Determination of Disulfide Bond Assignments and N-Glycosylation Sites of the Human Gastrointestinal Carcinoma Antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)*

Jae Min Chong and David W. SpeicherDagger

From the Wistar Institute, Philadelphia, Pennsylvania 19104

Received for publication, September 27, 2000, and in revised form, November 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The GA733-2 antigen is a transmembrane glycoprotein that migrates on SDS-PAGE1 as a 40-kDa protein. It is present in the majority of human epithelia (1-5) and is highly expressed in gastrointestinal carcinomas (6-9). Moreover, recent studies correlate high GA733-2 expression with malignant tumor development in other types of cancer lesions, raising intriguing questions about the role of this antigen in tumor progression (for review see Ref. 10).

The GA733-2 antigen was originally defined by the CO17-1A monoclonal antibody (mAb) and GA733 mAb (11, 12). Because a randomized phase II trial with mAb CO17-1A in colorectal carcinoma patients has demonstrated a significant decrease in recurrence and mortality of mAb-treated patients versus control patients (13, 14), GA733-2 antigen has attracted substantial attention as a target for immunotherapy for treating human carcinomas. More recently, it has been reported that a recombinant adenovirus expressing the full-length GA733-2 antigen has the capability to induce humoral, cellular, and protective immunity to the antigen resulting in significant and specific inhibition of the colorectal carcinoma cell growth in mice, which implies that this recombinant adenovirus may have potential as a vaccine for colorectal carcinoma patients (15).

The GA733-2 antigen has been shown to function as a calcium-independent homophilic cell adhesion molecule that does not exhibit any apparent homology to the four known cell adhesion molecule superfamilies (16). Although the functional consequence of GA733 antigen-mediated adhesion on tumorigenesis is unclear, emerging evidence correlates increased GA733-2 antigen expression with increased cell proliferation and a less differentiated cell phenotype as demonstrated in cervical intraepithelial neoplasia (17).

The GA733-2 gene encodes for a 314-amino acid polypeptide that includes a 23-amino acid signal sequence, a 242-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 26-amino acid cytoplasmic domain (18). The extracellular domain of GA733-2 antigen (GA733-2EC) has three potential N-linked glycosylation sites and an N-terminal cysteine-rich region containing 12 cysteines. It has been proposed that this N-terminal portion is comprised of an epidermal growth factor (EGF)-like motif followed by a thyroglobulin type 1A motif based on relatively weak homology (19, 20). However, structural evidence to support this hypothesis has not yet been reported. In addition, analysis of glycosylation sites on the GA733-2 antigen is of interest because changes in glycoprotein glycosylation typically accompany malignant transformation (for review see Ref. 21). For example, a recent study showed that modulation of the glycosylation of CD44 regulated cell adhesion during tumor growth and metastasis (22). Two of the three possible N-glycosylation sites in GA733-2EC are located in the N-terminal cysteine-rich region, which appears to contain the epitope for the CO17-1A mAb used in the clinical trial described above (10). Hence, it is likely that N-glycosylation of GA733-2 antigen may play roles in cell adhesion and/or tumor targeting. A better understanding of the special features of the GA733-2 antigen may provide insights into the function(s) of this antigen and should facilitate the rational design of mutants for structure-function studies.

In this study, we determined the linkages of the six disulfide bonds and the sites of N-linked glycosylation in GA733-2EC using MALDI-MS and N-terminal sequencing of purified peptides. The data show that the cysteine-rich region has two distinct domains with six cysteines forming three disulfides in each domain. Surprisingly, the first cysteine-rich domain does not belong to the EGF-like domain family as previously predicted (10, 19, 20). Instead, it has a unique disulfide linkage pattern. In contrast, assignment of disulfides in the second cysteine-rich domain is in agreement with the disulfide pattern of a thyroglobulin type 1A motif, recently determined in insulin-like growth factor-binding protein-1 and 6 (23), and consistent with its apparent sequence homology (20, 24). Of the three potential N-glycosylation sites, Asn51 was partially glycosylated, Asn88 was completely glycosylated, and Asn175 did not contain any carbohydrate moieties.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Immobilized tosylphenylalanine chloromethylketone-trypsin F7m columns were purchased from MoBiTec (Marco Island, FL). Trypsin (sequencing grade) was purchased from Promega (Madison, WI). N-Glycosidase A was obtained from Roche Molecular Biochemicals. Tris-(2-carboxyethyl)-phosphine (TCEP) and 2- (2'-nitrophenylsulfonyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were obtained from Pierce. Pyroglutamate aminopeptidase was obtained from Panvera (Madison, WI). Reagents for PAGE were obtained from Bio-Rad. All other reagents were either high performance liquid chromatography (HPLC) grade or the highest quality analytical reagent grades available.

Purification of GA733-2EC-- Recombinant GA733-2EC was produced in Hi Five insect cells using the baculovirus system as previously described (25, 26). The secreted GA733-2EC was purified from the culture supernatant using a GA733 mAb-Sepharose affinity column. The column was washed with PBS (10 mM sodium phosphate, 130 mM NaCl, pH 6.0), and proteins were eluted with 50 mM glycine-HCl, pH 2.5. The pH of the peak fractions was immediately raised to approximately pH 6 using 2 M Tris-HCl, pH 6.0, followed by dialysis against PBS. Protein samples were concentrated to 7-8 mg/ml using Centriprep and Centricon concentrators with a 10-kDa molecular mass cut-off membrane (Amicon, Beverly, MA). The concentrated protein solution was then injected into two TSK columns G3000 SWXL and G2000 SWXL (Toso-Haas, Japan) connected in series and separated at 0.8 ml/min using PBS, pH 6.0. The quality of purification was monitored by SDS-PAGE and Western blot analysis using the GA733 mAb. The concentration of GA733-2EC was determined by A280 in a 1-cm pathlength cell, using a molar absorption coefficient (epsilon g/l) of 0.926, calculated from the GA733-2EC sequence according to Pace et al. (27).

Protease Digestion-- For disulfide assignments, a tosylphenylalanine chloromethylketone-trypsin F7m column was used for initial fragmentation of GA733-2EC after equilibrating the column with 10 ml of 50 mM sodium phosphate, 3 M urea, pH 6.5, at 22 °C. Purified GA733-2EC (400 µg/134 µl) was treated with urea (final concentration, 3 M) and loaded onto the trypsin column. The protein solution percolated through the column by gravity over the period of ~1 h, and the eluate was reloaded a total of six times followed by overnight incubation in the column without flow at 22 °C. The column was then centrifuged at 2,000 rpm for 5 s to collect the digested peptide. Residual peptides were removed with two successive elutions using 200-µl aliquots of 50 mM sodium phosphate, 3 M urea, pH 6.5, followed by centrifuging at 2,000 rpm for 5 s.

Mild Trypsin Digestion in Solution for Carbohydrate Analysis-- GA733-2EC was digested with trypsin in solution under physiological conditions at a ratio of 1:500 (w/w) enzyme to substrate at 37 °C for 1 h. The reaction was stopped by adding phenylmethylsulfonyl fluoride (final concentration, 0.15 mM). The sample was denatured and reduced by adding 7 M urea and 10 mM TCEP (final concentration) followed by incubation at 37 °C for 30 min. The fragments were separated by HPLC gel filtration using two TSK columns G3000 SWXL and G2000 SWXL connected in series with 0.6 ml/min flow rate in M urea, 20 mM Tris-Cl, 1 mM TCEP, pH 7.0. Collected fractions were immediately dialyzed against 10 mM sodium phosphate, pH 7.0, to remove urea and TCEP.

Reverse Phase HPLC Separation of Peptides-- Peptides were separated by reverse phase (RP)-HPLC on a ZORBAX 300SB-C18 column (2.1-mm inner diameter × 150 mm, Hewlett Packard Co.) using a System Gold HPLC (Beckman, Fullerton, CA) at a flow rate of 0.2 ml/min. Various gradient conditions were used as described in figure legends with solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.085% trifluoroacetic acid in 95% acetonitrile). Where required, tryptic digests were reduced prior to RP-HPLC by adding 20 mM TCEP in 100 mM ammonium bicarbonate, pH 8.0. The mixture was incubated at 37 °C for 1 h, and 2% trifluoroacetic acid (final concentration) was then added prior to injection onto the HPLC column.

Deglycosylation and BNPS-skatole Cleavage-- The purified tryptic fragment containing glycosylation (T1) was treated with N-glycosidase A. The peptide solution (3 µg/250 µl in 50 mM sodium acetate, pH 5.0) was mixed with 14 µl of N-glycosidase A (0.1 µg/µl stock solution). The mixture was then incubated overnight at 37 °C followed by RP-HPLC to separate deglycosylated peptide. Deglycosylated T1-N (1.4 µg) in the presence of 5 M guanidine-HCl was cleaved with BNPS-skatole essentially as previously described (28).

Partial Reduction with TCEP and Cleavage of N-terminal Pyroglutamic Acid-- TCEP partial reduction of complexes with multiple disulfides was performed as previously described (29). The purified peptide complex (100 pmol/70 µl in 0.1% trifluoroacetic acid) was mixed with the same volume of 50 mM TCEP in 50 mM citrate, pH 3.5, and incubated for 3 min at 22 °C. Alkylation of peptides was performed by adding the TCEP-reduced peptide solution into the same volume of 1 M iodoacetamide in 100 mM HEPES, 1 mM EDTA, pH 7.5, followed by incubation for 30 min at 37 °C. The reaction was stopped by adding 1.3% trifluoroacetic acid (final concentration). Pyroglutamate aminopeptidase (1.3 milliunits/6.3 µl) was added to the peptide solution (33 pmol/50 µl in 50 mM sodium phosphate, 1 M guanidine-HCl, 10 mM dithiothreitol, 1 mM EDTA, pH 7.0) and incubated overnight at 60 °C to remove N-terminal pyroglutamic acid from the purified partially reduced and alkylated peptides.

N-terminal Sequence Analysis-- Automated Edman degradation was performed using an Applied Biosystems model 494 protein sequencer as previously described (30).

Mass Spectrometry-- Molecular mass determination was performed by MALDI-time of flight mass spectrometry using a Voyager DE-PRO mass spectrometer (PerSeptive Biosystems, Framingham, MA). The accelerating voltage was set to 20 kV. Data were acquired either in the linear or reflector mode of operation. Spectra were externally calibrated with protein A (44,614 Da), chymotrypsinogen (25,657.1 Da), ubiquitin (8,567.49 Da), insulin beta  chain (3,496.96 Da), and bradykinin (1,061.24 Da). Intact GA733-2EC and the tryptic 22-kDa fragment from partial digestion in solution (10 pmol/2 µl) were mixed 1:1 with a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, Sigma) in 33% acetonitrile and 0.1% trifluoroacetic acid. The 6-kDa fragment from partial tryptic digestion of GA733-2EC in solution was analyzed using alpha -cyano-4-hydoxy cinnamic acid (Sigma) in 33% acetonitrile and 0.1% trifluoroacetic acid as the matrix. Peptides with molecular masses smaller than 5 kDa were directly applied to the MS sample plate that was precoated with a saturated solution of nitrocellulose and alpha -cyano-4-hydoxycinnamic acid (1:4 w/w) in 2-propanol and acetone (1:1 v/v) as previously described (31).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Purification, Signal Removal, and Characterization of GA733-2EC-- The recombinant extracellular domain of GA733-2 (GA733-2EC) was purified from Hi Five insect cell culture supernatant by GA733 mAb-Sepharose affinity chromatography followed by HPLC gel filtration. The purified protein migrated on SDS-PAGE as a diffuse doublet with apparent masses of ~31 and 33 kDa (Fig. 1). MALDI-MS analysis of purified GA733-2EC showed two broad peaks with average masses (MH+) of 28,332 and 29,349 Da. Because the calculated mass of the GA733-2EC is 27,372 Da after removal of the signal peptide, these data suggest that the molecule is heterogeneously glycosylated, resulting in two major populations with average carbohydrate masses of about 960 and 1,977 Da. The broad peak shapes of the doublet in the mass spectrum are indicative of additional mass heterogeneity of these two major populations of molecules. N-terminal sequence analysis of GA733-2EC indicated that the majority of the protein has a blocked N terminus, although a low level sequence representing about 1% of the protein amount loaded to the sequencer was detected. The low level sequence (AAQEEC ... ) indicated a minor alternative cleavage of the signal peptide after residue 21 of the unprocessed full-length protein. Subsequent MALDI-MS analysis and pyroglutamate aminopeptidase digestion of tryptic T2 peptide complex (Tables I and III) showed that the predominant signal peptide cleavage occurred after residue 23 and the N terminus of the mature protein is pyroGlu (see below). Hence the amino acid residues of the mature protein are numbered starting with this pyroGlu residue (Fig. 2). The GA733-2EC eluted from the HPLC gel filtration column as a single symmetrical peak between the 44- and 17-kDa protein standards. Rechromatogrphy of the protein at high and low protein concentrations yielded consistent elution volumes (data not shown). These data suggest that recombinant GA733-2EC is a globular monomer with a native-like fold.



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Fig. 1.   SDS-PAGE analysis of purified GA733-2EC. Recombinant GA733-2EC was separated on 7% Tris-Tricine gels under nonreducing conditions and stained with Coomassie Blue. Lane 1, crude Hi Five insect cell culture supernatant; lane 2, GA733-2EC after immunoaffinity purification; lane 3, after HPLC gel filtration. The positions of the standard proteins in kDa are shown on the left.


                              
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Table I
MALDI-MS and N-terminal sequence analyses of tryptic peptide complexes
Data obtained by MALDI-MS for the major disulfide-containing tryptic peptides complexes isolated by RP-HPLC before and after reduction with TCEP.



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Fig. 2.   Amino acid sequence of GA733-2EC showing experimentally determined disulfide linkages and N-glycosylation sites. Amino acids are numbered starting with the N-terminal residue of the major species of the mature protein as determined in this study; the predominant N terminus of GA733-2EC is pyroglutamate (E*), which represents removal of a 23-amino acid residue signal peptide. The N terminus of a small amount of the protein involves cleavage of 21 amino acid residues with retention of an additional two residues (Ala-Ala); the N terminus of this minor component is not blocked. Bold lines indicate disulfide bonds. Glycosylated N-linked consensus sites have solid underlines, whereas the unoccupied site at Asn175 has a dashed underline. The major sites of cleavage by trypsin to produce disulfide-linked peptides used to define disulfide linkages are indicated by downward arrows.

Isolation of Disulfide-linked Tryptic Peptide Complexes-- A representative RP-HPLC chromatogram of a GA733-2EC digest using an immobilized trypsin column after reduction (Fig. 3A) is compared with the chromatogram of a replicate aliquot that was not reduced prior to RP-HPLC (Fig. 3B). All peaks in the nonreduced chromatogram and most peptides in the reduced chromatograms were analyzed by MALDI-MS. Peptides that could not be unambiguously identified by mass analysis were subjected to Edman sequencing (data not shown). Three major peaks and several minor peaks observed in the nonreduced digest (T1-T3, T2*, and T3* in Fig. 3B) were not seen in the reduced digest, indicating that they contained disulfide-linked peptides. In addition, several new peaks appeared in the reduced tryptic digest chromatogram that corresponded to cysteine-containing peptides released from disulfide linkages by reduction. The identities of these peptides are indicated by inclusive residue numbers of the GA733-2EC sequence (Fig. 3, A and B). Peaks T1-T3 from the nonreduced tryptic digest were further analyzed to determine the disulfide linkages in GA733-2EC. T2* and T3* were identified as incomplete cleavages of T2 and T3, respectively.



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Fig. 3.   Chromatographic identification of disulfide-linked tryptic peptides and summary of GA733-2EC disulfide bond determination. A, chromatographic separation of GA733-2EC tryptic digest (45 µg) after reduction with TCEP on a ZORBAX 300SB-C18 column as described under "Experimental Procedures" using the following gradient conditions; 2% B for 5 min; 2-32% B over 75 min; 32-60% B over 35 min. B, chromatographic separation obtained with 90 µg of GA733-2EC tryptic digest (not reduced) using the same gradient as in A. Major peaks, which disappeared following reduction, are indicated by T1-T3. T1 is comprised of two disulfide-linked polypeptide chains, 84-102 and (103)104-115. T2 consists of two disulfide-linked polypeptide chains, 1-10 and 11-38, and T3 consists of two disulfide-linked polypeptide chains, 43-47 and 59-83. T2* and T3* indicate incomplete tryptic cleavages of T2 and T3, respectively. Single peptides that appear as a result of reduction are indicated in A by their inclusive residue numbers. C, summary of strategy used to complete GA733-2EC disulfide bond assignments. T1 was deglycosylated with N-glycosidase A and further cleaved chemically with BNPS-skatole. Partial reduction with TCEP and alkylation was performed for T2 followed by enzymatic deblocking of the N-terminal pyroglutamate. Each peak is named after the reagent and protease used as well as with the peak number after RP-HPLC. T, trypsin (cleavage after K or R); N, N-glycosidase A; S, BNPS-skatole (cleavage after W); R, partial reduction and alkylation; P, pyroglutamate aminopeptidase (cleavage after pyroGlu).

Overview of Disulfide Linkage Determination-- MALDI-MS and Edman sequencing analyses of the tryptic peptide complexes shown in Fig. 3B are summarized in Table I. The T1 peptide complex contains four cysteines in two disulfide-linked polypeptide chains, Ala84-Arg102 and Thr104-Arg115. Asn88 is entirely in a glycosylated form with some heterogeneity of the carbohydrate moiety, as indicated by the broad and multiple peaks in the RP-HPLC chromatogram (Fig. 3, A and B). The complex also exhibited heterogeneous trypsin cleavage, i.e. a complex of Ala84-Arg102 with either Arg103-Arg115 or Thr104-Arg115. The T2 peptide complex contains six cysteines in two polypeptide chains: pyroGlu1-Lys10 and Leu11-Lys38. The T3 complex has two cysteines in two polypeptide chains, Cys43-Lys47 and Ala59-Lys83, giving one direct disulfide assignment of Cys43-Cys76. Fig. 3C shows a summary of additional processing of the T1 and T2 complexes to complete disulfide assignments of these complexes. To minimize complexity in data analysis, T1 was deglycosylated with N-glycosidase A. BNPS-skatole cleavage of deglycosylated T1 (T1-N in Fig. 3C) was subsequently performed to obtain peptides containing one disulfide bond each. Partial reduction with TCEP and alkylation with iodoacetamide were performed for T2, which contained three disulfide bonds. One disulfide bond in T2 was assigned by Edman sequencing of the T2-R5 peak (Fig. 3C). Subsequently, pyroglutamate aminopeptidase was used to remove the N-terminal pyroglutamate from T2-R3, making it possible to carry out Edman sequencing and complete disulfide assignments of T2. Using this strategy it was possible to establish the disulfide bonds as: Cys4-Cys23, Cys6-Cys36, Cys15-Cys25, Cys43-Lys76, Cys87-Cys93, and Cys95-Cys112 (Fig. 2). A more detailed description of the analyses of peaks T1 and T2 is presented below.

Analysis of T1-- The T1 peptide complex has four cysteines in disulfide-linked polypeptide chains, Ala84-Arg102 and Thr104-Arg115, with heterogeneous masses indicative of glycosylation. The carbohydrate in T1 were completely removed by N-glycosidase A, indicating that all the carbohydrate in this peptide complex was located on Asn88 (Table II). RP-HPLC purified deglycosylated T1 (T1-N) was used for all subsequent experiments. Because T1-N has two disulfide bonds with one of these two disulfide bonds linking the two tryptic peptides, a further cleavage was necessary to generate peptide complexes containing only one disulfide bridge each for direct assignment. BNPS-skatole cleavage was carried out to cleave T1-N at Trp94. Fig. 4B shows that BNPS-skatole treatment generated multiple products (T1-N-S1 to S7). MALDI-MS and Edman sequencing analyses were used to characterize all peaks. T1-N-S1 was shown to be Ala84-Trp94. T1-N-S2 and T1-N-S3 were identified as Cys95-Arg102 disulfide-linked to Thr104-Arg115 and Arg103-Arg115, respectively (Table II). Because T1-N-S1 and T1-N-S2 each have only a single disulfide bond, it was possible to assign the disulfide bonds Cys87-Cys93 and Cys95-Cys112 for the T1 complex. T1-N-S1 showed a mass 13.5 Da greater than that expected for Ala84-Trp94 (Table II), consistent with the formation of an oxolactone by oxidative halogenation of the C terminus of the tryptophanyl-cleaved peptide as reported by Rahali and Gueguen (32).


                              
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Table II
MALDI-MS and N-terminal sequence analyses of deglycosylated and BNPS-skatole-treated T1



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Fig. 4.   RP-HPLC analysis of BNPS-skatole cleavage of tryptic and deglycosylated peptide T1-N complex. Peptides were chromatographed on a ZORBAX 300SB-C18 column as described under "Experimental Procedures" using the following gradient conditions: 2% B for 5 min (A) or for 15 min (B and C); 2-10% B over 5 min; 10-30% B over 50 min. A, T1-N before treatment with BNPS-skatole. B, T1-N after BNPS-skatole cleavage. Peaks marked with asterisks were in a BNPS-skatole reaction control (no protein). The analysis of each peptide peak is summarized in Table II. C, rechromatogram of T1-N-S4 fraction in B after incubation at 37 °C for 30 min, showing two peaks. T1-N-S4' and T1-N-S4" eluted at the same retention times as T1-N-S4 and T1-N-S6 in B, respectively.

To characterize T1-N-S4 to S7, peak fractions were analyzed by MALDI-MS after reduction with TCEP to get more precise molecular masses. Although T1-N-S4 and T1-N-S6 eluted as separate peaks in RP-HPLC (Fig. 4B), MALDI-MS spectra of these two fractions were identical within expected error and indicated that both fractions were the uncleaved T1-N species with a mass increase of 15.7 Da on the Ala84-Arg102 peptide (Table II). This is consistent with oxidation of the indole ring of tryptophan in incomplete BNPS-skatole cleavage of peptides, which results in a 16-Da mass increase (32). T1-N-S5 and T1-N-S7 were also uncleaved T1-N molecules with a 49.4-Da mass increase on the Ala84-Arg102 peptide (Table II), which is most likely due to oxidation of Trp94 by addition of three oxygen atoms. The differing elutions of T1-N-S4 and T1-N-S6 on RP-HPLC and their identical masses suggested that they represented two different constrained conformations of the oxidized indole ring of Trp94. To test this hypothesis, fraction T1-N-S4 was isolated following RP-HPLC, incubated at 37 °C for 30 min, and then rechromatographed using the same gradient. Indeed, the RP-HPLC chromatogram showed partial conversion to a later elution peak T1-N-S4" that correspond to the elution position of T1-N-S6 (Fig. 4C). MALDI-MS analysis confirmed that there was no mass difference between T1-N-S4' and T1-N-S4" (data not shown).

Analysis of T2-- The disulfide assignments for the T2 complex were obtained by characterization of partially reduced and alkylated peptides using Edman sequencing. The T2 peptide complex is comprised of pyroGlu1-Lys10 and Leu11-Lys38, having one intrapeptide disulfide bond in Leu11-Lys38 and two inter-peptide disulfide bonds (Table I). This peptide fraction was subjected to partial reduction using TCEP followed immediately by alkylation with iodoacetamide and subsequent separation by RP-HPLC. The use of the strong reducing agent, TCEP, which reduces disulfides at low pH within a short incubation time, had been shown to minimize both thiol-disulfide exchange and disulfide rearrangement in the partially reduced peptides (29). The RP-HPLC analysis of multiple peaks of T2 following partial reduction and alkylation is shown in Fig. 5 and results from MALDI-MS and Edman sequencing analyses are summarized in Table III. Peptide T2-R2 is the original unmodified peptide. The T2-R1 and T2-R4 peptides have been completely reduced and alkylated, whereas peptides T2-R3 and T2-R5 were only partially reduced prior to alkylation. T2-R5 is the single peptide, Leu11-Lys38, with alkylation of Cys23 and Cys36 and no modification of Cys15 and Cys25, indicating that Cys15 and Cys25 form an intrapeptide disulfide bond. T2-R3 is comprised of two disulfide linked peptides, pyroGlu1-Lys10 and Leu11-Lys38, with one alkylated cysteine on each peptide. Because the N-terminal pyroglutamate prevented Edman sequencing, it was necessary to remove this group prior to Edman sequencing to determine which cysteine side chains had been modified. Pyroglutamate aminopeptidase treatment of T2-R3 was performed under reducing condition using 10 mM dithiothreitol, generating Glu2-Lys10 (T2-R3-P1) and Leu11-Lys38 (T2-R3-P2). After this treatment both peptides were completely reduced, but the single cysteine in each peptide that was alkylated prior to pyroglutamate aminopeptidase treatment could be identified by Edman sequencing (Fig. 5C). When dithiothreitol was omitted from the enzyme reaction buffer, no cleavage of the N-terminal pyroglutamate occurred. Edman sequencing of T2-R3-P1 and T2-R3-P2 showed that Cys6 and Cys36 were alkylated respectively. Because the Cys15-Cys25 linkage was found in the analysis of T2-R5, these results indicate that these two peptides are linked by the disulfide bond Cys4-Cys23 (Table III). Similarly, the two reduced and alkylated residues, Cys6 and Cys36 identified in T2-R3, represent the remaining disulfide bond. Although the peak fraction labeled with an asterisk in Fig. 5B also showed a molecular mass indicative of T2 with two alkylated cysteines, the amount of the species was negligible compared with the major species (T2-R3), and it was not analyzed further. Therefore, the partial reduction/alkylation of the T2 peptide complex indicated that three disulfide bonds assignments are Cys4-Cys23, Cys6-Cys36, and Cys15-Cys25.



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Fig. 5.   RP-HPLC analysis of partial reduced, alkylated and pyroglutamate aminopeptidase treated tryptic peptide T2 complex. Peptides were chromatographed on a ZORBAX 300SB-C18 column as described under "Experimental Procedures" using the following gradient conditions: 2% B for 15 min; 2-10% B over 5 min; 10-35% B over 62 min. A, T2 control before partial reduction and alkylation. B, T2 after partial reduction with TCEP and alkylation. The analysis of each peak is summarized in Table III. The small peak marked with asterisks showed the molecular mass of T2 with two alkylated cysteines by MALDI-MS, although the amount of this species was negligible compared with the major species with two alkylated residues, T2-R3. C, pyroglutamate aminopeptidase treatment of T2-R3 generating free N-terminal T2-R3-P1 peptide. The peak marked with an arrow was in the pyroglutamate aminopeptidase control.


                              
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Table III
MALDI-MS and N-terminal sequence analyses of partially reduced, alkylated, and pyroglutamate aminopeptidase-treated T2

N-Linked Glycosylation Sites in GA733-2EC-- The N-linked glycosylation sites in GA733-2EC were determined by MALDI-MS and N-terminal sequence analyses of partial or complete tryptic digests. As described above, Asn88 was found to be completely glycosylated during analysis of the T1 peptide complex. Mass analysis of reduced Ala84-Arg102 peptide showed that the carbohydrate moieties at this site were heterogeneous ranging from ~893 to 1,259 Da (Fig. 6A and Table I). Edman sequencing of T1 showed no signal for Asn88, further confirming the complete glycosylation at this site (data not shown). Because peptides containing nonglycosylated Asn88 were not found in RP-HPLC fractions from trypsin column digestion (Fig. 3, A and B), Asn88 is completely glycosylated. The variable masses of the carbohydrate moiety on Asn88 are consistent with the average mass increase of 960 Da observed for the major GA733-2EC species compared with its sequence mass.



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Fig. 6.   Characterization of N-glycosylation sites in GA733-2EC. A, MALDI-MS spectrum of Ala84-Arg102 after reducing T1 with TCEP. Mass differences between the observed series of peaks and the peptide sequence mass (2000.3 Da) are due to glycosylation on Asn88 (summarized in Table I), which explains the 960-kDa mass difference between the sequence mass and the major observed mass of GA733-2EC. B, partial tryptic digests were separated on 15% Tris-Tricine gels under reducing conditions and stained with Coomassie Blue. Lane 1, purified GA733-2EC; lane 2, partial digestion with trypsin in PBS (E: S = 1: 500) at 37 °C for 1 h. The positions of standard proteins in kDa are shown on the left. The solid and dashed arrows indicate the apparent 6- and 8-kDa bands, respectively. C, HPLC gel filtration chromatography of GA733-2EC after partial tryptic digestion using a TSK G3000 SWXL and a G2000 SWXL column connected in series under reducing and denaturing conditions. Peaks I and II were pooled separately and analyzed by MALDI-MS after dialysis to remove the buffer, reducing reagent, and urea. D, MALDI-MS spectrum of fractions I and II from C. The average mass of fragment I agrees within experimental error with the sum of the sequence mass of Ala59-Lys242 and the average mass of glycosylation on Asn88 deduced from A. The MALDI-MS spectrum of fragment II showing series of peaks with masses that differ by ~1,034 Da, indicating partial glycosylation on Asn51.

The other two potential N-glycosylation sites were evaluated using fragments from mild tryptic digestion of GA733-2EC in physiological solution. This partial digestion resulted in three products when separated under reducing conditions on a SDS gel: a 22-kDa band, an 8-kDa band, and a 6-kDa band (Fig. 6B). This cleavage site is located at Arg57, Arg58 in a protease-sensitive loop that has been previously reported to lead to release of an ~6-kDa peptide from the N terminus of the protein (33, 34). These fragments were separated by HPLC gel filtration under reducing and denaturing conditions (Fig. 6C). MALDI-MS analysis of the larger fragment (fraction I) showed a mass of 21,953 Da, which is within experimental error of the sum of the calculated mass of Ala59-Lys242 and the average mass of the carbohydrate moiety on Asn88 as described above (21,968 Da expected versus 21,953 Da observed). These data indicate that the partial tryptic 22-kDa fragment cannot contain additional glycosylation on either Asn175 or Ser/Thr side chains.

The MALDI-MS spectrum of fraction II in Fig. 6C showed two clusters of masses (Fig. 6D). The masses at 6,232.7 and 6,388.1 Da correspond to pyroGlu1-Arg57 and pyroGlu1-Arg58, respectively, with no N-linked or O-linked carbohydrate. The third peak (6,533.7 Da) in the first cluster apparently represents a small amount of protein with an alternative N terminus caused by variable cleavage of the signal peptide that results in an additional two alanines at the N terminus (see above). The second cluster of peaks shows masses 1,034-1,037 Da higher than the corresponding peaks in the first cluster, indicating that the smaller mild tryptic fragment is only partially glycosylated on Asn51. Because MS is not quantitative, the relative staining intensities of the 6- and 8-kDa bands, although consistent with the MS signal intensities, are more reliable indicators that only a small proportion of the protein is glycosylated at Asn51. Interestingly, the carbohydrate moiety on this site appears to be less variable in mass than the carbohydrate groups on Asn88. Finally, the sum of observed masses for glycosylations on Asn88 (960 Da) and Asn51 (1,034 Da) is 1,994 Da, which is close to the 1,977-Da difference between calculated mass and observed mass for the minor higher mass peak in the intact GA733-2EC MALDI-MS spectrum.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The disulfide bonds in recombinant GA733-2 antigen have been determined by a combination of MALDI-MS, peptide mapping, and N-terminal sequencing. All peptide cleavage, deglycosylation, and purification steps were carried out below pH 6.5 to prevent disulfide scrambling. The disulfide-bonding pattern in the GA733-2 antigen is summarized in Fig. 2. A 200-µl immobilized trypsin column was chosen for the primary protease treatment in these studies because its highly concentrated immobilized enzyme on the matrix surface facilitated proteolysis with no appreciable contamination of reaction solutions with enzyme degradation products.

Assignments of the disulfide linkages in the T1 (Ala84-Arg102/Thr104-Arg115) and T2 (pyroGlu1-Lys10/Leu11-Lys38) tryptic peptide complexes were not straightforward because they contained four and six cysteines, respectively. In initial experiments, proteolytic cleavages of deglycosylated T1 (T1-N) and T2 were performed using various proteases including chymotrypsin, pepsin, subtilisin, elastase, and thermolysin in the presence of 1 M guanidine-HCl. However, both complexes were fairly resistant to further proteolysis under all conditions evaluated, and informative cleavages between neighboring cysteines were not obtained. Partial reduction of T1-N with TCEP followed by alkylation was then used to attempt determination of disulfide bond assignments. The results of these experiments established that Cys87 was disulfide linked to either Cys93 or Cys95, but disulfide scrambling was extensive even when reaction times and pH were lowered (data not shown). Subsequently, BNPS-skatole cleavage provided unambiguous disulfide assignments for T1-N. Although several competing side reactions occurred that resulted in modifications of the peptides without cleavage at the tryptophan, there was no indication of disulfide rearrangement. Partial reduction with TCEP followed by alkylation was performed for T2, and no significant disulfide scrambling occurred with this complex. After removing the N-terminal pyroglutamate with pyroglutamate aminopeptidase, Edman sequencing of both alkylated peptides were performed, and the disulfide linkages in T2 were unambiguously determined.

Prior reports concluded that the extracellular domain of GA733-2 antigen consisted of a N-terminal cysteine-rich region comprised of an EGF-like domain followed by a thyroglobulin type 1A motif and a cysteine-free region with no apparent homology to other proteins outside of the GA733 protein family based on sequence homology and cysteine spacing (19, 20). The disulfide linkages of GA733-2 antigen determined experimentally in the present study are compared with those of other cysteine-rich domains in Fig. 7. The first domain of the GA733-2 cysteine-rich region is not an EGF-like motif. Instead, it has a unique disulfide linkage pattern of Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5. We propose that this novel disulfide structure be called a GA733 type 1 motif. The second GA733-2EC cysteine-rich motif has Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6 linkages, which match the disulfide pattern of the thyroglobulin type 1A motif recently determined for human insulin-like growth factor-binding proteins 1 and 6 (24). In contrast, the EGF-like motifs contain six cysteine residues, with a Cys1-Cys3, Cys2-Cys4, and Cys5-Cys6 linkage pattern (35, 36). The tumor necrosis factor receptor also contains motifs with six cysteines. The generally accepted disulfide pattern of the tumor necrosis factor receptor cysteine motif is Cys1-Cys2, Cys3-Cys5, and Cys4-Cys6 (37), but this pattern is equivalent to those of the EGF-like and the laminin type EGF-like motifs after a circular permutation (36). The laminin type EGF-like motifs contain eight cysteine residues, with a Cys1-Cys3, Cys2-Cys4, Cys5-Cys6, and Cys7-Cys8 pattern determined for human EGF receptor and mouse laminin gamma 1 domain (36, 38).



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Fig. 7.   Cysteine spacings in cysteine-rich motifs. Comparison of disulfide bonding patterns of cysteine-rich motifsis shown. Disulfide linkages and spacings for the GA733-2 cysteine-rich motifs are based on the results of this study. The generic ranges of spacing for thyroglobulin (Tg) type 1A motif were derived from alignments of human thyroglobulin (hThyroglobulin) type 1A motifs (20). Disulfide linkages for this protein have not been experimentally determined (shown as dashed lines). However, the linkages are expected to be the same as for the human insulin-like growth factor-binding proteins 1 and 6 (hIGFBP-1,6) domains. Note that the spacings between linked cysteines in hthyroglobulin motifs are highly variable whereas the spacings between adjacent disulfide linked pairs are highly conserved. Experimentally determined disulfide linkages and cysteine spacings for human insulin-like growth factor-binding proteins 1 and 6 (23) are shown for comparative purposes. A generic EGF-like cysteine-rich motif was derived from alignments using the EGF-like sequences for which three-dimensional structures were available (36). Experimentally determined disulfide linkages for human tumor necrosis factor-binding protein (hTNFBP) are also shown (37). The laminin type EGF-like (LE) motifs have similar disulfide linkages to those of EGF-like motifs, although they contain eight cysteines instead of six cysteines. Disulfide linkages of human epidermal growth factor receptor (hEGFR) and mouse laminin gamma 1 chain (mLaminin gamma 1 domain) were experimentally determined previously (36, 38).

It has been reported that treatment of colon cancer cells in culture with N-glycosylation inhibitor, tunicamycin, resulted in a reduced migration on SDS-PAGE of GA733-2 antigen that was consistent with the sequence predicted mass, whereas treatment with an O-glycanase showed no effects, suggesting that carbohydrates in GA733-2 are predominantly or exclusively N-linked (33, 34). Our data now confirm that the carbohydrate moieties on the GA733-2 antigen are N-linked with no evidence of O-linked carbohydrate moieties. The extracellular domain of GA733-2 antigen has three potential N-glycosylation sites on asparagine residues 51, 88, and 175 (Fig. 2). Through analysis of T1, we found that Asn88 is completely glycosylated with carbohydrate moieties ranging in size from about 893 to 1,259 Da, which accounts for the average 960-Da mass difference between the calculated mass of the GA733-2EC sequence and the major peak observed using MALDI-MS. Because GA733-2EC has a proteolytic cleavage sensitive site on Arg58 (18), purified GA733-2EC was subjected to mild tryptic digestion to generate the N-terminal 6-8-kDa fragment containing Asn51 and the C-terminal 22-kDa fragment containing Asn88 and Asn175. MALDI-MS analysis showed evidence of partial glycosylation on Asn51 but no glycosylation on Asn175. A prior analysis of GA733-2 antigen from keratinocytes using radioimmunoprecipitation concluded that the N-glycosylation was exclusively on the larger proteolytic fragment (34), which implied that Asn51 did not possess glycosylation. However, it is quite likely that a minor diffuse 8-kDa band on a SDS gel might have been overlooked in this earlier study. The degree of posttranslational modification at Asn51 may be functionally and clinically important because it is close to the CO17-1A mAb epitope (10) and to the proteolysis sensitivity site. Interestingly, different ratios of cleaved/noncleaved species of GA733-2 antigen were observed from different epithelial cell lines (33). Although proteolytic regulation of the function of GA733-2 antigen has not been directly demonstrated, the possible correlation between proteolytic cleavage, glycosylation, and adhesion characteristics of the protein is an intriguing question for future studies.

A schematic model of the GA733-2 antigen based upon the data in this study is compared with previous models in Fig. 8. In all models, the location of the protease sensitive site is indicated with an arrow. The early model by Schon et al. (34) predicted that multiple disulfide bonds linked the small N-terminal fragment to the larger fragment after cleavage in the protease sensitive loop. In addition, three glycosylation sites on the larger fragment were predicted with no glycosylation of the small fragment. Recently, Balzar et al. (10) predicted that the cysteine-rich region is comprised of two tandem EGF-like motifs rather than the previously predicted EGF-like motif followed by a thyroglobulin type 1A motif (19, 20). In contrast, our current model (Fig. 8) is based upon the disulfide bond structure and glycosylation sites established in the present study and delineate at least three distinct structural domains.



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Fig. 8.   Alternative schematic models of the GA733-2 antigen. A, loop-like conformation model adapted from Schon et al. (34). B, tandem EGF-like motif model adapted from Balzar et al. (10). C, current model based on disulfide linkages and glycosylation sites determined in the present study. Black arrows indicate the proteolytic cleavage sensitive site at Arg57/Arg58 in all models. The complete and partial N-glycosylation sites in the current model are indicated with solid hexagons and dashed hexagons, respectively. The amino acid backbone of the protein in the current model is shown approximately to scale to illustrate relative sizes of loops and domains.

In conclusion, we have determined the disulfide bond linkages and glycosylation sites in the human gastrointestinal carcinoma GA733-2 antigen. The extracellular portion of this protein is comprised of at least three distinct motifs; an N-terminal six-cysteine motif with a unique disulfide linkage pattern (GA733 type 1 motif), a six-cysteine motif with the same disulfide linkage as thyroglobulin type 1A motifs (GA733 type 2 motif = thyroglobulin type 1A motif), and a larger cysteine-free motif with no apparent homology to other proteins outside the GA733 protein family. Among the three potential N-glycosylation sites, Asn88 and Asn51 were found to have complete and partial glycosylation, respectively, whereas no glycosylation was found on Asn175. There was no evidence of O-linked glycosylation. Knowledge of the disulfide bonds and locations of the carbohydrate moieties of GA733-2 antigen provides a basis for further structural and functional studies of this protein and will aid in rational design of mutations in this protein.


    ACKNOWLEDGEMENTS

We thank David Reim for performing N-terminal sequence analyses and helpful comments on the manuscript, Olivera Kolbas for assistance with RP-HPLC, Kaye Speicher for advice concerning MALDI-MS, and Peter Hembach for assistance in preparation of the figures. We also thank Drs. Gavin Manderson and Ronen Marmorstein for helpful comments on the manuscript. We are grateful to the Wistar Institute Recombinant Protein Production Facility for providing baculovirus-infected cells.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA74294 and CA10815.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Wistar Inst., 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3972; Fax: 215-898-0664; E-mail: speicher@wistar.upenn.edu.

Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M008839200


    ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; EC, extracelluar; EGF, epidermal growth factor; MALDI, matrix-assisted laser desorption/ionization; mAb, monoclonal antibody; TCEP, tris-(2-carboxyethyl)-phosphine; BNPS-skatole, 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine; RP, reverse phase; HPLC, high performance liquid chromatography; MS, mass spectrometry; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Momburg, F., Moldenhauer, G., Hammerling, G. J., and Moller, P. (1987) Cancer Res. 47, 2883-2891[Abstract]
2. Klein, C. E., Cordon-Cardo, C., Soehnchen, R., Cote, R. J., Oettgen, H. F., Eisinger, M., and Old, L. J. (1987) J. Invest. Dermatol. 89, 500-506[Abstract]
3. Tsubura, A., Senzaki, H., Sasaki, M., Hilgers, J., and Morii, S. (1992) J. Cutan. Pathol. 19, 73-79[Medline] [Order article via Infotrieve]
4. Zorzos, J., Zizi, A., Bakiras, A., Pectasidis, D., Skarlos, D. V., Zorzos, H., Elemenoglou, J., and Likourinas, M. (1995) Eur. Urol. 28, 251-254[Medline] [Order article via Infotrieve]
5. Cirulli, V., Crisa, L., Beattie, G. M., Mally, M. I., Lopez, A. D., Fannon, A., Ptasznik, A., Inverardi, L., Ricordi, C., Deerinck, T., Ellisman, M., Reisfeld, R. A., and Hayek, A. (1998) J. Cell Biol. 140, 1519-1534[Abstract/Free Full Text]
6. Ross, A. H., Herlyn, D., Iliopoulos, D., and Koprowski, H. (1986) Biochem. Biophys. Res. Commun. 135, 297-303[Medline] [Order article via Infotrieve]
7. Gottlinger, H. G., Funke, I., Johnson, J. P., Gokel, J. M., and Riethmuller, G. (1986) Int. J. Cancer 38, 47-53[Medline] [Order article via Infotrieve]
8. Shetye, J., Frodin, J. E., Christensson, B., Grant, C., Jacobsson, B., Sundelius, S., Sylven, M., Biberfeld, P., and Mellstedt, H. (1988) Cancer Immunol. Immunother. 27, 154-162[Medline] [Order article via Infotrieve]
9. Shetye, J., Christensson, B., Rubio, C., Rodensjo, M., Biberfeld, P., and Mellstedt, H. (1989) Anticancer Res. 9, 395-404[Medline] [Order article via Infotrieve]
10. Balzar, M., Winter, M. J., de Boer, C. J., and Litvinov, S. V. (1999) J. Mol. Med. 77, 699-712[CrossRef][Medline] [Order article via Infotrieve]
11. Herlyn, M., Steplewski, Z., Herlyn, D., and Koprowski, H. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1438-1452[Abstract]
12. Herlyn, D., Herlyn, M., Ross, A. H., Ernst, C., Atkinson, B., and Koprowski, H. (1984) J. Immunol. Methods 73, 157-167[CrossRef][Medline] [Order article via Infotrieve]
13. Riethmuller, G., Schneider-Gadicke, E., Schlimok, G., Schmiegel, W., Raab, R., Hoffken, K., Gruber, R., Pichlmaier, H., Hirche, H., Pichlmayr, R., Buggisch, P., Witte, J., and the German Cancer Aid 17-1A Study Group. (1994) Lancet 343, 1177-1183[Medline] [Order article via Infotrieve]
14. Riethmuller, G., Holz, E., Schlimok, G., Schmiegel, W., Raab, R., Hoffken, K., Gruber, R., Funke, I., Pichlmaier, H., Hirche, H., Buggisch, P., Witte, J., and Pichlmayr, R. (1998) J. Clin. Oncol. 16, 1788-1794[Abstract]
15. Li, W., Berencsi, K., Basak, S., Somasundaram, R., Ricciardi, R. P., Gonczol, E., Zaloudik, J., Linnenbach, A., Maruyama, H., Miniou, P., and Herlyn, D. (1997) J. Immunol. 159, 763-769[Abstract]
16. Litvinov, S. V., Velders, M. P., Bakker, H. A., Fleuren, G. J., and Warnaar, S. O. (1994) J. Cell Biol. 125, 437-446[Abstract]
17. Litvinov, S. V., van Driel, W., van Rhijn, C. M., Bakker, H. A., van Krieken, H., Fleuren, G. J., and Warnaar, S. O. (1996) Am. J. Pathol. 148, 865-875[Abstract]
18. Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewski, Z., Koprowski, H., and Linnenbach, A. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3542-3546[Abstract]
19. Simon, B., Podolsky, D. K., Moldenhauer, G., Isselbacher, K. J., Gattoni-Celli, S., and Brand, S. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2755-2759[Abstract]
20. Molina, F., Bouanani, M., Pau, B., and Granier, C. (1996) Eur. J. Biochem. 240, 125-133[Abstract]
21. Dennis, J. W., Granovsky, M., and Warren, C. E. (1999) Biochim. Biophys. Acta 1473, 21-34[Medline] [Order article via Infotrieve]
22. Cichy, E., and Pure, E. (2000) J. Biol. Chem. 275, 18061-18069[Abstract/Free Full Text]
23. Neumann, G. M., and Bach, L. A. (1999) J. Biol. Chem. 274, 14587-14594[Abstract/Free Full Text]
24. Linnenbach, A. J., Wojcierowski, J., Wu, S., Pyrc, J. J., Ross, A. H., Dietzschold, B., Speicher, D. W., and Koprowski, H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 27-31[Abstract]
25. Strassburg, C. P., Kasai, Y., Seng, B. A., Miniou, P., Zaloudik, J., Herlyn, D., Koprowski, H., and Linnenbach, A. J. (1992) Cancer Res. 52, 815-821[Abstract]
26. Zaloudik, J., Basak, S., Nesbit, M., Speicher, D. W., Wunner, W. H., Miller, E., Ernst-Grotkowski, C., Kennedy, R., Bergsagel, L. P., Koido, T., and Herlyn, D. (1997) Br. J. Cancer 76, 909-916[Medline] [Order article via Infotrieve]
27. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein Sci. 4, 2411-2423[Abstract/Free Full Text]
28. Crimmins, D. L., Mische, S. M., and Denslow, N. D. (2000) in Current Protocols in Protein Science (Coligan, J. E. , Dunn, B. M. , Ploegh, H. L. , Speicher, D. W. , and Wingfield, P. T., eds) , pp. 11.4.1-11, John Wiley & Sons, Inc., New York
29. Gray, W. R. (1993) Protein Sci. 2, 1732-1748[Abstract/Free Full Text]
30. Reim, D. F., and Speicher, D. W. (1997) in Current Protocols in Protein Science (Coligan, J. E. , Dunn, B. M. , Ploegh, H. L. , Speicher, D. W. , and Wingfield, P. T., eds) , pp. 11.10.1-38, John Wiley & Sons, Inc., New York
31. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858[CrossRef][Medline] [Order article via Infotrieve]
32. Rahali, V., and Gueguen, J. (1999) J. Protein Chem. 18, 1-12[Medline] [Order article via Infotrieve]
33. Thampoe, I. J., Ng, J. S., and Lloyd, K. O. (1988) Arch Biochem. Biophys. 267, 342-352[Medline] [Order article via Infotrieve]
34. Schon, M. P., Schon, M., Mattes, M. J., Stein, R., Weber, L., Alberti, S., and Klein, C. E. (1993) Int. J. Cancer 55, 988-995[Medline] [Order article via Infotrieve]
35. Appella, E., Weber, I. T., and Blasi, F. (1988) FEBS Lett. 231, 1-4[CrossRef][Medline] [Order article via Infotrieve]
36. Abe, Y., Odaka, M., Inagaki, F., Lax, I., Schlessinger, J., and Kohda, D. (1998) J. Biol. Chem. 273, 11150-11157[Abstract/Free Full Text]
37. Jones, M. D., Hunt, J., Liu, J. L., Patterson, S. D., Kohno, T., and Lu, H. S. (1997) Biochemistry 36, 14914-14923[CrossRef][Medline] [Order article via Infotrieve]
38. Stetefeld, J., Mayer, U., Timple, R., and Huber, R. (1996) J. Mol. Biol. 257, 644-657[CrossRef][Medline] [Order article via Infotrieve]


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