THEJOURNAL OF BIOLOGICAL ‘%EMlSTRY Vol. 265, No. 15, Issue of May 25, pp. 8616~6626,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Sequence and Glycosylation Site Identity of Two Distinct Glycoforms of Nonspecific Cross-reacting Antigen as Demonstrated by Sequence Analysis and Fast Atom Bombardment Mass Spectrometry*

(Received for publication, July 6, 1989)

Stanley A. Hefta, Raymond J. Paxton, and John E. Shively From the Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010

Nonspecific cross-reacting antigen (NCA) is a highly glycosylated membrane protein which is immunologi- cally and structurally related to carcinoembryonic an- tigen, an important tumor-associated antigen. Two glycoforms of NCA were purified from a single liver metastasis of a colonic carcinoma and characterized with respect to their primary sequence and position of glycosylation sites. The two glycoforms (designated TEX (tumor-extracted antigen), M, 75,000, and NCA, M, 45,000) each showed a deglycosylated M, of 35,000 and yielded identical peptide maps. The structural characterization of TEX and NCA and the assignment of glycosylation sites was performed by fast atom bom- bardment mass spectrometry and microsequence analysis of the resulting peptides. This approach showed that TEX and NCA were identical with respect to primary sequence and provided direct evidence that 11 of the 12 predicted asparagine-linked glycosylation sites were glycosylated in both TEX and NCA. Indirect evidence was obtained for glycosylation at the other site. Both glycoforms also contain ethanolamine linked to Gly-286, a finding consistent with the conclusion that these proteins are anchored to the plasma mem- brane through a glycosyl-phosphatidylinositol tail. The large difference in the molecular weights of glycosy- lated TEX and NCA suggests significant variations in their oligosaccharide structures.

Structural analysis of highly glycosylated membrane pro- teins presents several problems to the protein chemist. Pres- ence of carbohydrate complicates purification, characteriza- tion, and sequence determination. Glycoproteins often give broad, poorly defined bands by SDS’-gel electrophoresis or gel permeation chromatography and tend to give multiple peaks by ion exchange chromatography or isoelectric focusing. Sequence analysis of glycoproteins is fraught with problems including resistance to digestion with proteases and hetero- geneity of the resulting glycopeptides. In addition to these

* This work was supported by National Institutes of Health Grants CA37808 and CA33572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 503550.

’ The abbreviations used are: SDS, sodium dodecvl sulfate; CEA, carcinoembryonic antigen; NCA, nonspecific cross-reacting antigen; TEX, tumor-extracted antigen: TFMSA. trifluoromethanesulfonic acid; FAB, fast atom bombardment; G-PI; glycosyl-phosphatidylino- sitol; HPLC, high performance liquid chromatography; mAb, mono- clonal antibody; PTH, phenylthiohydantoin.

concerns, the functional relevance of the carbohydrate portion of many glycoproteins is becoming increasingly evident, ne- cessitating assignment of glycosylation sites and oligosaccha- ride structures. To overcome these problems and to develop a microanalytical technique to assign glycosylation sites, we developed an approach utilizing the increased sensitivity of reversed-phase high performance liquid chromatography (HPLC) for glycoprotein purification, and chemical deglyco- sylation, sequence analysis, and fast atom bombardment (FAB) mass spectrometry for structural characterization. In earlier work (Paxton et al., 1987) we described the use of this strategy for analysis of carcinoembryonic antigen (CEA). In this paper we demonstrate the versatility of this approach by characterization of two distinct glycoforms of nonspecific cross-reacting antigen (NCA), members of the CEA gene family.

CEA was originally identified in epithelial-derived tumors of the gastrointestinal tract and in fetal tissue (Gold and Freedman, 1965a, 1965b). Although further studies showed that CEA was not tumor specific, the increased expression of CEA in >90% of colorectal carcinomas makes it a valuable tumor marker for diagnosing and following the progression of cancer (reviewed by Shively and Beatty (1985)). Protein struc- tural analysis (Paxton et al., 1987) and molecular cloning studies (Oikawa et al., 1987a) showed that CEA is a 180-kDa glycoprotein (approximately 50% carbohydrate by weight) comprising an amino-terminal domain followed by three highly homologous domains, each containing two immuno- globulin-like domains and a glycosyl-phosphatidylinositol- containing membrane anchor (Hefta et al., 1988). A number of glycoproteins have been identified that are immunologically cross-reactive with and structurally similar to CEA (reviewed in Thompson and Zimmermann (1988)).

Nonspecific cross-reacting antigen, the most prevalent CEA-cross-reacting antigen, was initially found in both nor- mal and neoplastic tissues (Mach and Pusztaszeri, 1972; von Kleist et al., 1972). More recently, multiple forms of NCA with masses of 50-55, 75,90, 95-97, and 160 kDa have been identified in epithelial cells and polymorphonuclear leuko- cytes (Buchegger et al., 1984; Grunert et al., 1985; Audette et al., 1987). Molecular cloning techniques have identified a single gene for NCA (Thompson et al., 1987), the identity of which was established by a comparison of the translated protein sequence with partial protein sequence data for NCA (Paxton et al., 1987; Grunert et al., 1988). A primary sequence for NCA has been proposed based on cDNA sequencing data (Neumaier et al., 1988; Tawargi et al., 1988). The apparent discrepancy between the protein and cloning studies suggests that the various forms of NCA may arise from alternative posttranslational modifications, or perhaps alternative splic- ing of a precursor RNA, although at present there is no

8618

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Glycoforms of Nonspecific Cross-reacting Antigen 8619

evidence for this. Recently, an NCA-like cDNA has been cloned from chronic granulocytic leukemia cells.2 Northern and Southern analyses performed with this cDNA give unique hybridization patterns when compared with the NCA cDNA, but the protein encoded for by this cDNA has not been identified. Given that this protein is predicted to be 85% homologous to NCA, it may correspond to one of the forms of NCA identified in normal leukocytes.

This laboratory has previously isolated NCA from spleen (Engvall et al., 1978) and a tumor-associated glycoprotein from liver metastases of colonic adenocarcinomas (Kessler et al., 1978). Structural and immunological characterization of these proteins showed many common features, although some differences were noted (Kessler et al., 1978). As a result of these differences, the tumor-associated glycoprotein was given the provisional designation of TEX for tumor-extracted an- tigen (Kessler et al., 1978). As part of a continuing effort to describe the relationships between members of the CEA gene family, we have undertaken a study to compare the structures of TEX (Mr 75,000) and NCA (Mr 45,000). In this report we show that TEX and NCA are distinct glycoproteins that can be purified from a single colon tumor liver metastasis. We compare their primary structures and show that TEX and NCA contain identical sequences and numbers of glycosyla- tion sites, and we present evidence that both forms are an- chored to the plasma membrane through a glycosyl-phospha- tidylinositol linkage (G-PI tail).

MATERIALS AND METHODS

Tumor Extraction and Antigen Purification-TEX and NCA were isolated from a single colon tumor liver metastasis using previously described methods (Colligan et al., 1972; Kessler et al., 1978). Briefly, the tumor was homogenized in water using a Waring blender, and the homogenate was treated with an equal volume of 2 M percholoric acid. Following centrifugation, the supernatant was dialyzed against water, concentrated by ultrafiltration using a PM-10 membrane (Amicon), and lyophilized. The tumor extract was reconstituted in pH 5.5 phosphate-buffered saline (0.05 M sodium phosphate contain- ing 0.15 M NaCl and 0.1% (w/v) NaN3) and chromatographed on a 5 x 170-cm Sepharose 4B column (Pharmacia LKB Biotechnology Inc.) equilibrated with phosphate-buffered saline. Fractions were analyzed by radioimmunoassay using goat anti-CEA sera (Egan et al., 1972). Appropriate fractions were pooled, dialyzed against water, and lyophilized. The Sepharose 4B pool was reconstituted in pH 5.7 uhosuhate-buffered saline containina 0.01 M EDTA (PBSE) and chromatographed on a 5 x 140~cm Sephadex G200 column (Phar- macia LKB Biotechnology Inc.) equilibrated with PBSE. Fractions were analyzed as above except rabbit anti-TEX sera were used in the radioimmunoassay. TEX and NCA containing fractions were pooled separately, dialyzed against water, and lyophilized. Final purifications of TEX and NCA were performed by reversed-phase HPLC on a 3.9 x 300-mm WBondapak phenyl column (Waters). Solvent A was triflu- oroacetic acid/water, 0.1:99.9 (v/v) and solvent B was trifluoroacetic acid/water/acetonitrile, 0.1:9.9:90 (v/v/v).

SDS-Electrophoresis-TEX and NCA samples were heated for 5 min at 100 “C in 63 mM Tris-HCl buffer, 2% (w/v) SDS, 20% (v/v) glycerol, pH 6.8, with 5% (v/v) 2-mercaptoethanol, and analyzed on polyacrylamide gels (Laemmli, 1970). The proteins were stained with Coomassie Blue R-250 or transferred to nitrocellulose sheets (Towbin et al., 1979). The transferred proteins were stained either for protein with amido black or for immunoreactivitv with anti-CEA monoclonal antibody (mAb) T84.1E3 (Wagener et al., 1983) or anti-NCA mAb NCA4 (Chavanel et al., 1983). mAb T84.1E3 recognizes several CEA- cross-reacting antigens, including TEX and NCA. mAb NCA4 was generated by immunization with normal lung NCA and was kindly provided by Dr. Pierre Burtin (Institute de Recherches Scientifiques sur le Cancer). Horseradish peroxidase-conjugated goat anti-mouse IgG, I-chloro-1-naphthol for color development, and molecular weight markers were from Bio-Rad.

Deglycosylation-TEX and NCA (50-200 pg) were lyophilized to

” J. A. Thompson, personal communication.

dryness and treated with 100 ~1 of trifluoromethanesulfonic acid (TFMSA)/anisole (2:1, v/v) at 4 “C for 5 h (Edge et al., 1981). The reaction mixtures were then cooled with dry ice/methanol and neu- tralized by slowly adding 300 ~1 of a 50% (v/v) aqueous pyridine solution. The pyridinium-TFMSA salt that formed upon addition of pyridine was redissolved by warming the solution to 0 “C. Following neutralization, the samples were extracted four times with diethyl ether, and the deglycosylated proteins purified by reversed-phase HPLC on a 4.6 x 30-mm RP300 column (Brownlee) using a flow rate of 0.5 ml/min. After the injection, the column was held in 100% solvent A for 15 min followed by a linear gradient to 100% solvent B in 30 min using the solvents described above. The deglycosylated proteins eluted at approximately 50% solvent B.

Chymotryptic Digestion and Peptide Mapping-Deglycosylated TEX and NCA were reduced with 10 mM dithiothreitol in 0.25 M Tris-HCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.0, for 4 h at 55 “C. The reduced samples were S-carboxymethylated at room temperature for 30 min by addition of 2-fold molar excess (per reductant) of iodoacetic acid (Waxdal et al., 1968). The carboxymethylated, degly- cosylated proteins were purified by reversed-phase HPLC as described above and digested with reversed-phase HPLC purified chymotrypsin (Worthington) in 0.1 M NH,.HCO, buffer, pH 8.2, at a substrate/ enzyme ratio of 5O:l (w/w) for 18 h at 37 “C. The chymotryptic peptides were separated by reversed-phase HPLC on a Vydac C,s column (2.1 X 250 mm) using the trifluoroacetic acid/acetonitrile solvent system described above.

Cyanogen Bromide Cleauage-Native TEX and NCA (250 fig) were reduced with 70 mM 2-mercaptoethanol in 0.25 M Tris-HCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.5, for 1 h at 55 “C followed by 1 h at room temperature. The reduced samples were S-alkylated at room temperature for 2 h by addition of a 5-fold molar excess (per reduc- tant) of 4-vinylpyridine (Amons, 1987). The reduced and alkylated proteins were purified by gel permeation HPLC using a 4.6 x 125- mm column packed with Bio-Rad TSK-250 resin, concentrated to dryness, and resuspended in 20 ~1 of 75% trifluoroacetic acid contain- ing 300 rg/pl cyanogen bromide (CNBr). The cleavage reactions were performed at room temperature for 36 h after which the samples were concentrated to dryness. The CNBr fragments were resuspended in hexafluoroacetone trihydrate, diluted with H,O, and purified by re- versed-phase HPLC using the solvent system described above.

Microsequence and Amino Acid Compositional Analyses-Peptides were spotted on polyvinylidenedifluoride membranes (Millipore) and subjected to automated Edman degradation on a gas-phase sequencer built at the City of Hope (Hawke et al., 1985) and equipped with a continuous flow reactor (Shively et al., 1987). The phenylthiohydan- toin amino acid derivatives were identified by on-line reversed-phase HPLC. Amino acid compositions were determined using a Beckman 6300 amino acid analyzer following vapor-phase hydrolysis with 6 M HCl containing 0.2% 2-mercaptoethanol at 110 “C for 24 h.

Fast Atom Bombardment Mass Spectrometry-Samples were con- centrated to dryness in polypropylene microcentrifuge tubes using a vacuum centrifuge, redissolved in 2 ~1 of 5% acetic acid, and added to 2 gl of a liquid matrix on a 1.5 X 6-mm stainless steel sample stage. For most analyses the liquid matrix consisted of dithiothreitol/di- thioerythritol (5:1, w/w) containing 6 mM camphorsulfonic acid; analyses of smaller peptides (<800 a.m.u.) were performed using thioglycerol as the liquid matrix. Positive ion FAB mass spectra was obtained using a JEOL HXlOOHF high resolution, double focusing, magnetic sector mass spectrometer operating at 5 kV accelerating potential and a nominal resolution of 1000 or 3000. Sample ionization was accomplished using a 6 keV Xe atom beam. Data were collected with a JEOL DA5000 data system. Calibration of the instrument was accomplished with a &l/K1 (1:2, w/w) standard mixture.

RESULTS

Purification and Initial Characterization of TEX and NCA-TEX and NCA were isolated from a single liver me- tastasis of a primary colorectal carcinoma. The antigens were extracted from the tumor with 1 M perchloric acid and initially purified by Sepharose 4B chromatography (Fig. 1A). A single peak of immunoreactivity was identified with two shoulders on the trailing edge. Fractions 111-180 were pooled and found to contain CEA. Fractions 181-240, which contained TEX and NCA, were pooled and rechromatographed on Sephadex G-200 (Fig. 1B). Two protein peaks were obtained, each of

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8620 Glycoforms of Nonspecific Cross-reacting Antigen

Fraction

FIG. 1. Purification of TEX and NCA by gel permeation chromatography. A, Sepharose 4B chromatography of the perchlo- ric acid-treated colon tumor extract. B, Sephadex G-200 chromatog- raphy of Sepharose 4B fractions 181-240. Chromatograohv conditions __ _ _ - were as described under “Materials and Methods!’ Fractions from each column were analyzed for protein by absorbance at 280 nm and for immunoreactivity by radioimmunoassay (MA) with either anti- CEA (A) or anti-TEX (B) sera. Sephadex G-200 fractions 141-190 and 191-230 were pooled and used for the further purification of TEX and NCA, respectively. Solid line, A280; dashed line, immuno- reactivity (fig/ml).

which showed immunoreactivity when assayed with anti-NCA sera. Fractions 141-190 corresponded to the antigen desig- nated TEX (Kessler et al., 1978), and fractions 191-230 corresponded to NCA.

TEX and NCA were purified further by reversed-phase HPLC. As shown in Fig. 2A, TEX was resolved into three overlapping peaks eluting at 37-40% solvent B. Analysis of peaks l-3 by SDS-electrophoresis under reducing conditions showed broad, overlapping protein bands with M, values centered at approximately 85,000, 75,000, and 65,000, respec- tively (Fig. 2B, left and right panels). These proteins reacted with equal intensities when immunostained with anti-CEA mAb T84.1E3 (Fig. 2B, right panel) or with anti-NCA mAb NCA4 (data not shown). Peak 1 also showed a 180-kDa immunoreactive protein by SDS-electrophoresis, which prob- ably represents a small amount of dimeric TEX that was incompletely dissociated during the sample preparation pro- cedure. The immunostaining results suggest that peaks l-3 represent subspecies of TEX.

Reversed-phase HPLC of NCA also yielded three overlap- ping peaks eluting at 40-43% solvent B (Fig. 2C), slightly later than that observed for TEX. Unlike TEX, the NCA sample contained proteins that eluted later in the gradient, at 54-58% solvent B. SDS-electrophoretic analysis of peaks l-3 showed broad, overlapping proteins bands with M, values centered at approximately 55,000,45,000, and 40,000, respec- tively (Fig. 20, left and right panels). These proteins reacted with similar intensities when immunostained with anti-NCA mAb NCA4 (Fig. 20, right panel) or with anti-CEA mAb T84.1E3 (data not shown), suggesting that peaks 1-3 are subspecies of NCA. SDS-electrophoresis of peaks 5 and 7 from the NCA chromatogram (Fig. PC) showed proteins with M, values of 48,000 and 45,000, respectively (Fig. 20, left panel). Neither protein was reactive when stained with anti- NCA mAb NCA4 (Fig. 20, right panel) suggesting that they were not immunologically related to NCA. Amino acid com-

positional and NHz-terminal sequence analyses of these pro- teins suggested that they were cul-antichymotrypsin and 01~- antitrypsin, respectively (data not shown).

Amino acid compositions for TEX peaks l-3 and NCA peaks l-3 are shown in Table I. Minor differences were observed for several amino acids within and between the TEX and NCA subspecies, but for the most part their compositions were equivalent. NH&erminal sequencing through residue 35 was performed on each peak, and the sequences were found to be identical (data not shown). The amount of material isolated (microgram) precluded performing extensive carbo- hydrate analyses. The carbohydrate contents for TEX and NCA glycoforms could be estimated, however, by the amount of glucosamine quantitated during amino acid analysis (Del Valle and Shively, 1979) and from the results of previous studies which show ratios of other sugars to glucosamine (Kessler et al., 1978; Engvall et al., 1978). This method of quantitation does not provide precise values for percentage of carbohydrate, but nevertheless is useful for comparing the relative amounts. For TEX peaks l-3 the percentage carbo- hydrate by weight was estimated to be 51, 46, and 40%, respectively, and for NCA peaks l-3 was estimated as 36, 30, and 23%, respectively. These results suggest that the differ- ences observed in the molecular weight and retention time of the three subspecies of TEX and NCA are due to differences in carbohydrate content.

Peptide Mapping, FAB Mass Spectrometry, and Microse- quence Analysis-The initial characterization of TEX and NCA showed not only that each protein was partially sepa- rated into three structurally related subspecies, but that TEX and NCA were themselves structurally related, despite an average molecular weight difference of 30,000. For further structural characterization of TEX and NCA, the glycoforms were deglycosylated with TFMSA and digested with chymo- trypsin. As shown in Fig. 3, the M, of each of the three TEX and three NCA subspecies was reduced to 35,000 by TFMSA treatment, suggesting that they contained equivalent polypep- tide chains. Staining with Coomassie Blue (panel A) or im- munostaining with mAb T84.1 (panel B) gave equivalent results. The major TEX and NCA subspecies, peak 2 in each case, were selected for peptide mapping. Fig. 4 compares the chymotryptic map of TEX (top) with NCA (bottom). The maps are nearly identical with only minor differences in peak intensity and resolution. The earlier eluting peaks were gen- erally well separated and could be characterized directly. Later eluting peaks required some rechromatography prior to analy- sis. For clarity of presentation, a singular numbering system for the corresponding TEX and NCA peptides is employed as shown in the TEX map (Fig. 4), and only those peptides necessary for defining the TEX and NCA sequences are numbered and discussed below. Although not discussed fur- ther, all of the peptides in the TEX and NCA maps were characterized including those not numbered. The peak labeled ad. corresponds to undigested protein.

The TEX and NCA peptides were initially analyzed by FAB mass spectrometry. As summarized in Table II, the observed masses for the corresponding TEX and NCA pep- tides deviated by <0.4 a.m.u., with the majority having devia- tions of to.2 a.m.u. Based on these results, the corresponding peptides appear to be identical, strongly suggesting that the primary sequences of TEX and NCA are the same. A com- parison of the observed TEX and NCA peptide masses with the calculated chymotryptic peptide masses for the cDNA- derived NCA sequence also showed excellent agreement (Table II), with deviations of ~0.3 a.m.u. for 27 of 31 peptides (for this analysis it was assumed that potential asparagine-

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Glycoforms of Nonspecific Cross-reacting Antigen

C

8621

B MrxlO.3 D h4rxlfJ3

30-

1234567 12357 HPLC Fraction

I 2 3 1 2 3 HPLC Froclion

FIG. ‘2. Reversed-phase HPLC purification and SDS-electrophoresis of TEX and NCA. A, approxi- mately 50 pg of Sephadex G-200-purified TEX was applied to a 3.9 x 300-mm FBondapak phenyl column and eluted with a linear gradient from 0 to 60% solvent B in 90 min using the solvents described under “Materials and Methods.” The elution was monitored at 280 nm, and the peaks were collected manually as indicated on the chromatogram. Solid line, AZXO; dashed line, % solvent B. B, peaks Z-3 (Fig. 2A) were electrophoresed under reducing conditions in duplicate on a 10% (w/v) SDS-polyacrylamide gel, transferred to nitrocellulose, and stained for protein with Amido Black (left panel) and for immunoreactivity with anti-CEA mAb T84.1E3 (right panel). Molecular weight markers were visualized by Amido Black staining and are indicated with tick marks. C, approximately 200 pg of Sephadex G-200-purified NCA was applied to a 3.9 x 300.mm PBondapak phenyl column and eluted with a linear gradient from 20 to 60% solvent B in 60 min. The elution was monitored at 280 nm, and the peaks were collected manually as indicated on the chromatogram. Solid line, AgHO; dashed line, % solvent B. D, peaks l-7 (Fig. 2C) were electrophoresed under reducing conditions in duplicate (except for peaks 4 and 6) on a 10% (w/v) SDS-polyacrylamide gel, transferred to nitrocellulose, and stained for protein with Amido Black (left

panel) and for immunoreactivity with anti-NCA mAb NCA4 (right panel). Molecular weight markers were visualized by Amido Black staining and are indicated with tick marks.

TABLE I Amino acid compositions of TEX and NCA purified

by reverse-phase-HPLC Aliquots (l-2 rg) of HPLC peaks l-3 from Fig. 2, A and C, were

hydrolyzed in sealed tubes containing 50 ~1 of 6 N HCl with 0.2% (v/ v) P-mercaptoethanol at 110 “C for 48 h. Cysteine was determined as cysteic acid in separate analyses after oxidation with performic acid, and tryptophan was not determined.

Amino acid mol oio

Amino acid TEX peaks NCA peaks

1 2 3 1 2 3

CYSf2 1.6 1.6 1.5 1.6 1.4 1.6 Asx 12.6 12.7 13.4 12.8 12.2 13.3 Thr 8.0 8.4 7.9 8.3 8.3 8.3 Ser 11.7 9.4 9.6 9.3 9.3 9.3 Glx 10.4 11.3 11.3 11.3 11.6 11.4 Pro 7.1 7.7 7.3 7.4 7.4 7.7 GUY 8.2 6.9 7.7 7.9 8.1 7.4 Ala 6.3 6.2 6.1 6.2 6.2 6.2 Val 6.3 7.0 6.7 6.8 7.0 7.0 Met 0.9 1.0 1.1 1.5 1.3 1.1 Ile 4.0 4.4 4.2 4.2 4.3 4.1 Leu 8.1 8.8 8.7 8.3 8.6 8.5 Tyr 4.3 4.8 4.7 4.9 4.8 4.7 Phe 2.3 2.7 2.3 2.5 2.5 2.4 His 2.1 1.9 1.9 1.4 1.3 1.3 Lys 2.8 2.8 2.8 3.0 3.2 3.0 Arg 2.9 2.6 2.7 2.7 2.8 2.7

linked glycosylation sites each contained a single residue of N-acetylglucosamine; see before for details). The observed masses of CT 54 and CT 22b deviated from the calculated values by -1 a.m.u. This difference is believed to be due to the conversion of asparagine to aspartic acid caused by the

Mr x 1O-3 A

200 -

‘A?- . *

66 * .

43 m *

22 .

14

123456 123456

FK. 3. Comparison of deglycosylated TEX and deglycosy- lated NCA by SDS-electrophoresis. Reversed-phase HPLC puri- fied TEX and NCA were chemically deglycosylated, purified by reversed-phase HPLC, and analyzed in duplicate on a 12% (w/v) polyacrylamide gel. Lanes l-3, TEX peaks 1-3 (Fig. 2A) after degly- cosylation. Lanes 4-6, NCA peaks Z-3 (Fig. 2C) after deglycosylation. The proteins were stained with Coomassie Blue R-250 (panel A) or transferred to nitrocellulose and stained for immunoreactivity with anti-CEA mAb T84.1E3 (panel B). Molecular weight markers are partially shown on the edges of panel A.

cyclization and subsequent imide hydrolysis of the asparagi- nyl-glycine bond present in these peptides. Such a cyclization/ hydrolysis reaction is expected to produce a P-aspartyl-glycine sequence (Han et al., 1983). Repeated attempts to obtain masses for CT 25a and CT 49 were unsuccessful. For CT 25a, the molecular ion signal was obscured by matrix ions when using the dithiothreitol/dithioerythritol/camphorsulfonic acid or the thioglycerol matrix, and we were unable to detect a molecular ion signal using glycerol. We were also unable to obtain a molecular ion for CT 49 regardless of the matrix employed. Further discussion of the sequence assignment for

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8622 Glycoforms of Nonspecific Cross-reacting Antigen

061 TEX

1.0 i NCA

20 40 60 80 Time (mid

100 120

FIG. 4. Chymotryptic map of TFMSA-treated TEX (top) and NCA (bottom). Carboxymethylated deglycosylated TEX and NCA samples were treated with 2% (w/w) chymotrypsin at 37 “C for 18 h. The chymotryptic peptides were separated with a linear gradient of 100% (v/v) solvent A to 40% solvent A, 60% (v/v) solvent B in 120 min. Solvent A is 0.1% (v/v) trifluoroacetic acid in H,O. Solvent B is 0.1% trifluoroacetic acid, 9.9% H,0/90% acetonitrile (v/v/v). . Solid bnes, A2i4; dashed lines, % solvent B. The peak labeled u.d. refers to undigested protein. Microsequence analysis of the peak eluting at 98 min failed to produce a sequence suggesting that it is an organic contaminant.

these peptides is provided below. By using the mass spectral data presented in Table II, the

TEX and NCA peptides were aligned relative to the cDNA- predicted NCA sequence. Fig. 5 shows that 271 of the 310 residues predicted by the NCA cDNA sequence were identified by mass spectrometry; only residues 32-34 and 275-310 were unidentified. To confirm the mass spectral alignment, pep- tides were selected from either the TEX or NCA maps and subjected to automated microsequence analysis. The sequenc- ing results (indicated by Protein in Fig. 5) showed complete agreement with the mass spectral alignment and identified 244 of the 310 residues predicted for the NCA sequence. Sequence analysis of CT 25a and CT 49, the two peptides for which mass spectral data was not obtained, yielded sequences corresponding to residues 32-34 and 215-286, respectively, although Asn-275, a potential glycosylation site was not as- signed (see below). As predicted from the mass spectral data, the sequence of CT 54 and CT 22b ended prematurely at the residues preceding their asparaginyl-glycine bonds. These re- sults are consistent with the formation of P-aspartyl-glycine bonds which are intractable to Edman degradation but which would lead to an increase of 1 a.m.u. in the observed mass of these peptides. A polymorphism predicted by the two cDNA sequences was also confirmed by both mass spectrometry and sequence analysis, with peptides CT 43b and CT 30 being derived from Gly-205 (Neumaier et al., 1988) and Val-205 (Tawaragi et al., 1988) forms of NCA, respectively. Despite analyzing all peptides of the TEX and NCA chymotryptic maps (even those not discussed), sequences for residues 287- 310 of the predicted NCA sequence were not detected by either mass spectrometry or sequence analysis suggesting a possible posttranslational modification (see below). Although

TABLE II

Muss spectral analysis of NCA and TEX chymotryptic peptides Mass measurements were made as described in text. All measure-

ments reported reflect the monoisotopic mass, except those sur- rounded by brackets which are average masses. ND, not determined, talc., calculated, obs., observed.

Chymotryptic peptide

CT 43a CT 24a CT 37 CT 50 CT 24b CT 25a CT 36a CT 22a CT 41 CT 25b CT 47 CT 45 CT 18 CT 54 CT 22b CT 23a CT9 CT 34 CT 36b CT 42 CT 43b CT 30 CT 23b CT 11 CT 28 CT 55 CT 59 CT 29 CT 15 CT 13 CT 49

Mass determinations

CdC. NCA obs

1035.55 1035.54 958.50 958.58

1071.60 1071.54 1184.66 1184.48 1282.64 1282.76

455.19 ND 1506.76 1506.80 1302.67 1302.64 1623.78 1623.85 1805.77 1805.85 1992.00 1992.00 (3160) (3159)

1545.62 1545.58 1638.87 1639.60 1153.63 1154.69 1620.81 1620.70 1096.53 1096.60 2204.95 2204.84 (3284) (3284)

1934.94 1935.00 1403.64 1403.55 1445.69 1446.03 1500.66 1500.69

918.43 918.47 2400.04 2399.95 1174.53 1174.55 2135.97 2135.96 1684.77 1684.59

761.26 760.97 448.49 448.41

1482.73 ND -

TEX ohs.

Sequence position

1035.52 l-9 958.50 lo-18

1071.54 10-19 1184.48 10-20 1282.61 21-31

ND 32-34 1506.96 35-48 1302.69 49-61 1623.74 62-74 1805.69 75-86 1992.20 87-104 (3160) 105-131

1545.71 132-142 1639.83 143-156 1154.65 146-156 1620.66 157-169 1096.55 170-179 2205.23 180-197

(3284) 170-197 1934.81 198-215 1403.45 202-215 1445.88 202-215 1500.59 216-226

918.35 227-235 2400.23 216-235 1174.41 236-243 2135.88 236-251 1684.69 252-263

761.15 264-269 448.20 270-274

ND 275-286

gaps exist in both the mass spectral and sequence data, the combination of these techniques allowed elucidation of the complete primary structures of TEX and NCA. Because the TEX and NCA sequences are identical, only a single sequence is shown in Fig. 5.

Posttranslational Modifications-As previously demon- strated for CEA (Paxton et al., 1987), direct assignment of asparagine-linked glycosylation sites by both FAB mass spec- trometry and microsequence analysis is possible following treatment with TFMSA. This reagent cleaves 0-glycosyl link- ages leaving the asparagine-linked N-acetylglucosamine as a reporter group (Edge et al., 1981). Identification of the glyco- sylation site is possible by 1) observation of PTH-Asn(Gl:- NAc) during sequence analysis and/or by 2) an increase of 203.07 a.m.u. (per GlcNAc) in the observed mass of the glycopeptide (relative to the non-glycosylated peptide) during FAB mass spectrometry (Paxton et al., 1987). In the present study we utilized this approach to identify each of the glyco- sylation sites in TEX and NCA as illustrated below.

Shown in Fig. 6A is the separation of PTH-amino acids (10 pmol each) and PTH-Asn(GlcNAc) (15 pmol). As can be seen, PTH-Asn(GlcNAc) is well separated from the other PTH- derivatives. Sequence analysis of a TEX glycopeptide, CT 41, is shown in Fig. 6B. Three cycles (8-10) are shown to illustrate the identification of PTH-Asn(GlcNAc) in cycle 9. Note that the yield of PTH-Asn(GlcNAc) is reduced relative to neigh- boring cycles due to the reduced solubility of the Asn(GlcNAc) derivative in butyl chloride, the solvent used for extraction of cleaved residues during automated sequence analysis. In Fig. 6C, the comparative mass spectral analyses of CT 41 from the

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Glycoforms of Nonspecific Cross-reacting Antigen 8623

TEX and NCA maps are presented. The observed masses are 203.07 a.m.u. higher than that predicted for the peptidyl portion (SGRETIYPNASLL) of CT 41, consistent with the presence of N-acetylglucosamine.

A summary of the TEX and NCA glycosylation sites deter- mined by these methods is provided in Table III. Of the 12 glycosylation sites predicted by the NCA cDNA sequence, 8 were identified in TEX and NCA by both mass spectrometry and sequence analysis, and 3 were identified by mass spec- trometry only. The final glycosylation site predicted by the NCA cDNA sequence, Asn-275 present in CT 49, is discussed below in the context of the COOH terminus of TEX and NCA.

As noted above, no peptides from either the TEX or NCA maps were isolated that contained sequences corresponding to the 24-residue COOH-terminal domain predicted by the cDNA data. The sequence of CT 49 terminated with Gly-286, the residue immediately adjacent to the predicted COOH- terminal domain (Fig. 5). The atypical ending with glycine (not a predicted chymotryptic cleavage site) suggested that Gly-286 was the COOH-terminal residue in the mature anti- gens. Included in this peptide is Asn-275, a predicted glyco- sylation site. Despite repeated attempts using a variety of matrices, we were unable to confirm the sequence of CT 49 by mass spectrometry. In addition, we were unable to assign Asn-275 as either Asn or Asn(GlcNAc) during sequence analy- sis. Amino acid compositions were not determined due to a limited amount of sample.

To confirm Gly-286 as the COOH-terminal residue and to establish that TEX and NCA each contain G-PI anchors, which presumably would be linked to Gly-286, we reisolated TEX and NCA COOH-terminal peptides for further analyses.

FIG. 5. Primary sequence of TEX and NCA. The sequence predicted by the cDNA data is identified by cDNA. The protein sequencing data is indicated by Protein. A summary of the mass spectral data (Table II) is provided for convenience. Only one se- quence is shown for TEX and NCA since both proteins were found to be identical with respect to sequence and number of glycosylation sites. The asterisks designate glycosylation sites. Presence of etha- nolamine was determined as described in the text.

As an alternative to chymotryptic peptide mapping, which gave a relatively poor yield of the COOH-terminal peptide (CT 49), fully glycosylated TEX and NCA were fragmented with cyanogen bromide, taking advantage of the proximity of 2 methionine residues (Met-264 and Met-281) near the ex- pected COOH terminus of TEX and NCA. The third methi- onine residue in TEX and NCA is residue 164. The proteins were not deglycosylated for this treatment to maintain solu- bility of the resultant CNBr fragments. TEX and NCA CNBr digests were identical by reversed-phase HPLC, each having a minor peak (CNBr 1) eluting at -30% solvent B followed by a major peak (CNBr 2) eluting at -45% solvent B (chro- matograms not shown). CNBr 2 contained sequences begin- ning with the NH, terminus and residue 165, the result of cleavage at Met-164. The sequence of CNBr 1 for both TEX and NCA began with Cys-265 (detected as pyridylethyl cys- teine), resulting from cleavage at Met-264, and terminated with Gly-286 (Table IV). The expected cleavage at Met-281 was not observed, probably due to the resistance of the Met- Ile bond as previously documented (Corradin and Harbury, 1970), although partial conversion of methionine to homoser- ine was observed. The sequence termination of CNBr 1 at Gly-286 confirms that it is the COOH-terminal residue in TEX and NCA and demonstrates the absence of residues 287- 310 predicted by the cDNA data (Fig. 5). Cycle 11 of CNBr 1, predicted to be Asn-275, was blank. Because glycosylated TEX and NCA were used for the CNBr digests, this result is consistent with Asn-275 being a glycosylation site, as fully glycosylated asparagine residues are not detected during au- tomated sequence analysis due to the insolubility of the cleaved residue in the extraction solvent.

Due to the expected presence of both an oligosaccharide chain and a G-PI anchor in CNBr 1, which could potentially complicate its mass spectral analysis, amino acid composi- tional analysis was employed to confirm the identity of the peptide. There was excellent agreement between the observed and expected compositions for the hydrophobic residues in both TEX and NCA CNBr 1 (Table V), although differences were noted for the hydrophilic residues. The observation of 2.6 and 2.2 residues of Asx for TEX and NCA CNBr 1, respectively, further supports the assignment of Asn-275 as a glycosylation site.

The compositional data also demonstrates the presence of approximately one equivalent of ethanolamine in both TEX and NCA CNBr 1. Ethanolamine elutes with base-line reso- lution between Lys and NH, under the conditions used for these analyses (data not shown). Since ethanolamine is not a normal constituent of N-linked oligosaccharides or peptides, detection of ethanolamine during compositional analysis of the CNBr fragments is consistent with TEX and NCA con- taining a G-PI membrane anchor that is covalently coupled to Gly-286 through an ethanolamine-phosphodiester bond. Addition of such a complex is believed to occur posttransla- tionally (reviewed in Ferguson and Williams, 1988) and would be expected to involve removal of the 24-residue COOH- terminal domain predicted by the cDNA data. Typically, confirmation that proteins are G-PI anchored relies on the susceptibility of such proteins to release by phosphatidylino- sitol-specific phospholipase-C treatment. The source of TEX and NCA (liver metastasis) prevented us from accomplishing these experiments in this study. Other studies in this labora- tory involving expression of NCA in tissue culture, however, have confirmed the G-PI linkage by showing specific release of the expressed NCA by phosphatidylinositol-specific phos- pholipase-C (Hefta et al., 1990).

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8624 Glycoforms of Nonspecific Cross-reacting Antigen

FIG. 6. Identification of glycosyl- ation sites. A, separation of PTH-deriv- atives by reversed-phase HPLC using a Beckman ODS 2.1 X 250-mm column operated at 0.25 ml/min flow rate. Buffer A is 90% methanol, 10% acetonitrile (v/ v). Buffer B is 90% 10 mM NHIOAc, pH 5.8, 10% methanol (v/v). Following in-

jection, the PTH-derivatives were eluted by a linear gradient from 10% B to 46% B in 20 min followed by a second linear gradient from 46% B to 50% B in 9 min. B, reversed-phase chromatograms for se- quencer cycles 8-10 of a representative glycosylated peptide (SGRETIYP- NASLL) from TFMSA-treated TEX (52% injected). Cycle 9 was determined to be PTH-Asn(GlcNAc) and, hence, de- fines a site of glycosylation. Z.S., internal standard; DTE, dithioerythritol; DPTU, diphenylthiourea. C, comparative FAB mass spectrum of the TEX and NCA glycopeptides described in B. The ob- served masses (MH’) are shown for both glycopeptides and indicate an increase of approximately 203.07 a.m.u. from the mass predicted for the peptidyl portion (1420.71), consistent with the presence of N-acetylglucosamine.

TABLE III

A ,010 B .OlO

GlcNAc I/K

% m N 005 E 005

Q Q

IO 20 30 IO 20 30

8

)PTU

P

;I

;lcNAc

G

L-

Retention Time (Mid Retention Time (Mid

C

10

L

Posttranslational modijications identified in TEX and NCA Asn(GlcNAc) was identified as described in the text. Presence of

ethanolamine is consistent with the presence of a G-PI anchor as described in the text. ND, not detected; NA, not analyzed.

Identification method

Modification-residue Sequence FAB-MS Amino acid

analysis

Asn(GlcNAc)-70 Asn(GlcNAc)-77 Asn(GlcNAc)-81 Asn(GlcNAc)-118 Asn(GlcNAc)-139 Asn(GlcNAc)-163 Asn(GlcNAc)-190 Asn(GlcNAc)-222 Asn(GlcNAc)-240 Asn(GlcNAc)-254 Asn(GlcNAc)-258 Asn(GlcNAc)-275 Ethanolamine- 286

+ + + I’& ND ND

+ + +

i+D NA

+ + + + + + + + + + F-h ND

NA NA NA NA NA NA NA NA NA NA NA

+ +

DISCUSSION

In this paper we describe the isolation and structural char- acterization of two glycoforms of NCA, a member of the CEA gene family. Owing to its importance as a tumor marker, much of the past work in this field has involved characteriza- tion of CEA. Modern protein chemistry and molecular cloning techniques have greatly enhanced our knowledge of the struc- ture and genomic organization of CEA and its cross-reacting antigens and have firmly established a relationship with the immunoglobulin gene superfamily (Paxton et al., 1987; Oi- kawa et al., 1987b). The physiological role of the CEA gene family remains to be determined, although in vitro studies suggest that CEA and perhaps NCA function as intercellular adhesion molecules (Benchimol et al., 1989; Oikawa et al., 1989). Despite these advances, a number of questions remain unanswered concerning the relationships of CEA-related an- tigens isolated from human tissues, their immunoreactivities as defined by monoclonal antibodies, and the genes of the

Sequence analysis of CNBr 1 fragments The samples (So-100 pmol) were spotted on polyvinylidenedifluo-

ride membranes (1 x 10 mm), placed in a continuous flow reactor (Shively et al., 1987), and subjected to automated Edman degradation with a gas-phase sequencer built at the City of Hope (Hawke et al., 1985). pe-Cys, pyridylethyl-cysteine; ND, not determined.

TEX fragment NCA fragment

Cycle Residue pm01 Cycle Residue pm01

1 pe-Cys 22 1 pe-Cys 42 2 Gln 36 2 Gln 46 3 Ala 52 3 Ala 72 4 His 4 4 His 12 5 Asn 32 5 Asn 34 6 Ser 22 6 Ser 10 6 Ala 28 7 Ala 34 7 Thr 14 8 Thr 18 9 GUY 8 9 GUY 14 10 Leu 10 Leu 20 11 (Asn) I% 11 (Asn) ND 12 Arg 6 12 Arg 6 13 Thr 6 13 Thr 8 14 Thr 10 14 Thr 14 15 Val 24 15 Val 16 16 Thr 8 16 Thr 10 17 Met 8 17 Met 16 18 Ile 6 18 Ile 12 19 Thr 4 19 Thr 10 20 Val 18 20 Val 14 21 Ser 2 21 Ser 4 22 GUY 2 22 GUY 2

CEA family. This is especially true for NCA, for which mul- tiple forms with different molecular weights and immuno- reactivities have been identified.

NCA principally refers to a CEA-cross-reacting antigen (Mr - 50,000) which can be isolated from normal lung, spleen, and colon tumors (Mach and Pusztaszeri, 1972; von Kleist et al., 1972; Laferte and Krantz, 1983). More recently, multiple forms of NCA were identified in normal lung and peripheral blood granulocytes, including NCA-160 (Mr 160,000), NCA- 95 (1M, 95,000), NCA-90 (Mr 90,000), and NCA-55 (Mr 55,000)

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Glycoforms of Nonspecific Cross-reacting Antigen 8625

TABLE V Amino acid analysis of CNBr I fragments

The samples were hydrolyzed with 6 M HCl containing 0.2% 2- mercaptoethanol (v/v) at 110 “C for 24 h and analyzed with a Beck- man 6300 amino acid analyzer. pe-Cys, pyridylethyl-cysteine; ND, not determined.

Observed Residue Expected

TEX NCA pm01 pITlO

pe-Cys 1 ND ND Asx 2 2.6 (316) 2.2 (214) Thr 5 4.4 (534) 3.2 (306) Ser 2 1.9 (231) 1.4 (135) Glx 1 1.5 (182) 1.5 (146) GUY 2 1.7 (205) 3.0 (292) Ala 2 2.1 (256) 2.4 (232) Val 2 1.8 (223) 2.2 (208) Met 1 0.3 (35) 0.4 (43) Ile 1 0.9 (110) 1.0 (97) Leu 1 1.1 (130) 1.1 (106) His 1 0.9 (107) 0.8 (80) Ax 1 1.0 (121) 1.2 (112) Ethanolamine 1 0.5 (65) 1.0 (110)

(Buchegger et al., 1984; Audette et al., 1987). Similar studies identified M, 97,000 (NCA-97) Zt4, 75,000 (NCA-75), and Ii4, 50,000 (NCA-50) forms of NCA from colon tumor and normal lung (Grunert et al., 1985). A plasma form of NCA has also been identified, with an NH*-terminal sequence identical to that reported in the present study.3 These observations illus- trate not only the complexity of NCA but also the need to purify and characterize the multiple forms in order to estab- lish their relationships.

The purpose of the present study was to determine the primary sequence and position of glycosylation sites for two distinct forms of NCA, designated NCA (Mr 45,000) and TEX (Mr 75,000), which were isolated from a single colon tumor metastasis. Using a systematic approach for the structural characterization of highly glycosylated proteins, we estab- lished that TEX and NCA were identical with respect to deglycosylated molecular weight and amino acid sequence. Furthermore, we showed by direct methods that 11 of the 12 potential glycosylation sites in both TEX and NCA were glycosylated. Indirect methods showed that the twelfth gly- cosylation site in both proteins were also glycosylated. Lastly, we found evidence for posttranslational modification at the COOH terminus of TEX and NCA involving removal of a 24- residue hydrophobic region with subsequent addition of eth- anolamine, a component of G-PI anchors.

The observed difference in the glycosylated molecular weights for TEX and NCA is almost certainly due to varia- tions in their carbohydrate contents, implying that they are glycoforms. The estimated carbohydrate contents for TEX and NCA based on glucosamine determinations during amino acid analysis support this conclusion. The large difference in molecular weight of these proteins indicates gross differences in the structures of the oligosaccharide chains rather than minor differences which could arise from oligosaccharide mi- croheterogeneity. Proof of this will require a complete struc- tural characterization of the TEX and NCA oligosaccharides. Studies are currently in progress to determine complete car- bohydrate compositions for TEX and NCA. The ultimate goal is to isolate and characterize individual glycopeptides so that oligosaccharide structures can be assigned to each glycosyla- tion site in TEX and NCA.

The systematic approach to protein structural characteriza-

’ S. A. Hefta, R. J. Paxton, J. E. Shively, unpublished data.

tion presented in this paper is necessary to adequately de- scribe the molecular relationships of the multiple forms of NCA. The NCA described in this study, M, 45,000, is probably equivalent to purified NCA from normal lung and metastatic lung and colon tumors reported by other laboratories (Laferte and Krantz, 1983; Krop-Watorek et al., 1983; Grunert et al., 1988). The relationship of TEX with other reported NCA species is less clear. Based on molecular weight comparisons and tissue distribution, TEX would appear to be equivalent to NCA-75 from colon tumors and normal lung (Grunert et al., 1985). Alternatively, it is possible that TEX corresponds to NCA-90 from granulocytes (Audette et al., 1987), although no biochemical data is available to support this possibility. Immunochemical data shows that NCA-50/NCA-55 is dis- tinct from NCA-95/NCA-97 (Buchegger et al., 1984; Grunert et al., 1985). Together with our results, these data suggest that TEX is also distinct from NCA-95/NCA-97.

At present the functions of TEX and NCA have not been established, preventing a detailed examination of their struc- ture-function relationships. In these studies we purified TEX and NCA from a single liver metastasis, containing a homog- enous cell population. The production of two glycoforms of NCA from tumor cells is analogous to the production of multiple forms of NCA by granulocytes. The dramatic differ- ence in carbohydrate composition, demonstrated by the large difference in molecular weight of the glycosylated proteins, suggests that the oligosaccharide chains are functionally im- portant in NCA. We are currently exploring possible functions of TEX/NCA with the goal of correlating alterations of func- tion with changes in the carbohydrate structure.

Acknowledgments-We thank Dr. Terry D. Lee and Kassu Legesse for assistance with FAB mass spectrometry and Jim Sligar for tech- nical assistance.

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