregation of the D181A⅐34-mer DNA flap complex and is ϳ10% above the I o value for the FEN-1⅐34-mer DNA flap complex samples, suggesting that aggregation is present, but not severe. In addition, the d max value calculated for the mutant complex samples is ϳ34 Å longer than that calculated for the wild-type complex. It is possible that the D181A mutant complex is a slightly more extended structure; however, drawing this conclusion is precarious due to the slight aggregation problem. The scattering data from the complexes and from the free proteins taken together suggest that the D181A mutant binds and interacts differently with the DNA fragment compared with wild-type FEN-1 and is evident by the induction of aggregation of the mutant complex. The 5Ј-nuclease domains of E. coli and Taq DNA polymerases are functional homologs of human FEN-1 that possess two Mg 2ϩ -binding sites. The amino acid sequences of 10 5Јnuclease domains from DNA polymerases in the polymerase I family and viral nucleases were compared by Gutman and Minton (43). The results showed six highly conserved sequence motifs containing 10 conserved acidic residues. In earlier work, we extended this sequence comparison to eight additional sequences of XPG/FEN-1 nuclease family and confirmed that seven acidic amide residues are very conserved in all 18 sequences (35). According to the crystallographic structures of Taq DNA polymerase (44)  radius around the metal ions. Some of these residues ligate one Mg 2ϩ ion, whereas others ligate the second Mg 2ϩ ion. Our mutagenesis work (34) further confirmed that Asp-181 is a critical amino acid that ligates the Mg 2ϩ and is involved in the cleavage process of the flap endonuclease activity only, but does not appear to affect DNA binding. In this report, we found from SAXS that Asp-181 is actually important for proper binding of the DNA and is not only required for cleavage activity. In fact, proper binding proceeds enzymatic activity whereby the proper orientation of the DNA in the catalytic site is required before cleavage can ensue.
Measurement of the time course change in scattering after initiating cleavage activity by adding magnesium to FEN-1⅐34mer DNA flap complex samples required slowing down the kinetics. Typically, for the protein and substrate concentrations used in this report, activity rates are such that it would require only minutes for DNA cleavage completion, too fast to monitor with our scattering instrument, which requires at least 30 min of measurement time to obtain good enough statistics for calculating extrapolated I o values. Addition of monovalent ions is know to slow kinetics for this system by ϳ100fold (4); therefore, we added 100 mM NaCl and performed measurements at the low temperature of 10°C. After initiating activity, we observed a decrease in the scattering intensity as shown in Fig. 7a. The decrease in scattering intensity contin-ued for up to 6 h, after which the profiles remained constant. When activating nuclease activity by adding Mg 2ϩ , we expect a decrease in I o scattering if the activated protein and the DNA components dissociate after DNA cleavage. Assuming complete dissociation of the components after cleavage, we can write the total scattering in terms of each species in solution as follows (Equation 2), where ssDNA and dsDNA are single-and doubled-stranded DNA, respectively. No scattering cross-term would be present between the species in solution. It is possible that the protein remains bound to either the single-or double-stranded DNA fragment after cleavage, in which case, we would have the total scattering given by Equation 3, or Equation 4.
The protein⅐DNA complex terms in Equations 3 and 4 would include a scattering cross-term as similarly expressed in Equation 1. From Equations 1-4, we would expect 0, 43.9, 32.5, and 15% decreases in I o scattering, respectively. The "apparent" I o scattering decreased by ϳ24 -29% for two independent measurements. This apparent decrease was determined by comparing the first measurement made within 1 h of activation and a final measurement made after 6 h of activation after which no further decrease in I o was observed. At first glance, this measured decrease in intensity appears most consistent with FEN-1 remaining bound to the single-stranded portion of the DNA fragment after cleavage. However, because our measurements were for 30 min, the initial t ϭ 0 measurement is underestimated. A crude estimate on the order of magnitude of the error in underestimating the t ϭ 0 measurement can be made by assuming that the reaction is completed by 6 h and that it proceeds exponentially. A nonlinear least-squares "best" fit to a plot of I o with respect time can be made using the form I o ϭ A ϩ B exp(Ϫt/tЈ ), where A and B are constants to be determined in the fit and 1/tЈ is the reaction rate, also to be determined in the fit. We assume a considerably larger error for the earliest measurement (average t ϭ 15 m) relative to the later measurements and iterate the fit by successively replacing the earliest measurement until the fit converges and then extrapolate to t ϭ 0. Using this simple and crude approximation gives the exponential fit to the I o data as shown in Fig. 7b. We find that, under the conditions of this time course experiment, I o at t ϭ 0 is underestimated by at least 21%. Taking this into account, the correction in the decrease in I o would then be ϳ37-42% due to nuclease activity, more consistent with total dissociation of the protein from its DNA substrate. It is worth pointing out that, regardless of error estimates, we can conclude with certainty that the protein is not bound solely to the remaining nicked double-stranded DNA component since the measurement at t ϭ 0 can be only underestimated, not overestimated. Similar measurements were made after Mg 2ϩ was added to D181A⅐34-mer DNA flap complex samples. In this case, no change in the scattering intensity with respect to time was observed. FEN-1 nuclease is critical in the maintenance of genome stability and mutational avoidance. Yeast null mutants displayed a unique mutational spectrum derived from the failure of RNA primer removal during the lagging strand DNA synthesis. The SAXS study reported here provides a global structure of this unique DNA replication and repair enzyme and confirms the interaction of enzyme/flap DNA substrate/Mg 2ϩ FIG. 7. Time course SAXS measurement showing Mg 2؉ activation of the FEN-1 activity. Mg 2ϩ was added to the FEN-1⅐34-mer DNA flap complex (1:1) at t ϭ 0 h, and scattering profiles were record at intervals for a 30-min duration. Profile measurements at t ϭ 0 (measurement between 0 and 30 min; black circles) and at t ϭ 6 h (gray squares) are shown in a. We added 100 mM NaCl to the buffer and performed measurements at 10°C to slow down the nuclease activity rate. After 6 h, I o did not decrease any further. In b, we show I o (black circles) versus time fitted to an exponential function of the form I o ϭ A ϩ B exp(Ϫt/tЈ , where A and B are constants to be determined in the fit and 1/tЈ is the reaction rate, also to be determined in the fit. The fitting procedure was iterative so as to account for the underestimation of the extrapolated t ϭ 0 measurement. Note the underestimation of the first measurement, which lies below the iterative exponential fit.
cofactor. It also suggests the critical role of Asp-181 in the enzyme/flap DNA substrate interaction to ensure a proper conformation and a cleavage-ready status of the enzyme. A time course change in the scattering profiles arising from magnesium activation of the FEN-1⅐DNA flap substrate complex is consistent with the enzyme being freed from both the singleand double-stranded DNA product portions after cleavage. Given the critical role of FEN-1 in DNA replication and repair, this study illustrates initial understanding of molecular dynamics common to the structure-specific nuclease family that are central to processes influencing genome stability and early events that modulate cancer susceptibility and tumorigenesis.


s2fieldsofstudy: Biology Chemistry

text:
Ca2+ Sensitization and Potentiation of the Maximum Level of Myofibrillar ATPase Activity Caused by Mutations of Troponin T Found in Familial Hypertrophic Cardiomyopathy*

Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed inEscherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+–sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+–sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+–sensitive ATPase activity, Ca2+–sensitization and potentiation of the maximum level of the ATPase activity.

This study was carried out to investigate the functions of five HCM-linked TnT mutants in cardiac myofibrils under physiological conditions. We prepared recombinant human cardiac Tn subunits (wild-type TnC, TnI, and TnT) and five HCM-linked TnT mutants, and these recombinant proteins were then incorporated into rabbit cardiac myofibrils using our previously reported TnT treatment procedure (15)(16)(17). The Ca 2ϩ -sensitive ATPase activity of these reconstituted myofibrils was examined. We thus found the HCM-linked TnT mutants to have at least two different effects on ATPase activity, which are Ca 2ϩsensitization (I79N, R92Q, and R278C) and the potentiation of the maximum level (F110I and E244D). The Ca 2ϩ -sensitizing effect of I79N and R92Q TnT mutants is consistent with our previous study using skinned fibers (18). These findings suggest that an enhanced contractility of the cardiac muscle, which occurs as a result of mutations in both groups, may be a common mechanism for the pathogenesis of HCM associated with these TnT mutations.

EXPERIMENTAL PROCEDURES
Cloning and Mutagenesis of Human Cardiac Tn cDNAs-Human cardiac Tn (TnC, TnI, and TnT) cDNAs were amplified by reverse transcriptase-polymerase chain reaction of human heart mRNA purchased from CLONTECH (Palo Alto, CA). The polymerase chain reaction products were subcloned into the pUC 119 vector for screening by a restriction analysis and DNA sequencing. The obtained wild-type Tn cDNAs were then constructed into a pET-3d vector for expression. To obtain mutant TnTs, mutagenesis was carried out by polymerase chain reaction according to the method described by Horton (19). Oligonucleotides employed for mutagenesis were: 5Ј-CCTCCCAAGAACCCCGAT-GG-3Ј for the I79N mutation, 5Ј-GACATCCACCAGAAGCGCATG-3Ј for the R92Q mutation, 5Ј-GAGGCTCACATTGAGAACAGG-3Ј for the F110I mutation, 5Ј-TATAACTTGGATGCAGAGAAG-3Ј for the E244D mutation, and 5Ј-CTCCAAGACCTGCGGGAAGG-3Ј for the R278C mutation. The changed bases were underlined. The results of the mutations in TnT cDNA were confirmed by DNA sequencing.
Expression and Purification of Recombinant Tns-The wild-type Tn and mutant TnTs were expressed in Escherichia coli BL21(DE3) and purified as described previously (18) with slight modifications. The induction of protein expression by isopropyl-␤-D-thiogalactopyranoside was continued for 5.0 h, and then the cells were harvested and lysed. The lysate was then centrifuged at 15,000 rpm for 25 min at 4°C. For the purification of TnT and TnC, 10 mM Tris/HCl (pH 8.0), 6 M urea, 5 mM CDTA, and 15 mM 2-ME were added to the supernatant and the samples were applied to RESORCE Q column (Pharmacia Biotech Inc.) using a fast protein liquid chromatography system. For the purification of TnI, the pellet was resuspended with the same solution described above and the sample was applied to a Poros HS/M column (PerSeptive Biosystem).
Preparation of Myofibrils-To prepare myofibrils, fresh rabbit car-diac left ventricular muscle was cut in 5-mm slice and soaked in a solution containing 1% Triton X-100, 100 mM KCl, 20 mM MOPS (pH 7.0), and 0.5% 2-ME for 6 h at 4°C. The solution was then changed to another solution containing 50% glycerol, 100 mM KCl, 20 mM MOPS (pH 7.0), and 0.5% 2-ME and the slices were kept in it overnight at 4°C. The following morning, these slices were transferred to the new solution and stored at Ϫ20°C. Myofibrils were prepared from the kept samples according to the method of Solaro et al. (20). Tn Exchange in Myofibrils-Tn exchange was carried out according to our previously reported method (15)(16)(17) with slight modifications. Myofibrils (300 g/ml) were incubated in a solution containing 20 mM MOPS/KOH (pH 6.2), 265 mM KCl, 2 mM EDTA, 2 mM MgCl 2 , 0.5 mM 2-ME and 37.5 g/ml recombinant human cardiac TnT at 25°C for 1 h while shaking. These myofibrils were washed with a solution containing 0.5% Briji-58, 20 mM MOPS (pH 7.0), 265 mM KCl, 2 mM MgCl 2 , 0.5 mM 2-ME, and the myofibrils were resuspended in a solution (300 g/ml) containing 60 mM KCl and 1 mM sodium bicarbonate after washing with the same solution. The myofibrils were then reconstituted with recombinant TnI (15.2 g/ml) and TnC (11.6 g/ml) on ice for 1 h.
ATPase Activity Measurement-The reaction mixture (200 l) for the ATPase assay consisted of 90 mM KCl, 5 mM MgCl 2 , 20 mM MOPS (pH 7.0), 1 mM Ca 2ϩ -EGTA, 4 mM ATP, 40 g of myofibrils. The reaction was started by adding ATP at 25°C and was terminated by adding 1.6 ml of a mixture of 50% acetone, 2.5 mM (NH 4 ) 6 Mo 7 O 24 ⅐4H 2 O and 1.25 N H 2 SO 4 . The contents were mixed carefully and 160 l of 1 M citric acid were added to tubes and the yellow color was measured at OD 355 (21). Ca 2ϩ concentrations in the reaction mixtures were calculated as described previously (22).
Electrophoresis-SDS-PAGE was performed according to the procedure of Laemmli (23), with an acrylamide concentration of 12%. Alkaline gel electrophoresis was carried out according to the procedure described previously (24). Alkaline urea gel electrophoresis was done by the procedure of Pan and Solaro (25), with an acrylamide concentration of 13.5% at pH 8.5. The gel was stained with Coomassie Brilliant Blue R-250 and an optical densitometric scan was performed using Phoretix gel analysis software (Phoretix International Ltd.).

Comparison of Recombinant Human Cardiac Tn Subuint
and Native Rabbit Cardiac Tn Subuint-Three subunits of human cardiac Tn (TnT, TnI, and TnC) were cloned by the reverse transcriptase-polymerase chain reaction technique and expressed using a pET-3d vector. As shown in Fig. 1A, recombinant human cardiac TnT and TnC had exactly the same electrophoretic mobility as rabbit cardiac TnT and TnC on 12% SDS-PAGE gel. Recombinant human cardiac TnI, however, had a little faster mobility than rabbit cardiac TnI. An alkaline gel electrophoresis analysis showed the recombinant human cardiac Tn subunit combined with the native rabbit cardiac Tn subunit to form binary or ternary complexes under nondenaturing conditions (Fig. 1B). As a result, the recombinant human cardiac Tn subunits had the ability to form a Tn complex in the same way as the native rabbit cardiac Tn subunits.
Effect of Wild-type TnT and I79N TnT Treatment on the ATPase Activity of Rabbit Cardiac Myofibrils-We previously reported the endogenous Tn complexes in myofibrils were replaced with exogenous TnT after treatment with an excessive amount of purified TnT in acidic solution and the ATPase activity of the treated myofibrils becomes insensitive to Ca 2ϩ (15)(16)(17). Fig. 2 shows the ATPase activity of rabbit cardiac myofibrils treated with different amounts of recombinant TnT in the presence (pCa 5.18) and absence (pCa 7.05) of Ca 2ϩ . The ATPase activity in the presence of Ca 2ϩ did not change after the treatment with TnT at any concentrations, whereas the ATPase activity in the absence of Ca 2ϩ increased in a concentration-dependent manner and reached almost a maximum level after treatment with 20 g/ml TnT (Fig. 2). The maximum ATPase activities of wild-type TnT-treated myofibrils were 22 Ϯ 1.6 nmol⅐P i /mg/min in the absence of Ca 2ϩ and 32 Ϯ 0.5 nmol⅐P i /mg/min in the presence of Ca 2ϩ (mean Ϯ S.E., n ϭ 4).
The maximum ATPase activities of I79N TnT-treated myofibrils were 22 Ϯ 1.5 nmol⅐P i /mg/min in the absence of Ca 2ϩ and 33 Ϯ 3.0 nmol⅐P i /mg/min in the presence of Ca 2ϩ (mean Ϯ S.E., n ϭ 4). As a result, the maximum ATPase activity in the absence of Ca 2ϩ increased up to approximately 65% of the maximum activity in the presence of Ca 2ϩ by the treatments with either wild-type or I79N TnT at 37.5 g/ml. In the following experiments, myofibrils were treated with this concentration of TnT.

Determination of the Exchange Rate of Tn Complex in Myofibrils Treated with Wild-type TnT and I79N
TnT-To determine the extent of the exchange of Tn complex in the myofibrils, both SDS-PAGE and alkaline urea gel electrophoresis were carried out. The densitometry of TnI bands in the SDS-PAGE pattern of the myofibrils showed that 65.3 Ϯ 3.2% (mean Ϯ S.E., n ϭ 5) and 63.8 Ϯ 4.0% (mean Ϯ S.E., n ϭ 5) of the native TnI was removed by wild-type TnT treatment and I79N TnT treatment, respectively (Fig. 3A). Although native rabbit cardiac TnT and recombinant human cardiac TnT had exactly the same electrophoretic mobility on 12% SDS-PAGE gel (Fig. 3A, lanes 1 and 2), the recombinant human cardiac TnT had a slightly faster mobility than rabbit cardiac TnT on alkaline urea gel (Fig. 3B, lanes 1 and 2). A densitometry scan on alkaline urea gel was performed to estimate the amount of TnT exchanged into myofibrils. On the typical scanning patterns of TnT-treated and untreated myofibrils (Fig. 3C), the density of both native rabbit TnT and TnC bands decreased by treatment with wild-type TnT or I79N TnT of which mobility were the same as actin. Although a quantitative analysis of TnTs could not be carried out because of the high background density, the increase in the density of the actin peak after TnT treatment indicated the incorporation of human recombinant TnTs into the myofibrils. The densitometry of the TnC bands showed the percentage of native rabbit TnC removed from myofibrils to be 65.0 Ϯ 4.0% (mean Ϯ S.E., n ϭ 5) by wild-type TnT treatment and 63.9 Ϯ 2.1% (mean Ϯ S.E., n ϭ 5) by I79N TnT treatment. These gel scan analyses, taken together with the ATPase assays, provided strong evidence that the extent of the recombinant human cardiac TnT incorporated into the  myofibrils were approximately 65% and that the wild-type and I79N mutant TnTs were also equally effective in replacing the endogenous Tn complex in myofibrils. Fig. 4, the addition of the recombinant human cardiac TnI resulted in a decrease in the Ca 2ϩ -insensitive ATPase activity of the myofibrils treated with wild-type or I79N TnT in a dose-dependent manner and were completely inhibited at 15.2 g/ml (Fig. 4A). The amounts of TnI required for a 50% inhibition (IC 50 ) were 0.80 Ϯ 0.05 g/ml and 1.74 Ϯ 0.21 g/ml (mean Ϯ S.E., n ϭ 3) for wild-type TnT-treated and I79N TnT-treated myofibrils, respectively. This indicated that the inhibitory effect of TnI on actin-tropomyosin was significantly smaller in the I79N mutant TnT-treated myofibrils than the wild-type TnT-treated myofibrils. In the presence of Ca 2ϩ , recombinant human cardiac TnC activated the suppressed ATPase activity of the TnI-reconstituted myofibrils in a dosedependent manner and fully neutralized the ATPase activity at the concentration of 11.6 g/ml (Fig. 4B). No significant difference in the neutralizing effect of TnC was detected between the myofibrils containing wild-type TnT and I79N TnT.

pCa-ATPase Activity Relationships of the Myofibrils Treated with Wild-type TnT and I79N TnT, and Reconstituted with Recombinant Human Cardiac TnI and TnC at Different pH
Values (pH 6.5, 7.0, and 7.5)-Although no significant difference was seen in the pCa-ATPase activity relationships at pH 7.5, the Ca 2ϩ sensitivity (pCa 50 , pCa at half-maximum activation) of the myofibrils reconstituted with I79N mutant TnT was significantly higher than the Ca 2ϩ sensitivity of myofibrils reconstituted with wild-type TnT at pH 7.0 and 6.5 (Fig. 5). The difference in the Ca 2ϩ sensitivity of the reconstituted myofibrils was thus larger at pH 6.5 than at pH 7.0 (Table I). However, no significant difference was observed in the maximum ATPase activity and the Hill coefficient (n H , an index of cooperativity) between the myofibrils reconstituted with wildtype TnT and reconstituted with I79N mutant TnT at any pH.
An SDS-PAGE Analysis of TnT Mutants, R92Q, F110I, E244D, and R278C-Four other missense mutations, R92Q, F110I, E244D, and R278C mutant TnTs, were generated by the overlap extension methods. An SDS-PAGE analysis showed all of these mutants to have the same electrophoretic mobility on 12% SDS-PAGE gel (Fig. 6A). The densitometric scan of the SDS-PAGE gel was performed to determine the amount of native TnI removed from the myofibrils after treatment with mutant TnTs (Fig. 6B, Table II). No significant difference was found in the amounts of native TnI removed from myofibrils by treatment with mutant TnTs. This result indicated that the extent of the removal of native TnT and hence the incorporation of these mutant TnTs into the myofibrils were almost the same as the wild-type and I79N TnTs.
The Effect of TnT Mutants on the Ca 2ϩ -sensitive ATPase Activity-The pCa-ATPase activity relationships were examined at pH 7.0 using myofibrils which were treated with these TnT mutants and then reconstituted with recombinant human cardiac TnI and TnC. As shown in Fig. 7, an increase in the Ca 2ϩ sensitivity of the ATPase activity was observed with R92Q and R278C TnT mutants, with no significant effect on

Comparison of the effects of mutant TnTs on myofibrils
Myofibrils were treated with wild-type or mutant TnT, and an SDS-PAGE analysis or ATPase activity measurement in the presence of Ca 2ϩ (pCa5.18) was carried out before and after reconstitution. The gels stained with Coomassie Brilliant Blue R-250 were analyzed by an optical densitometric scan using Phoretix gel analysis software to determine the amount of TnI removed from myofibrils by the treatment with TnTs. Data are the mean Ϯ S.E. of five or four independent experiments. pCa values at half-maximum ATPase activity (pCa 50 ) and Hill coefficient (n H ) were calculated from the data shown in Fig 7. The data are the mean Ϯ S.E. of three or four experiments done in triplicate. the maximum ATPase activity. On the other hand, an increase in the maximum ATPase activity was observed with F110I and E244D TnT mutants, without any change in the Ca 2ϩ sensitivity of the ATPase activity (Fig. 8). No significant change in the cooperativity was observed between the wild-type TnT-treated myofibrils and mutant TnT-treated myofibrils (Table II). The summary in Table II clearly indicates these HCM-related TnT mutants to have the same ability as wild-type TnT to exchange native TnT in the myofibrils and are thus classified into at least two groups according to their effects on the Ca 2ϩ -sensitive ATPase activity.

DISCUSSION
This is the first report to evaluate the properties of recombinant human cardiac TnT⅐I⅐C complex in myofibrils under physiological conditions. The results of the present study indicated the recombinant human cardiac Tn subunits and native rabbit cardiac Tn subunits to be almost identical. These findings led us to analyze the effects of HCM-linked TnT mutants on the Ca 2ϩ -sensitive ATPase activity of rabbit cardiac myofibrils. We already reported that I79N and R92Q TnT mutants in HCM have a Ca 2ϩ sensitizing effect on the tension development of skinned fibers prepared from the rabbit ventricular muscle (18). In the present study, the Ca 2ϩ sensitizing effects of I79N and R92Q mutant TnTs on the myofibrillar ATPase activity were thus confirmed.
Regarding the molecular mechanism of action of these TnT mutants, the inhibitory effect of TnI on the Ca 2ϩ -insensitive ATPase activity of myofibrils with I79N mutant TnT was found to be smaller than that with wild-type TnT, while the neutralizing effect of wild-type TnC was not affected by this mutation (Fig. 3, A and B). These findings suggest that the I79N mutant TnT reduced the affinity of TnI to actin-tropomyosin. The same mechanisms on the action of R92Q TnT mutant was also suggested in our previous study using skinned fibers (18). In this report, we showed that a mutation at Arg 278 in TnT, in addition to the mutations at Ile 79 and Arg 92 in the TnT mentioned above, thus caused an increased Ca 2ϩ sensitivity of the ATPase activity without any change in the maximum level. It was also recently reported that the HCM-linked ␣-tropomyosin mutant, D175N, is associated with an increase in the Ca 2ϩ sensitivity (26). It is therefore highly conceivable that an increase in the Ca 2ϩ sensitivity is one of the characteristic features of the HCM caused by the regulatory proteins associated with the thin filament.
The results of both the present and previous studies showing the Ca 2ϩ sensitizing effects of R92Q mutant on the ATPase activity of myofibrils and the tension development of skinned fibers apparently conflict with the results of a recent study by Marian et al. (27). They reported that the expression of R92Q mutant human cardiac TnT in cultured adult feline cardiac myocytes impaired the contractile performance as indicated by both fractional cell shortening and the peak velocity of shortening of the myocytes (27). Although the reason for this discrepancy in the results is not known, it might be due to the difference of the design of the experiments. No significant difference has been reported between the effects of I91N mutation in rat embryonic cardiac TnT, thus corresponding to I79N mutation in human, and wild-type TnT on Ca 2ϩ sensitivity of the thin filaments examined by S-1 Mg-ATPase activity at pH 7.5 (28). While we found this mutant to behave in a manner similar to a wild-type at pH 7.5, the mutant had a Ca 2ϩ sensitizing effect on the actomyosin ATPase activity at pH 7.0 and 6.5, which was more significant at an acidic pH 6.5, which is also consistent with the previous observations on skinned fibers (18). This finding suggests that the I79N mutant may have a strong effect on the contractile performance during myocardial ischemia, although the effects of acidic pH on other TnT mutants have not yet been examined.
The most significant finding of the present study was that the maximum ATPase activity of the myofibrils was potentiated by the mutations at Phe 110 and Glu 244 in TnT ( Fig. 8 and Table II). The potentiation effects of F110I and E244D TnT mutants were not observed after the TnT treatment but were observed only after subsequent reconstitution with TnC and TnI (Table II). This strongly suggests that the potentiation of the maximum ATPase activity is caused through a change in the interaction of TnT and TnC⅐I.
In the present study, we found that the effects of HCMlinked TnT mutations can be classified into at least two different types. The I79N, R92Q, and R278C TnT mutants enhanced Ca 2ϩ sensitivity of myofibrillar ATPase activity without affecting the maximum level of ATPase activity, whereas the F110I and E244D TnT mutants enhanced a maximum level of ATPase activity without affecting the Ca 2ϩ sensitivity. These two groups of missense mutations in TnT might cause HCM in different manners. However, it is noteworthy that the mutations which cause an increase in the maximum ATPase activity alone also have an apparent Ca 2ϩ sensitizing effect at a submaximal concentration of Ca 2ϩ due to a scale-up of the overall ATPase activity. Since the intact cardiac muscle has been reported to never be activated beyond the half-maximal level (29), an enhanced myofilament response to the submaximal Ca 2ϩ may be a common phenomenon for the pathogenesis of HCM associated with these missense mutations in TnT. In several reports HCM-linked ␤-myosin heavy chain mutations and TnT mutations have been shown to impair cardiac performance and cause compensatory cardiac hypertrophy (30 -32). However, our findings suggest that certain mutations might determine a hypercontractile state and thus appear to induce hypertrophy directly, instead of causing it indirectly via the mechanism of compensatory hypertrophy.


extfieldsofstudy: Biology Medicine

text:
Heparan Sulfate-modified CD44 Promotes Hepatocyte Growth Factor/Scatter Factor-induced Signal Transduction through the Receptor Tyrosine Kinase c-Met*

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt’s lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.

The CD44 family of cell surface glycoproteins is broadly expressed by cells of epithelial, mesenchymal, and hematopoietic origin and is involved in cell-matrix adhesion, hematopoiesis, and lymphocyte homing and activation (1). Furthermore, a large body of experimental and clinical studies support a role for CD44 in tumor progression and metastasis (2)(3)(4). The CD44 gene consists of 19 exons (5). Due to alternative splicing, which involves at least 10 exons encoding domains of the extracellular portion of the CD44 molecule, a large number of CD44 isoforms is generated (6 -10). Post-translational modification generates further diversity, yielding both N-linked and O-linked glycan forms of CD44 in addition to proteoglycan variants containing chondroitin, keratan, or heparan sulfate (11)(12)(13)(14). The expression pattern of these CD44 variants is tissue-specific. On lymphocytes the short 80 -90-kDa standard form of CD44 (CD44s) 1 is most abundant, while larger variants (CD44v) predominate on some normal and neoplastic epithelia and are also found on activated lymphocytes and on malignant lymphomas (15)(16)(17)(18)(19). This selective expression suggests specific biological functions for the various splice variants, but at present, these are poorly defined. Similarly, the mechanism(s) through which CD44 functions in tumorigenesis is not known.
An obstacle toward understanding the functions of the CD44 family is the limited knowledge of its molecular partners. The cytoplasmic tail of the CD44 molecule has been shown to interact with the actin cytoskeleton via ankyrin and proteins of the ERM family, and is associated with Src family tyrosine kinases (20 -23). This suggests a role in signaling as well as in the regulation of cell shape and motility. Although several potential CD44 ligands have been identified, the only interaction of the extracellular domain of CD44 that has been extensively studied is that with hyaluronate. CD44s acts as a major receptor for this glycosaminoglycan which is highly abundant in mesenchymal tissues and is believed to play a role in cell migration and differentiation (24,25).
A novel and potentially highly significant function of CD44 is its ability to interact with heparin-binding growth factors (26,27). These growth factors bind to a HS side chain attached to the evolutionary conserved consensus motif SGSG encoded by exon v3 (13,27). Heparan sulfate proteoglycans (HSPGs) are believed to play an important regulatory role in cell growth and motility by binding growth factors and by presenting these factors to their high affinity receptors. This process has been particularly well explored for the fibroblast growth factors 1 and 2 (FGF-1 and -2). For these factors, binding to HSPGs has been shown to be required for their biological function, presumably by promoting FGF dimerization required for efficient receptor cross-linking and activation (28 -32).
In the present study, we explored the physical and functional interaction between heparan sulfate-modified forms of CD44 (CD44-HS) and hepatocyte growth factor/scatter factor (HGF/ SF). HGF/SF is a heparin-binding growth factor (33) that induces growth, motility, and morphogenesis of target epithelial and endothelial cells by binding to the receptor tyrosine kinase c-Met (34,35). In addition, recently HGF/SF was shown to be involved in hematopoiesis, and lymphocyte adhesion and migration (36 -42). Apart from these physiological functions, there is ample evidence for a key role of the HGF/SF-c-Met pathway in tumor growth, invasion, and metastasis. For example, HGF/SF induces epithelial cells to invade collagen matrices in vitro, and NIH 3T3 cells co-transfected with c-met and HGF/SF acquire an invasive and metastatic phenotype (43)(44)(45). Furthermore, in HGF/SF transgenic mice, tumors develop in many different tissues including mammary glands, skeletal muscles, and melanocytes (46). In human cancer, both HGF/SF and c-Met are often overexpressed, and in hereditary renal cancer germline mutations in the c-met gene have recently been reported (47)(48)(49)(50)(51)(52). Here, we show that CD44-HS strongly promotes signal transduction through the HGF/SF-c-Met pathway, which is demonstrated to occur in a heparan sulfate-dependent fashion.
Purification of Wild Type and Mutant HGF/SF-The construction of pVL1393 vectors (Pharmingen, San Diego, CA) containing wild type or mutant HGF/SF (HP1) cDNA was described elsewhere (57).
HGF/SF (wild type and HP1) was produced in a baculovirus system as described previously (58). In brief, Sf9 insect cells were transduced with an amplified virus stock and after 3 days media were pooled and analyzed for scattering activity in the Madin-Darby canine kidney cell dissociation assay (59). Then, HGF/SF was purified with Ni-NTA resin from the QIAexpress system (Qiagen, Hilden, Germany). HGF/SF concentrations were measured by enzyme-linked immunosorbent assay as described previously (41). In addition, HGF/SF (wt and HP1) was analyzed by Western blotting using goat anti-HGF/SF.
Enzyme Treatments-For enzymatic cleavage of glycosaminoglycans,

FIG. 2. Presence of heparan sulfate on CD44 isoforms.
A, FACS analysis of heparan sulfate expressed on representative mock, CD44s, CD44v8 -10, or CD44v3-10 Namalwa transfectants that were treated with PBS (filled histogram), 25 milliunits/ml heparitinase (solid line), or 25 milliunits/ml chondroitinase ABC (dotted line) at 37°C for 3 h. Heparan sulfate was detected by the mAb 10E4, followed by RPE-conjugated goat anti-mouse. B, a similar FACS analysis as shown in A, but with the use of mAb 3G10 which recognizes ⌬HS stubs which remain on HSPG core proteins after treatment with heparitinase. C, Western blot of CD44 immunoprecipitates. CD44 was precipitated from CD44 Namalwa transfectants using the anti-pan CD44 mAb Hermes-3. Precipitates were then treated with PBS (Ϫ), 200 milliunits/ml heparitinase (HT), or 1 unit/ml chondroitinase ABC (CH) at 37°C for 2 h. The Western blot was stained with the anti-pan CD44 mAb Hermes-3 (upper panel), stripped, and re-stained with the mAb 3G10 (lower panel) which recognizes ⌬HS stubs after treatment of HS with heparitinase. FACS Analysis-For FACS analysis cells were blocked with 10% pooled human serum (CLB, Amsterdam, The Netherlands), 1% bovine serum albumin (Fraction V) (Sigma, Bornem, Belgium) in PBS at 4°C for 15 min and washed with FACS buffer (1% bovine serum albumin in PBS), respectively. Then, the cells were incubated with the primary antibodies for 1 h, washed, and incubated with the secondary antibody for 30 min. Incubations were in FACS buffer at 4°C, and cells were analyzed by using a FACScan (Becton Dickinson, Mountain View, CA).
For binding of recombinant human HGF/SF (wild type or HP1) (R&D Systems or our own product), cells were incubated with this protein (18 nM or as indicated) for 1 h, prior to the antibody incubations. This step was followed by washing with FACS buffer.
Western blotting of immunoprecipitates and total cell lysates was essentially performed as described previously (23). A single modification was that, for analysis of phosphorylated proteins, membranes were blocked and stained in 2% bovine serum albumin, 20 mM Tris-HCl, 150 mM NaCl (pH 7.5), and 0.05% Tween 20 (Sigma). Films were scanned with an Eagle Eye II video system (Stratagene, La Jolla, CA) and band intensities were determined with ONE-Dscan software (Stratagene). c-Met phosphorylation was expressed as the ratio of phosphorylated c-Met to c-Met precipitated.
For analysis of phosphorylation of the ERK1 and -2 MAP kinases, after the indicated treatments, 5 ϫ 10 5 cells were directly lysed in sample buffer and analyzed by 10% SDS-polyacrylamide gel electrophoresis and blotted. Equal loading was confirmed by Ponceau S staining of the blot. The part of the blot below 50 kDa was stained with anti-phospho-MAPK antiserum, the upper part with anti-phosphotyrosine PY-20. Primary antibodies were detected by HRP-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse, respectively. Identification of the ERKs was confirmed by staining with anti-ERK1 or anti-ERK2.

RESULTS
Binding of HGF/SF to CD44 Isoforms-Binding of HGF/SF to different CD44 isoforms was assessed by using a panel of Namalwa Burkitt's lymphoma cell lines stably transfected with cDNAs encoding CD44s, CD44v8 -10, or CD44v3-10 ( Fig. 1A) (56). Prior to transfection, the cells were negative for CD44 and c-Met expression at both the protein and mRNA level (data not shown). All transfectants used for HGF/SF binding studies expressed comparable levels of CD44 (Table I). HGF/SF binding to the CD44 transfectants was measured by FACS analysis using an anti-HGF/SF mAb, an approach that avoids chemical modification of the ligand. As shown in Fig. 1B, CD44 negative control cells as well as CD44s and CD44v8 -10 transfectants showed a low saturable binding of HGF/SF. In contrast, cells expressing CD44v3-10 bound much larger quantities of HGF/ SF. These results suggest that CD44v3-10 contains a binding site(s) for HGF/SF.
Binding of HGF/SF to CD44 Is Heparan Sulfate-dependent-We next conducted a series of experiments aimed at determining the role of HS side chains in the binding of HGF/SF. First, the presence of total HS on the different transfectants was assessed by FACS analysis using the HS-specific mAb 10E4 ( Fig. 2A), and the mAb 3G10 (Fig. 2B) which recognizes the ⌬HS stubs remaining on HSPG core proteins after treatment with heparitinase (55). Both figures show that cells transfected with CD44v3-10 express approximately 20-fold higher levels of HS compared with those transfected with other CD44 isoforms. Next, we investigated the presence of HS on CD44 itself. This was done by using mAb 3G10. With this mAb, a single major HS band was detected in Western blots of CD44 precipitates from the CD44v3-10 cells, but not from the other transfectants (Fig. 2C). Staining the blot with an anti-pan CD44 mAb demonstrated that this band corresponded to CD44v3-10 (Fig. 2C).
To assess the role of HS in the interaction between HGF/SF and CD44v3-10, we studied the effect of heparitinase treatment and performed binding studies with HP1, a HGF/SF mutant which has a greatly decreased (more than 50-fold) affinity for heparan sulfate and heparin (57). As shown in Fig.   FIG. 3. The role of heparan sulfate in the binding of HGF/SF to CD44 Namalwa transfectants. A, FACS analysis to detect HGF/SF bound to CD44 Namalwa transfectants that were treated with PBS, 10 milliunits/ml heparitinase, or 50 milliunits/ml chondroitinase ABC at 37°C for 2 h prior to incubation with 18 nM HGF/SF at 4°C for 1 h. B, FACS analysis of wild type or mutated (HP1) HGF/SF bound to CD44 Namalwa transfectants. HGF/SFs were detected with mouse anti-HGF/SF followed by RPE-conjugated goat anti-mouse. Results are expressed as relative mean fluorescence intensity (MFI) (as compared with PBS treated mock transfectants). Error bars represent the standard deviation from three independent experiments. 3A, heparitinase treatment resulted in a near complete loss of HGF/SF binding, while treatment with chondroitinase ABC had no effect. The essential role of HS moieties on CD44v3-10 in HGF/SF binding was further confirmed by the observation that HP1 did not bind to CD44v3-10 (Fig. 3B). These data demonstrate that CD44v3-10 is a heparan sulfate-modified CD44 isoform (CD44-HS) that binds HGF/SF via its HS side chain.
CD44-HS Promotes c-Met Activation-To explore the functional impact of HGF/SF bound to CD44-HS on the c-Met signaling pathway, we generated double transfectants expressing c-Met in combination with either CD44v3-10 or CD44s. We selected stable transfectants expressing equal amounts of c-Met to be used in the subsequent studies (Fig. 4). Using these cell lines, we assessed in the first instance HGF/SF induced c-Met phosphorylation. As shown in Fig. 5, triggering with HGF/SF led to a vast and rapid increase in the phosphorylation of c-Met on tyrosine residues in the cells expressing CD44v3-10. By contrast, phosphorylation of c-Met was only weakly increased in the cells with CD44s (Fig. 5) and was absent in the parental cell line (data not shown), confirming the lack of endogenous c-Met in these cells. The dose-response studies demonstrated that CD44v3-10 promotes c-Met phosphorylation over a broad dose range (Fig. 5A) with an approximately 7-fold relative increase at plateau level. The time curve (Fig.  5B) showed that phosphorylation was maximal between 2 and 10 min after addition of the growth factor and declined thereafter. Moreover, this strong enhancing effect of CD44v3-10 on c-Met phosphorylation was dependent on HS moieties since it was lost upon heparitinase treatment (Fig. 6A). The importance of HS for HGF/SF signaling was further strengthened by studies using the HGF/SF heparin-binding domain mutant HP1. This mutant induced an equal (weak) phosphorylation of c-Met in both the CD44v3-10 and CD44s transfectants (Fig.  6B). Thus, these data suggest that CD44v3-10 binds HGF/SF via its HS side chains and then presents it to the high affinity receptor c-Met.

CD44-HS Promotes Downstream Signaling through c-Met in a Heparan
Sulfate-dependent Fashion-The pivotal role of CD44-HS in promoting the action of HGF/SF was further supported by analyzing the cell lysates of HGF/SF-stimulated cells for tyrosine-phosphorylated proteins. We observed tyrosine phosphorylation of several substrates, the two most prominent phosphoproteins of unknown identity are found at 115-125 kDa. A minor phosphoprotein is found at 145 kDa which likely represents c-Met (Fig. 6C). In addition, several smaller phosphoproteins of unknown origin were observed (not shown) including a 42-kDa phosphoprotein which may represent the p42 ERK2 MAP kinase.
In order to establish whether signal transduction by c-Met is potentiated by the HS moieties on CD44v3-10, we further investigated the activation of downstream targets of c-Met signaling. Since HGF/SF has been shown to activate the ERK MAP kinases in Madin-Darby canine kidney, HT29, and A549 cells (60 -64), we assessed whether HGF/SF is also able to induce MAP kinase activation in Namalwa B cells. For this purpose we used an antibody recognizing only the active, phosphorylated form of the ERK1 and -2 (p44 and p42) MAP kinases. As shown in Fig. 6C, HGF/SF treatment results in phosphorylation of the MAP kinases ERK1 and -2 in Namalwa transfectants expressing c-Met. The phosphorylation of the ERK2 MAP kinase upon HGF stimulation of the cells was also confirmed by MAP kinase gel-shift analysis. 2 We observed stronger phosphorylation of ERK1 and -2 in the CD44v3-10 expressing cells as compared with the CD44s expressing cells (Fig. 6C, bottom panel). Moreover, heparitinase treatment resulted in a decrease of HGF/SF-induced ERK phosphorylation in the CD44v3-10 cells, resulting in a level of ERK phosphorylation that is similar to the level of HGF/SF-induced ERK phosphorylation in CD44s transfectants. HGF/SF-induced phosphorylation of the ERKs in CD44s transfectants remained unaffected by heparitinase treatment. Taken together, our data demonstrate that signal transduction elicited by HGF/SFinduced c-Met activation is strongly promoted by CD44-HS, and depends on the presence of the HS moiety on CD44-HS. DISCUSSION We observed that cells transfected with CD44v3-10 efficiently bind HGF/SF (Fig. 1) and that this CD44 isoform is decorated with HS moieties (Fig. 2). By contrast, transfectants that express CD44s or CD44v8 -10, CD44 isoforms which are not modified with HS (Fig. 2), were not able to bind HGF/SF above background (parental) levels (Fig. 1). This selective HS modification of CD44v3-10 is in line with the recent study by Jackson et al. (13) which demonstrated that HS side chains bind to CD44 at the SGSG motif encoded by exon v3. Indeed, we demonstrated that the interaction of HGF/SF with CD44v3-10 is HS-dependent. Binding was completely abrogated by heparitinase treatment, and HP1, a HGF/SF mutant with greatly decreased affinity for heparan sulfate and heparin (57), failed to bind CD44v3-10 (Fig. 3). Interestingly, it has been demonstrated that specific chemical modifications of HS side chains on proteoglycans appear to regulate their affinity for selected heparin-binding growth factors, including HGF/SF and FGF-2, and hence determine growth factor binding specificity (65)(66)(67)(68)(69). This suggests that the HS moiety covalently attached to CD44v3-10 contains specific binding sites for HGF/SF.
The key finding of our study is that CD44-HS has a major functional effect on HGF/SF-induced signal transduction. Expression of CD44-HS at the cell surface led to a vast increase in HGF/SF-induced phosphorylation of c-Met on tyrosine residues 2 M. Spaargaren and G. J. T. Zwartkruis, unpublished observation. followed by HRP-conjugated rabbit anti-mouse (upper panel). Next, the blot was stripped and re-stained with rabbit anti-c-Met followed by HRP-conjugated goat anti-rabbit (lower panel). C, Western blot from total cell lysates from equal numbers of the cells described in A. The upper part of the blot was stained with the anti-phosphotyrosine mAb PY-20, followed by HRP-conjugated rabbit anti-mouse. The lower part of the same blot was stained with anti-phospho-MAPK antibody, followed by HRP-conjugated goat anti-rabbit. The arrows indicate a phosphorylated protein at 145 kDa and two major phosphoproteins at 115-125 kDa (upper panel), and the phosphorylated ERK1 and ERK2 MAP kinases (lower panel). Several independent clones were tested and gave comparable results. (Fig. 5). Furthermore, it resulted in a strong tyrosine phosphorylation of two as yet unidentified 115-125-kDa proteins that were hardly phosphorylated in the absence of CD44-HS (Fig.  6C). One of these proteins might represent p110/115-Grb2 associated binder (Gab)-1, an adaptor protein that has recently been found to associate with the multifunctional docking site of c-Met (70). Alternatively, the observed bands might be p120-Cbl and/or p125-FAK. Both protein tyrosine kinases participate in signal transduction via receptor protein tyrosine kinases and integrins (71,72). This is particularly interesting given our previous results that HGF/SF stimulation of Namalwa Burkitt's lymphoma cells results in enhanced integrin ␣4␤1-mediated adhesion (41) and the recent observation that Cbl is involved in integrin activation and spreading of macrophages (73). Furthermore, Cbl was recently reported to be required for efficient cellular transformation through the Tpr-Met oncoprotein (74). In addition to the 120 -125-kDa proteins, we demonstrated for the first time that HGF/SF induces phosphorylation of the MAP kinases ERK1 and -2 in B cells (Fig.  6C). Even more intriguing was the observation that CD44-HS promoted the HGF/SF-induced phosphorylation of ERK1 and -2. ERK1 and -2 are intermediates in signaling pathways linking extracellular signals to gene transcription in the nucleus and have been implicated in a wide variety of biological responses including cell proliferation. Interestingly, several recent studies have implicated the ERKs in integrin activation (75) as well as in HGF/SF-induced motility (i.e. scattering), and tubulogenesis of the epithelial Madin-Darby canine kidney cell line (60,62,63). Because of our previous data concerning the involvement of HGF/SF in integrin-mediated adhesion of B cells (41), we are currently investigating the possible role of the ERKs in B cell adhesion and migration.
We demonstrated that the enhancing effects of CD44-HS on signal transduction via c-Met were critically dependent on the interaction of HGF/SF with the HS moieties on CD44-HS, as they were not observed after heparitinase treatment, or when the cells were triggered with the heparin-binding domain HGF/SF mutant HP1 (Fig. 6). Importantly, the specific effects of the heparitinase treatment and the mutations in HP1 on HGF/SF-induced signal transduction in the CD44v3-10 expressing cells as compared with the CD44s cells demonstrates that the difference in HGF/SF-elicited responses in these cells is not due to any possible clonal variation in these stable cell lines. We speculate that CD44-HS promotes the action of HGF/SF through concentration of HGF/SF on the cell surface and by presenting it to the high affinity receptor c-Met (Fig. 7). Similar mechanisms were proposed for the role of high and low affinity receptors in FGF functioning (32,76,77). In addition, CD44-HS might also protect HGF/SF from proteolytic degradation as endothelial cell-derived HS was shown to do for FGF-2 (78).
It should be noted, that, apart from growth factor presentation, CD44 may have additional functions in HGF/SF-c-Met mediated signaling. For example, CD44 might recruit molecular partners into a multi-molecular complex with c-Met. This possibility is suggested by the fact that two recently identified cytoplasmic molecules associated with CD44 have also been implicated in c-Met signaling. First, studies by Ponzetto et al. (64) have shown that c-Met is a substrate for Src family tyrosine kinases, while our own studies have revealed a physical and functional association between CD44 and Src family member p56 lck (23). Second, studies by Jiang et al. (79) and Crepaldi et al. (80) have demonstrated that HGF/SF stimulates the tyrosine phosphorylation of the ERM protein ezrin. As reported by Tsukita et al. (22), ERM proteins serve as molecular linkers between CD44 at the cell surface and the actin cytoskeleton. This interaction is believed to be involved in the regulation of cell shape and motility.
We propose that collaboration between CD44-HS and growth factor receptors, viz. c-Met, as shown in our present study, might be an important factor in tumor growth and metastasis. By overexpressing CD44-HS, tumor cells would acquire a strongly increased sensitivity to HGF/SF-mediated growth signals, leading to a growth advantage and promoting metastasis (Fig. 7). This hypothesis is supported by the fact that c-Met and HGF/SF are (over)expressed in conjunction with CD44 in several types of tumors. In colorectal cancer, for example, c-Met is frequently overexpressed (48,49,81), while HGF/SF is expressed within the tumor tissue microenviroment. 3 Interestingly, in these tumors CD44 splice variants, including variants decorated with HS, are often overexpressed and predict metastatic spread and tumor related death (82,83). A similar scenario may hold for breast cancer and non-Hodgkin's lymphoma, as in these tumor types overexpression of CD44v3 as well as c-Met has also been reported (19,42,51,84).
In conclusion, we demonstrated that through binding and presenting HGF/SF, CD44-HS promotes signal transduction via the receptor tyrosine kinase c-Met. Consequently, overexpression of CD44-HS might give tumor cells a growth and metastatic advantage and, in this way, might influence disease outcome.