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[CANCER RESEARCH 38, 2199-2208, 0008-5472/78/0038-OOOOS02.00 August 1978] Amino-terminal Sequences of the Major Tryptic Peptides Obtained from Carcinoembryonic Antigen by Digestion with Trypsin in the Presence of Triton X-1001 John E. Shively,2 Michael J. Kessler,3 and Charles W. Todd Division of Immunology, City of Hope National Medical Center, Duarte, California 91010 ABSTRACT Sequence studies of Carcinoembryonic antigen (CEA) were initiated to obtain direct chemical evidence regard ing the structure of CEA for comparison with that of CEA cross-reacting antigens. To obtain, purify, and perform amino acid sequencing on mg amounts of CEA (a glycoprotein with a molecular weight of 180,000, composed of 60% carbohydrate by weight), we needed to develop new approaches and refine existing techniques. This report describes the procedures developed during the course of this study and presents initial results. Trypsin digestion in the presence of 0.25% Triton X-100 produced seven major glycopeptide fragments that were separated and purified by high-pressure liquid chromatography on ion-exchange resins followed by Sephadex gel chromatography. NH,terminal sequences were determined on 1- to 2-mg amounts of the glycopeptides by high-pressure liquid chromatography for the analysis of phenylthiohydantoin derivatives of amino acids. Four peptides gave sequences through 20 cycles of Edman degradation, one gave se quences through 13 cycles, and two gave sequences through 10 cycles. Three of these peptides also gave sequences of up to 8 to 13 cycles for a minor component. The abrupt halt in Edman degradation for each peptide was interpreted as the failure to sequence beyond a carbohydrate substitution on the polypeptide chain. Since each sequence obtained was unique, the results substan tiate the claim that the polypeptide chain of CEA is a definite chemical entity and that the microheterogeneity resides in the carbohydrate portion of the molecule. INTRODUCTION CEA4 is a glycoprotein (26) with a molecular weight of approximately 180,000 (41), composed of about 60% car bohydrate and 40% protein by weight (46). Extensive stud ies on CEA have revealed details of its physical structure by molecular sizing techniques and by electron microscopy (41), its carbohydrate structure by periodate oxidation (8, 21) and by methylation-linkage analysis (7, 21), and its NH21 Supported in part by National Cancer Institute Grants CA16434 and CA19163 from the National Large Bowel Cancer Program. 2 To whom requests for reprints should be addressed, at Division of Immunology, City of Hope National Medical Center, 1500 East Duarte Road Duarte, Calif. 91010. 3 Present address: S. U. N. Y. at Buffalo, Department of Cell and Molecular Biology, Gary Hall, Buffalo, N. Y. 14214. "The abbreviations used are: CEA, Carcinoembryonic antigen- SOS sodium dodecyl sulfate: PTH, phenylthiohydantoin. Received January 23, 1978; accepted April 20, 1978. AUGUST 1978 terminal amino acid sequence (4, 45) by Edman degrada tion. In addition these studies and others (22, 31, 52, 53) support the current view that the antigenic determinants recognized in the radioimmunoassay for CEA reside in the protein portion of the molecule. Since CEA was reportedly shown to be heterogeneous in terms of charge and molecu lar weight polydispersity (3, 5, 17, 23, 42), a structural feature attributable to its extensive carbohydrate microhet erogeneity, it is reasonable to assume that the antigenic determinants should reside in an invariant portion of the molecule, namely, the polypeptide chain. CEA was first described as a tumor-associated antigen by Gold and Freedman (18, 19) because it was detected in tumors of the digestive system and in fetal gastrointestinal tissue. Subsequently, several investigators (9, 20, 22, 30, 50) have isolated CEA cross-reacting antigens from normal adult tissues. Recent evidence (33, 34, 48) has shown that many of these substances differ in chemical composition and molecular size from CEA. These findings have demon strated a real need to characterize chemically the CEAreactive molecules from different sources. Significant progress in the structural studies of CEA and CEA cross-reacting antigens has been made in this labora tory. CEA isolated from normal adult colon washings (14) has been shown to be structurally identical to that isolated from liver métastasesof colon adenocarcinoma (40), but significant differences were found in the lower-molecularweight CEA cross-reacting antigens isolated from normal (16) and malignant tissues (25). Structural studies on the protein portion of CEA and CEArelated antigens are necessary to establish the basis for their observed antigenic differences. The results of such studies may lead to the development of a more specific test for CEA in malignant and other diseased tissues that show elevated CEA levels. Protein sequence studies on CEA are hampered by unusual technical problems since CEA con tains over 600 amino acids and contains over 50% carbo hydrate. The carbohydrate, all of which is linked to protein through /V-acetylglucosamine to asparagine linkages, is difficult to remove by chemical cleavage (7, 8, 52). Specific cleavage of peptide bonds by cyanogen bromide treatment has been unsuccessful due to the lack of methionine in CEA and the occurrence of hydrolytic cleavage, which occurs at low pH (presumably aspartylproline bonds). The use of the cyanylation method of cleaving cysteine peptide bonds by the method of Jacobson ef al. (24) formed A/ blocked peptides that could not be sequenced (12, 28). In the present study it was found that glycopeptides capable of sequence analysis could be obtained from CEA 2199 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. J. E. Shively et al. by trypsin digestion in the presence of 0.25% Triton X-100 and subsequent separation by ion-exchange high-pressure liquid chromatography. In some cases the resulting glycopeptides could be sequenced through 20 degradation cy cles before the analysis came to an abrupt halt, presumably due to the presence of a carbohydrate chain linked to an asparagine residue. Although this report constitutes only a start on the amino acid sequence analysis of CEA, it shows for the first time that CEA can be broken up into isolatale glycopeptide fragments that are amenable to NH2-terminal amino acid sequencing. These results support the conclu sion, suggested originally by the finding of a single, uniform NH,-terminal amino acid sequence for CEA (45), that indeed the polypeptide portion of CEA is a definite chemical entity. The authors intend to use the techniques developed here to answer specific questions about the structural features responsible for the antigenic differences observed among the various CEA and CEA cross-reacting entities. MATERIALS AND METHODS Preparation of CEA. Isolation and purification of CEA was carried out as previously described (6) and was mea sured by double-antibody radioimmune assay (13) with a "Co volume marker (15). In addition, contaminating traces of mucopolysaccharides were removed by concanavalin A Sepharose affinity chromatography as previously described (36); recovery of CEA from this step was 78%. CEA from 1 tumor source was used throughout these studies. Neuraminidase Digestion. Vibrio cholerae neuraminidase digestion was performed according to Coligan and Todd (8). A control sample was run in which CEA was omitted from the reaction. No material capable of sequence analysis was found in the control sample. Reduction and Alkylation. CEA (210 mg) treated with neuraminidase and dissolved in 30 ml of 9.0 M urea buffered with 0.1 M Tris-HCI, pH 8.3, and purged with N2was reduced with 0.3 mmol of dithiothreitol for 1 hr at room temperature and alkylated with 0.43 mmol of iodoacetamide (0.30 mCi of [14C]iodoacetamide diluted to a final specific activity of 0.70 mCi/mmol) for 1 hr at room temperature in the dark. The sample was dialyzed exhaustively against water, lyophilized, and dried over P2O5. Trypsin Digestion. CEA (218 mg) treated with neuramini dase, reduced, alkylated, and dissolved in 15 ml of 0.25% aqueous Triton X-100 (Sigma Chemical Co., St. Louis, Mo.) was adjusted to pH 8.1 with a dilute ammonia solution ana digested with 2.0 mg of solid u-l-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Bio chemical Corp., Freehold, N. J.) for 12 hr at 37°.pH 8.1 was maintained by further addition of dilute ammonia as neces sary. The reaction mixture was lyophilized and dried over P205 (yield, 254 mg). A sample of a,-acid glycoprotein was obtained from Dr. Yu-Lee Hao, American Red Cross Blood Research Laboratory, Bethesda, Md., and was used for model studies of trypsin digestion in the presence of Triton X-100. Amino Acid Analysis. Amino acid analysis was performed by a modification of the method of Liu and Chang (29). Samples of 100 to 200 ^g were hydrolyzed under vacuum in 2200 heavy-walled ignition tubes at 110°for 48 hr in duplicate with 0.5 ml of 3 N p-toluenesulfonic acid containing 0.2% 3(2-aminoethyl)indole. The hydrolyzed samples were ana lyzed on a Beckman 121H amino acid analyzer (Beckman Instruments, Inc., Palo Alto, Calif.) The basic amino acids and amino sugars were eluted from a 20- x 0.9-cm column of PA35 (Beckman) with sodium citrate buffer, pH 5.26 (0.4 N sodium). The acidic and neutral amino acids were eluted from a 56- x 0.9-cm column of AA15 (Beckman) with sodium citrate buffers of pH's 3.10 (0.16 N sodium), 3.60 (0.02 N sodium), and 4.20 (0.20 N sodium). Carbohydrate Analysis. Carbohydrate analysis was per formed on the trimethysilyl derivatives of sugar methyl glycosides (37). Sialic acid was determined by the previ ously described method and by the method of Warren (51). SDS/Gel Electrophoresis. SDS/gel electrophoresis was performed on either 6 or 12% polyacrylamide gels accord ing to the method of Swank and Munkres (44). Gels were routinely stained with Coomassie blue or periodic acidSchiff reagent (38). For location of 14C-labeled peptides with CEA activity, portions of the extracts of 1-mm-thick gel slices shaken overnight in 0.5 ml of phosphate-buffered saline (0.075 M sodium phosphate/0.075 M NaCI; pH 7.2) were counted. Isoelectric Focusing. Isoelectric focusing was performed on an LKB 8100 electrofocusing column of 110-ml capacity according to the method of Vesterberg and Svensson (49). LKB carrier ampholytes (1%; LKB Instruments, Inc., Rockville, Md.) were used in the pH ranges of 2.5 to 4, 4 to 6, and 5 to 8. Peptide Separation Procedures. Ion-exchange separa tions were performed on either DC4A cation or DA8X8 anión exchangers (Durrum Instrument Corp., Palo Alto, Calif.) with a DuPont Model 830 high-pressure liquid Chromato graph. Gel filtration was performed on either Sephadex G-50 or G-100 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.). Aliquots were analyzed for 14C-labeled peptides by liquid scintillation counting on a Beckman LS-330 scintilla tion counter with an Aquasol (New England Nuclear, Boston, Mass.) cocktail. Free amino groups were detected by reac tion with fluorescamine (Hoffmann-La Roche Inc., Nutley, N. J.). Portions of 10 /JL\were evaporated to dryness under N, in 0.6- x 5-cm tubes and mixed with 50 /¿Iof 0.2 M pyridine and 50 /¿Iof 2 mg fluorescamine per ml acetone (w/v); the relative fluorescence was read in a 0.10-ml cuvet on an Aminco fluorometer (excitation, 364 nm; emission, 458 nm). Two-Dimensional Mapping Procedures. Chromatogra phy in the vertical dimension with butyl alcohol/acetic acid/ water (4/1/5, v/v) for 24 hr and electrophoresis in the horizontal dimension with pyridine/acetic acid/water (20/2/ 378, v/v) for 24 hr at 3000 volts was performed according to the procedure of Bennett (2). Sequencing Procedures. NH,-termmal peptide se quences were performed with a modified dimethyl allylamine program (Beckman 102974) on a Beckman Model 890C sequencer equipped with a dry ice/acetone trap on its lowvacuum pump. The program modifications were the follow ing. Benzene/ethyl acetate (1/1, v/v) was used as S1. N2 delivery through S2 at Step 14 was 80 sec. N2 dry at Step 17 was 80 sec. Steps 38 and 39 were interchanged (new Step CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. Tryptic Peptides from CEA acrylamide gel electrophoresis, and ion-exchange chroma tography. The failure of trypsin alone to digest denatured CEA was attributed to the protection of lysyl and arginyl peptide bonds by the presence of extensive amounts of carbohydrate on CEA. Model peptide mapping studies on a, acid glycoprotein treated, reduced, and alkylated by neuraminidase established that trypsin digestion was more effective in the presence of 0.25% Triton X-100. Similarly, this result was confirmed with CEA. The tryptic map of CEA (not shown here) reveals the formation of 5 to 10 slowmoving, poorly resolved peptides from CEA. In contrast, trypsin digestion of CEA performed in the absence of Triton X-100 gave very few large peptides and no smaller peptides. The tryptic peptides of CEA were eluted and analyzed for [14C]cysteine by scintillation counting, for carbohydrate under N2, and evaporated to dryness on the Buchler Eva content by the phenol/sulfuric acid method of Dubois ef a/. pomix. The PTH's were extracted 3 times with 25 /¿I of 1,2- (11), and for NH2-terminal amino acids by the method of dichloroethane/methanol (7/3, v/v) according to the Neuhoff (32). The results of these analyses (not shown here) method of Wittman-Liebold (54), transferred to a 0.4- x 4- revealed that the slower-moving peptides were composed cm polyethylene microfuge tube (Analytical Aids, Wood- of variable amounts of carbohydrate and cysteine, the side, Calif.), mixed with 25 ¿¿I of water, and stored at -20° [14C]carboxyamidomethyl derivative. Each peptide con until analyzed (the PTH is in the bottom, organic phase). tained several NH2-terminal amino acids. In general the 2This procedure has the following advantages, (a) The incor dimensional mapping procedure was found inadequate for poration of an internal standard in the method gives more the separation of large glycopeptides. accurate quantitation and monitors transfer losses, (o) The The attempted separation of CEA tryptic peptides on mild conversion conditions prevent amide hydrolysis of SDS/gels is shown in Chart 1. Untreated CEA migrates as a glutamine and asparagine derivatives and give higher yields single, diffuse band on 6% gels and as a substantially of serine and threonine. (c) The water extraction step sharper band on 12% gels. An increase in the separation of removes traces of impurities that would otherwise contrib CEA tryptic peptides is apparent in 12 compared to 6% gels. ute to analysis background levels. The PTH's were sepa 38 was cleavage reaction for 80 sec, and new Step 39 was N2delivery through S3 for 200 sec). For improvement of the sensitivity of the analysis of PTH-amino acids by thin-layer chromatography, 1 mCi of phenyliso[35S]thiocyanate was added to 16 mmol of phenylisothiocyanate in R1 of the sequencer (final specific activity, 0.06 mCi/mmol). The thiazolinone derivatives of the amino acids were converted to their PTH derivatives by a modification of the procedure of Laursen (27). The PTH derivative of aminoisobutyric acid (4.5 nmol) was added to the chlorobutane solution of each thiazolinone as an internal standard. Samples were evapo rated to dryness on a Buchler Evapomix in 1.5- x 15-cm conical tubes fitted with ground-glass joints, mixed with 200 fji\ of 20% aqueous trifluoroacetic acid containing 0.05 mg dithiothreitol per ml, allowed to react 15 min at 55° rated and quantitated by gas chromatography according to the method of Pisano and Bronzert (35) on a HewlettPackard 571OA gas Chromatograph and by high-pressure liquid chromatography on a Waters Associates liquid Chro matograph (Waters Associates, Inc., Milford, Mass.), both equipped with an Autolab System IV peak integrator. Excel lent resolution of PTH derivatives of 17 amino acids was achieved on C1B^Bondapak (Waters Associates) reverse phase columns by either Program 1, a linear gradient from 0 to 35% Component B over 30 min, or Program 2, a concave gradient from 10 to 40% Component B over 30 min. In both cases the flow rates were 2 ml/min, Component A was 0.01 M^odium acetate/acetonitrile buffer (95/5, v/v; pH 7.6), and Component B was 100% acetonitrile. These separations are similar to those reported by Zimmerman ef al. (55) and Downing and Mann (10). In addition the thin-layer Chro matographie procedure of Summers ef al. (43) was used with 2,5-bis-2-(5-ferf-butyl benzoxazolyljthiophene (Pack ard Instrument Co., Inc., Downers Grove, III.) as the fluor. Thin-layer chromatograms were recorded and compared by UV photography and by autoradiography of the [35S]PTH derivatives on X-ray film. Reagents, solvents, and sperm whale apomyoglobin obtained from Beckman were used throughout this work. RESULTS Trypsin Cleavage. Initial treatment of CEA (treated with neuraminidase, reduced, and alkylated) with trypsin gave only a few peptides in low yields when it was monitored by 2-dimensional mapping, gel chromatography, SDS/poly- AUGUST 1978 400 4.0 6.0 Mobility 8.0 10.0 12.0 in cm Chart 1. SDS/polyacrylamide gel electrophoresis profiles of CEA tryptic peptides. Samples were run on either 6 or 12% polyacrylamide gels (0.5 x 18 cm). The standards were lysozyme (14,000), 0-lactoglobulin (18,000), car bonic anhydrase (32,000), ovalbumin (45,000), and bovine serum albumin monomer (68,000) and dimer (136,000). Arrow, migration of CEA treated with neuraminidase, reduced, and alkylated: bars, molecular weight ranges for unfractionated CEA tryptic peptides, as detected by either Coomassie blue staining or location of [14C]carboxyamidomethyl cysteine. The identi fication of the peptides (T1A1. etc.) corresponds to a separate experiment in which the peptides purified by ion-exchange and gel filtration chromatog raphy were run on separate gels and detected by Coomassie blue staining or location of ["C]carboxyamidomethyl cysteine. 2201 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. J. E. Shively et al. Results similar to these were obtained with a variety of peptide detection methods: iodination or fluorescent label ing before separation; Coomassie blue or periodate-Schifi staining after separation. Peptide bands were eluted from gels and analyzed for [14C]carboxyamidomethyl cysteine and NIVtermmal amino acids. A minimum of 10 tryptic fragments could be estimated from these experiments. Isoelectric focusing of CEA tryptic peptides is shown in Chart 2. Although CEA treated with neuraminidase, re duced, and alkylated does not give a single sharp peak on isoelectric focusing, it is clear that the profile of its trypsin digest is different and reveals at least 10 separate peaks. The acidic nature of the tryptic peptides was confirmed by the 2-dimensional tryptic map and by the results of ionexchange chromatography. Microanalysis (<5 mg) of the peptides obtained from either column or polyacrylamide gel isoelectric focusing proved difficult (complete removal of ampholytes and urea from the sample was the major prob lem). The most practical preparative method for separation and purification of the tryptic peptides of CEA was ion exchange followed by gel permeation chromatography. Trial separa tions of the tryptic fragments of sperm whale apomyoglobin on a DC4A cation exchanger by high-pressure liquid chromatography and fluorescamine detection of eluted peptides gave 16 peptides in a run time of only 2 hr. How ever, a similar separation of a, acid glycoprotein treated with neuraminidase, reduced, and alkylated gave a large breakthrough peak of unseparated acidic peptides and 6 well-resolved basic peptides. Further analysis revealed that the acidic a, acid glycoprotein peptides were glycopeptides of relatively high molecular weight, which could be eventually separated on DA8-X8 anion-exchange resin. Since the CEA glycopeptides were of higher molecular weight (75,000 dallons) and comprised larger amounts of carbohydrate, the separation of the CEA tryptic peptides was more difficult to achieve than were those ob tained from a, acid glycoprotein. The separation out lined in Chart 3 details the purification of 8 tryptic frag ments from CEA. The unfractionated digest gave a poorly defined separation on gel permeation chromatography alone but, when it was run first on a cation exchanger, it yielded a breakthrough peak (T1) and a retained peak (T2), each of which yielded a breakthrough and a retained peak on anión exchanger (T1A, T2A, T1B, and T2B, respec tively). Each of the 4 peptide mixtures separated by ion exchange gave 2 well-defined peaks on subsequent gel permeation chromatography. The gel permeation results shown in Chart 3 gave lower estimations of molecular weights than that obtained by SDS/gel electrophoresis (Chart 1). However, the large effect of carbohydrate substi tution in glycopeptides on either migration in Sephadex chromatography or SDS/gel electrophoresis precludes ac curate molecular weight determinations. The method of purification shown in Chart 3 was adopted for large-scale preparation of CEA tryptic peptides. Table 1 summarizes peptide yields for this procedure. The apparent low yields for the initial DC4A cation exchange step is partially due to the conversion of ammonium salts of pep tides formed during trypsin digestion in ammonia buffer to their respective free acid derivatives. Amino Acid and Carbohydrate Analyses. Table 2 pre sents the amino acid and carbohydrate analyses for CEA and its tryptic fragments. In general the high-molecularweight glycopeptides resemble intact CEA in overall com position, except Peptides T1B2 and T2B2, which are low in carbohydrate, and Peptide T2A2, which was obtained in such low yield and contaminated with foreign matter that further analysis was impossible. Sequence Results. It was necessary to refine our se quencing procedures to obtain meaningful quantitative data on 2- to 3-mg amounts of the CEA tryptic peptides. Improvements in the sequenator programs, conversion pro cedure, and PTH identification detailed in "Materials and Methods" made this possible. The high-pressure liquid Chromatographie separation and quantitation of PTH deriv atives was especially useful in distinguishing a major from a minor sequence in several of the peptides that were later found to be a mixture (in all cases the minor sequence comprised 30% or less of the mixture). An example of the resolution and sensitivity achieved with the high-pressure liquid Chromatographie system is shown in Chart 4, along Chart 2. Isoelectric focusing of CEA tryptic pep tides. Five mg of CEA tryptic digest mixed with ' " llabeled digest and '"l-labeled CEA treated with 100 SO neuraminidase, reduced, and alkylated were ap plied to a 1- x 40-cm column, and isoelectric focusing was performed over a pH range of 2.5 to 8.0 for 72 hr. M «O 5 «o ú 3.0 IO 50 60 TO 90 100 110 ml Eluted 2202 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. Tryptic Peptides from CEA A 4.0 2.0 ' \ A Fraction T l on DA6X8 4.0 â„¢¿ 2.0 i »hole D.g«. on OC44 Summary of isolation Separation methodDC4A 4.0 Table 1 and yields of CEA tryptic peptides (mg)11.24.115.321.618.640.26.4 yield411556585010836 applied27.4 cationexchanger"DA8-X8wholedigest37.2 of 2.0 Froclion T2 on DA8X8 2.0 ÃŒJO aniónexchanger*Sephadex T117.6 of 0.5 1.0 40 T216.3 of - ,BS Froction 2.0 TIA2 TÕA °"s«Ph G5° 1.0 G-50Sephadex T1A12.6ofT2A19.2 of 2.0 1.0 2.0 G-100mg 1.0 T1B6.9ofT2BPeptideT1T2TotalT1AT1BTotalT2AT2BTotalT1A1T1A2To of 2.0 1.0 05 IS 20 25 Effluent ml Chart 3. Separation of CEA tryptic peptides. A, the peptide mixture (27.4 mg) was applied to a 0.21- x 100-cm stainless steel column packed with DC4A cation exchanger at 60°,with 1100 psi and a flow rate of 1 ml/mm, and eluted in steps, first with 0.01 M acetic acid and then with pyridinium acetate (PyrAc) buffer (0.2 M pyridine/4.5 M acetic acid; pH 3.1). B and C the peptide mixtures T1 (37.2 mg) and T2 (17.6 mg) were separately applied to a 0.21- x 100-cm stainless steel column packed with DA8-X8 aniónexchanger at 40°, with 3000 psi and a flow rate of 0.5 ml/min, and eluted in steps, first with 0.1 M A/-ethyl morpholine/0.2 M a-picoline/0.1 M pyridine buffer (pH 8.0) and then with pyridinium acetate buffer (2.0 M pyridine/2.4 M acetic acid; pH 5.0). D to G, peptide fractions T1A, T2A, T1B, and T2B were applied to either Sephadex G-50 (Seph G50, fine) or G-100 (Seph G100; 0.60- x 48-cm) columns, with flow rates of 0.2 ml/min, and eluted with 0.10 M acetic acid. Arrows, elution volumes of the standards BSA (bovine serum albumin), RNase (ribonuclease), Ala-Val (L-alanyl-L-valine), and CEA (12*l-labeledCEA). In all cases 0.4ml fractions were collected, and two 0.01-ml aliquots were withdrawn for analysis. One fraction was counted for "C. whereas the other was reacted with fluorescamine and the relative fluorescence was determined. Bars in dicate fractions pooled for sequence determination. with several cycles of an actual sequence. Unidentified peaks in Chart 4 correspond to UV-absorbing impurities that do not change from cycle to cycle and that are generally encountered when sequencing 2 mg or less of protein. The PTH-amino acids that could not be separated by this system were identified by either gas or thin-layer chromatography. The use of [35S]PTH-amino acids on polyamide plates permitted the unambiguous determination of several PTH-amino acids that are usually obtained in reduced yields. Table 3 summarizes the results obtained for the first 15 cycles of Edman degradations performed on 7 tryptic pep tides from CEA. Peptides T1A1, T1A2, T1B1, and T2B1 gave sequences through position 15 (yields were extremely low past Cycle 15), but T1B2 and T2B2 gave abruptly lower yields at Cycle 10. Absolute yields of PTH-amino acids (e.g., Cycles 1 and 2) were 30 to 50%, based on the rough molecular weight estimates for each peptide on SDS/gel electrophoresis (Chart 1). Since the sequenator program used gave routine yields of 50% for sperm whale apomy- " Results were obtained for Batch 1 of whole tryptic digest. Similar results were obtained for Batches 2 and 3. The percent age of yield is based on weight. 6 Results were obtained for Batch 1 of T1 or T2. Similar results were obtained for the second yield is based on weight. batch of each. The percentage of oglobin, the previous yields are acceptable. The NH2-terminal amino acid determination of each of the peptides by the dansyl procedure of Neuhoff (32) gave the same NH2-termini as that shown in Table 3. In all cases the tryptic peptides showed varying amounts of e-dansylamino lysine. DISCUSSION Effect of Triton X-100. These experiments demonstrate for the first time that specific cleavage of CEA into peptide fragments capable of sequence analysis can be achieved. In principle the methodology adopted to produce, separate, and sequence the glycopeptides of CEA can be used on other high-molecular-weight, high-carbohydrate-content glycoproteins. The use of trypsin digestion in the presence of Triton X-100 gave higher, more uniform yields of tryptic peptides for CEA. The binding of this non-ionic detergent to hydrophobic regions of the protein perhaps unfolded the collapsed structure of CEA treated, reduced, and alkylated by neuraminidase [see Slayter and Coligan (41) for a discus sion of the electron microscopy of CEA] enough to allow better trypsin binding to the polypeptide chain. This ex planation of enhanced trypsin digestion is based partly on the known binding of detergents to hydrophobic portions of proteins and in part on the resulting NH2-terminal se quences of the tryptic fragments obtained, which are high in hydrophobic amino acids. Control experiments have shown no change in trypsin activity on a synthetic substrate in the presence of 0.25% Triton X-100. AUGUST 1978 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. 2203 J. E. Shively et al. Amino acid" and carbohydrate Table 2 h composition of CEA tryptic peptides %3.521.70 Tyrosine Phenylalanine Tryptophan Lysine HistidineArginine 2.76 1.17 2.85 1.893.50 Carboxymethyl cysteine Aspartic acid ThreonineSerineGlutamic 3.16 1.81 2.84 1.134.59 2.61 0 1.57 0.427.68 2.70 2.12 0 1.161.252.49 1.45 1.452.64 1.97 0 2.06 0.676.88 2.51 8.14 4.27 1.613.98 2.69 1.73 2.28 1.583.61 1.59 0 1.49 1.293.93 1.60 1.37 1.39 1.78 3.49 0.53 2.62 0.60 1.13 13.99 14.03 17.97 21.10 15.20 17.67 10.61 14.29 14.14 8.119.689.5210.275.25 4.238.4411.35011.01 11.2311.914.985.545.58 11.0512.355.6107.78 8.9711.979.509.304.81 6.1612.1711.168.036.92 9.5013.045.214.596.27 7.0710.9810.3511.204.47 6.6710.4711.747.0510.54 acidProlineGlycine 5.540 AlanineHalf-cystine ValineMethionineIsoleucineLeucine% 6.390 5.480 5.820 6.090 5.800 5.970 6.570 5.590 6.250.104.238.2534.69.79.07.6 4.6402.588.8917.56.610.76.9 4.9702.1413.3232.65.57.56.5 6.5604.666.8656.96.89.17.2 5.5509.056.5917.2% 5.0504.6312.7736.33.05.48.0 6.0709.066.801.70.1<1T2B14.69 6.6205.257.0041.05.45.15.4 6.8007.336.3218.81.1<1 recoveredFucoseGalactoseMannose of amino acid by weight1.12.22.0 D-GlucosaminerfD-Glucosaminep% of carbohydrate Apparent molecular " Samples 14.114.640.4T1A15.38 14.415.238.6T1A22.54 13.013.132.5T1B14.39 12.017.135.1T1B2Mol 11.812.528.2T2A23.33 2.83.68.1T2A13.36 recoveredCEAr5.04 wt' were hydrolyzed 180,000 50.000 40.000 110,000 12,000 in 0.5 ml of 3 N p-toluenesulfonic acid for 48 hr in duplicate Beckman 121H amino acid analyzer. 6 Samples were treated with 1.5 N methanolic HCI for 24 hr in duplicate Packard 7620 gas Chromatograph equipped with a flame ionization c CEA treated with neuraminidase, reduced, and alkylated. d Determined by gas chromatography. '' Determined by amino acid analysis. ! Molecular weight values calculated from migration on SDS/gels The potential interference of the carbohydrate chains of CEA in the trypsin cleavage is great since there are on the average 80 carbohydrate chains of about 7 residues/chain present and >600 amino acids/mol (39). With the assump tion that the carbohydrate substitution on asparagine resi dues is heterogeneous in terms of degree and extent of substitution, there is the likelihood that different CEA mol ecules can be cleaved by trypsin at different arginine or lysine residues, depending on the degree of interference caused by carbohydrate chains. The beneficial effect of Triton X-100 may be in part due to a lessening of the carbohydrate steric effects. Although CEA contains sufficient arginine and lysine residues to produce up to 40 tryptic peptides, only 8 were isolated. Thus it seems likely that many of these residues are inaccessible to cleavage by trypsin. Also, possibly, several small peptides (<10 amino acids) were lost due to the small amounts of the CEA fractionated and the fractionation procedure adopted. The relatively large-molecularweight (75,000 daltons) glycopeptides obtained contained variable amounts of carbohydrate. In each case the se quence was lost before 20 cycles of Edman degradation could be achieved. Most probably, the sequence was lost due to the drop in yields, which often occurs when an 2204 8.09.323.9T2B23.48 50,000 16.000 70.000 28,000 at 110°under vacuum and analyzed on a at 80°and analyzed as trimethylsilyl derivatives on a Hewlett- detector. shown in Chart 1 should be considered approximate. asparagine linked to a carbohydrate chain is encountered (47). Amino acid analysis of CEA gives an average of 90 asparagine plus aspartic acid residues/mol of CEA and, since there are 80 carbohydrate chains (all linked to protein through asparagine) per molecule, the likelihood of en countering carbohydrate linked to asparagine in sequence studies on CEA is high. Indeed, 4 of the 7 peptides sequenced encountered asparagine before 20 degradation cycles. Peptide T2A1 contains the sequence Asn-X-Thr, which appears to be a common recognition sequence for carbohydrate linked to asparagine in glycoproteins (1). The fact that these asparagines were identified in the usual chlorobutane washes obtained from the sequenator sug gests that these residues had little or no carbohydrate at tached, since the presence of hydrophilic carbohydrate substituents on PTH-asparagine would render these deriva tives insoluble in chlorobutane. The finding that Peptides T1A2, T1B1, and T2B2 gave both a major and a minor sequence demonstrates that these 3 peptides were not completely pure. However, in no case did the minor sequence of these 3 peptides corre spond to the sequence of the other peptides. Evidently, the minor sequences represent distinct peptides obtained in lower yield. A tentative reason for the lower yields of these CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research. Tryptic Peptides CO C£ CU "D $ ì{¡ili"O 1i i er1- ^~á1 co• :zz LU> ':'.¡ >- co tt-•z. -z.o s*1 p g >,Q) - to C —¿â€¢^ u) u||IN.— ^ fl) fl) ¡¡TÃ-ül*(/3 A y) Q} o_l _J< Q)« O flj ¿¿ Cïiiïl">, CO O. C To> ñ â„¢¿ ^ < S >LU > LUx _ -C _i_i CLTöCLnO-TBu-i-Q.33 C O CD > > _i_i (SO >- > LU LU UJ LU 5C3 <a<a<31 5 5 if <=< 5 52 52 -JL 2? •¿Â£ ÃŒ! ICOICOICOIMI Q) lati§ yi from CEA .£5 CO O) > LU LU xiCL 2x O -i-C -o! * aCL ^^ rf i^ CO c c "ôiBOO ^coC- CO ^ <><UJ <>< -JLa. 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Tryptic Peptides from CEA 03 PTH Ammo Acid Stondards linear (rom 0%B to 35% B over 30 mm, 2ml/mm A<Ph 76, 01MActtoit/CHjCN 95/5 U/») B- lOOIfcCMjCN N GA 02 MP u 01 LJ 03 Program 2 concave from K)% 0 to 40%B over 30 mm A* B same as above PTH Ammo Acid Standards o> o 02 01 CO I I Program 2 i Cycle 2 : CycteS : L.. I Cycl«5 ! Cyc* 6 Alt 1 , » 1 determine if the tryptic peptides could be used to make antisera against specific portions of the CEA molecule. CEA has been shown to require the presence of intact disulfide bridges to retain activity in the radioimmunoassay (21, 53). We have produced high-liter antiserum against reduced and alkylated CEA, which cross-reacts with intact CEA in a radioimmunoassay although it has no activity against trypsinized, reduced, and alkylated CEA. Evidently, the tryptic fragments obtained from CEA do not possess sufficient similarity in tertiary structures to reduced and alkylated CEA to yield significant cross-reactivity in this sensitive test. Alternatively, these fragments may not be derived from peptide regions involved in the antigenic sites recognized by this antiserum. This result was confirmed for the unfractionated and fractionated CEA tryptic peptides. The unfractionated tryptic fragments were injected into rabbits unconjugated and conjugated to either bovine serum albumin or methylated bovine serum albumin, but in no case was specific antibody formed against the injected antigens. Additionally, 500 /.¿g/injection(total, 3 injections) of purified CEA tryptic peptides gave no detectable antisera as judged by direct binding against their respective antigens. These negative results further suggest that tertiary structure is of paramount importance in determining CEA antigenicity. Possibly, the antigenic determinants in CEA recognized by anti-CEA are present in those portions of the molecule, which are also accessible to cleavage by trypsin. Although trypsin-cleaved CEA is not antigenically active, specific cleavage at cysteine residues in CEA does yield antigeni cally active peptides (12, 28) that are blocked at their amino termini. Structural studies on these peptides should yield meaningful information on the nature of the antigenic determinants of CEA. The success of these preliminary sequence studies holds considerable hope that CEA and CEA cross-reacting anti gens may be directly compared in terms of their protein chemistry. These studies are currently underway. ACKNOWLEDGMENTS Time (seconds) Chart 4. High-pressure liquid Chromatographie separations of PTH deriv atives. Five to 10 fil of the sample were injected onto a /¿BondapakC„ (0.04x 30-cm) column. Pan A. Program 1 separation of <1 nmol each of 16 PTH standards, except arginine (R) (6 nmol) and cysteine (C) (3 nmol). Under these conditions methionine (M) and proline (P) are not separated from valine (V). and tryptophan (W) is not separated from isoleucine (/) and leucine (L). The internal standard PTH derivative of aminoisobutyric acid is desig nated by y. Part B, Program 2 separation of P from M and V. Part C, Program 2 results for the first 6 cycles of Edman degradation performed on CEA Tryptic Peptide T2B1. D, aspartic acid; £, glutamic acid; N, asparagine; S, serine; 7, threonme; G, glycine; A, alanine; H, histidine: C?,glutamine; Y, tyrosine; F, phenylalanine; K, lysine. peptides may be that a given population of CEA molecules possesses a spectrum of specific trypsin cleavage sites that vary in their accessibility to the enzyme. One interesting and perhaps anomalous cleavage has occurred in CEA to produce peptide T1B2 with a NH2-terminal lysine. This somewhat surprising result suggests an unusual structural feature such as a cluster of 2 or more basic residues in the sequence of CEA. Antigen Studies. An important goal of this work was to AUGUST 1978 The authors are grateful to David Bills, Jaga Nath Singh Glassman, and Nancy Buker for their excellent technical help. We thank Jean Warren for performing all of the radioimmunoassays. REFERENCES 1. Aubert, J.-P., Biserte, G., and Loucheux-Lefebvre, M.-H. CarbohydratePeptide Linkage in Glycoproteins. Arch. Biochem. Biophys., 775: 410418,1976. 2. Bennett, J. Paper Chromatography and Electrophoresis, Special Proce dure for Peptide Maps. Methods Enzymol., 11: 330-339, 1967. 3. Chism, S. E.,-Bell. P. M., and Warner, N. L. Heterogeneity of CEA and CEA-like Preparations Determined by Farr Assays for Lectin Binding. J. Immunol. Methods, 13: 83-89,1976. 4. Chu, T. M., Bhargava, A. K., and Harvey, S. R. Structure Studies of the GlycoproteinsAssociatedwith Carcinoembryonic Antigen (CEA). Feder ation Proc., 33: 1562,1974. 5. Coligan, J. E., Henkart, P. A., Todd, C. W., and Terry, W. D. 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Amino-terminal Sequences of the Major Tryptic Peptides Obtained from Carcinoembryonic Antigen by Digestion with Trypsin in the Presence of Triton X-100 John E. Shively, Michael J. Kessler and Charles W. Todd Cancer Res 1978;38:2199-2208. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/8/2199 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1978 American Association for Cancer Research.