~ THE JOURNAL OF BIOLOGICAL CHEMlSTRV 1985 by The American Society of Biological Chemists, Inc. Vol. 26, No. 22, Issue of October 5, pp, 11944-11952 1985 Printed in ~s.a. The Oligosaccharide Moieties of the Epidermal Growth Factor Receptor in A-431 Cells PRESENCE OF COMPLEX-TYPE N-LINKED N-ACETYLGALACTOSAMINE RESIDUES* CHAINS THAT CONTAIN TERMINAL (Received for publication, April 16,1985) Richard D. CummingsS, Ann Mangelsdorf SoderquistQT, and Graham Carpenters11 From the $Department of Biochemistry, University of Georgia, Athens, Georgia 362 and the Department of Biochemistry ana! IIDiuision of DerrnatoLogy, Vanderbilt University School of Medicine, Nashuille, Tennessee 37232 The receptor for epidermal growth factor (EGF) in These data demonstrate that the complex-type Asnthe human epidermoid carcinoma cell line A-431 is a linked oligosaccharides in the EGF receptor from A- glycoprotein of apparent molecular weight = 17,. 431 cells contain sugar residues related to human blood During biosynthesis, the receptor is first detected as a type A. In light of other recent studies, these results precursor of apparent Mr = 16,. In this report we suggest that in A-431 cells blood group determinants describe our studies on the structures of the oligosac- in surface glycoproteins are contained in Asn-linked charide moieties of the mature receptor and its precur- but not O-linked oligosaccharides. sor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complextype and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosac- phosphorylation of other intracellular polypeptides (1, 8, 9). charides in both precursor and mature receptor can be Additionally, Downward et al. (1) have recently shown that cleaved by endo-b-n-acetylglucosaminidase H and oc- the human EGF receptor has amino acid sequences homolocur in the mature receptor as Man9GlcNAc2 (6%), gous to the u-erb-b transforming protein of avian erythro- MansGlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6- blastosis virus. GlcNAc2 (2%), whereas, in the receptor precursor the The realization of the central role of the receptor in cellular high mannose chains occur primarily as Mansresponses to EGF has prompted considerable research into GlcNAcz (7%). the molelcular structure of the receptor. Most studies have The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary speutilized A-431 cells, a line of human epidermoid carcinoma cies and are unusual in several respects. (i) Many of cells that have about 2 times more receptor than other cell the chains do not contain sialic acid, while the remain- lines examined (11, 12). In these cells the EGF receptor is * This work was supported by research Grants CA37626 (R. D. C.) and CAZ471 (G. C.) from the National Cancer Institute and Grant BC-294 (G.C.) from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 7 Supported by Training Grant CA9313 from the National Cancer Institute. 11 An Established Investigator of the American Heart Association. The surface receptor in human cells for the polypeptide hormone epidermal growth factor (EGFl) is a glycoprotein of apparent molecular weight = 17, (1, 2). Binding of the hormone to the receptor initiates a number of metabolic changes culminating in the induction of DNA synthesis and mitosis (3). After binding the hormone, the receptor mediates the internalization and eventual degradation of the hormone, and in this process there is a concomitant reduction or downregulation in the amount of available surface receptors (4-7). Stimulation by EGF of the tyrosine-kinase activity of the receptor causes the autophosphorylation of the receptor and ing chains contain 1-2 sialic acid residues. (ii) Half of synthesized as a precursor of apparent Mr = 16, that is the [3H]mannose-derived radioactivity was recovered converted to the mature receptor of apparent M, = 17, as [3H]fucose and the remaining half as r3h]mannose, (13, 14). indicating that there may be an average of 3 fucose The apparent molecular weight of the precursor is reduced residuesjchain. (iii) About one-third of the [3H]glucos- from 16, to 13, by treatment with endo-/3-n-acetylamine-derived radioactivity in these glycopeptides was glucosaminidase H (Endo H), an enzyme that releases certain recovered as N-acetylgalactosamine and these residues high mannose- and hybrid-type Asn-linked oligosaccharides are all a-linked and occur at the nonreducing termini. frompeptides (13,14). Likewise, treatment of A-431 cells with tunicamycin, a drug that acts to block formation of Asn- 11944 linked oligosaccharides, causes the accumulation of receptor with apparent M, = 13, (13, 14). These results suggest that the EGF receptor precursor contains a considerable number of Asn-linked oligosaccharides. The abbreviations used are: EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; Endo H, endo-8-n-acetylglucosaminidase H; ConA, concanavalin A; LDL, low density lipoprotein; HPLC, high performance liquid chromatography; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid.
Recently, Fredman et al. (15), Parker et al. (16), and Childs et al. (17) have demonstrated that the mature EGF receptor in A-431 cells is bound by antibodies reactive with oligosaccharides containing blood type A and H determinants. Since blood group determinants are commonly found on -linked oligosaccharides, these results raise the possibility that the mature receptor contains -linked chains. However, Childs et al. (17) found that the apparent molecular weight of radiolabeled glycopeptides from the receptor was not reduced by treatment with mild base, suggesting that the receptor lacks -linked sugar chains. In the present study we have examined the oligosaccharide moieties in purified EGF receptor from A-431 cells. We have found that about two-thirds of the 1-12 Asn-linked oligosaccharides are complex-type, while the remaining one-third are high mannose-type. Our experiments indicate that the receptor contains significant quantities of N-acetylgalactosamine, which is commonly associated with -linked oligosaccharides. However, all N-acetylgalactosamine residues in the EGF receptor are contained in the nonreducing termini of complextype Asn-linked oligosaccharides, and the receptor does not contain sugar chains linked through N-acetylgalactosamine to either serine or threonine residues. The presence of a- linked N-acetylgalactosamine residues at the nonreducing termini of the Asn-linked oligosaccharides may explain the reactivity of antibodies to blood group determinants on the EGF receptor in A-431 cells. EXPERIMENTAL PROCEDURES Materials-Sephadex G-25, quaternary (QAE)-Sephadex, Dowex 1 (formate), Dowex 5-H+, sodium borohydride, bovine epididymal a- L-fucosidase, Vibrio cholera neuraminidase and Amberlite MB-3 were obtained from Sigma. ConA-Sepharose was purchased from Pharmacia Fine Chemicals. Lentil lectin-sepharose was prepared as described previously (18). Bio-Gel P-1 was obtained from Bio-Rad. Pronase was purchased from Calbiochem-Behring and a-n-acetylgalactosaminidase (C. lampus) and endo--n-acetylglucosaminidase H (S. griseus) were obtained from Miles Laboratories Inc. a-man- nosidase, @-N-acetylglucosaminidase, and @-galactosidase were prepared from jack bean meal by the method of Li and Li (19). E. freundii endo-p-galactosidase was kindly supplied by Dr. Minoru Fukuda (La Jolla Cancer Research Foundation, La Jolla, CA). Standard N-acetyl- [14C]galactosamine was prepared by acetylation of the radioactive EGF Receptor Glycosylation 11945 fied EGF Receptor-The radiolabeled bands corresponding to the EGF receptor of either M, = 17, (mature form) or M, = 16, (precursor form) were excised from the dried polyacrylamide gels and incubated at 6 "C for 3 h in 1 ml of.1 M Tris, ph 8., containing 1 mm CaClz and 1 mg of Pronase (25). After incubation, 3 ml of water was added, the sample was boiled for 1 min, and the supernatant was removed. An additional 3 ml of water was added to the gel, the mixture was again boiled, and the supernatant removed. The pooled supernatants were evaporated to 1 ml, and this was applied directly to columns of ConA-Sepharose. Column Chromatography-Radiolabeled glycopeptides were applied to 1-ml columns of ConA-Sepharose or lentil lectin-sepharose, and 2-ml fractions were collected as described previously (26, 27). Chromatography of glycopeptides on Bio-Gel P-1 was conducted on a 1.5 X 5-cm column equilibrated in.1 M NH4HC3, and 1-ml fractions were collected. Glycopeptides were desalted and separated from monosaccharides on 1 X 5-cm columns of Sephadex G-25 in 7% 1-propanol. High mannose-type oligosaccharides released by endo-p-n-acetylglucosaminidase H were separated by amine adsorption high performance liquid chromatography (HPLC) on a Beckman Model lloa dual pump system using a MicroPak AX-5 column (Varian) as described previously (28). Paper Chromatography-Descending paper chromatography of oligosaccharides was performed for 4&72 h on Whatman No. 1 filter paper sheets in solvent A, ethyl acetate:pyridine:acetic acidwater (5:5:1:3). Mannose and fucose were separated by descending paper chromatography in solvent B, ethyl acetate:pyridine:water (82:l). N- Acetylgalactosamine and N-acetylglucosamine were separated by descending paper chromatography on borate-impregnated paper using solvent C, 1-butankpyridine:water (6:43) (29). N-Acetylneuraminic acid was analyzed by descending paper chromatography in solvent A. Acid Hydrolysis of Glycopeptides-Glycopeptides were hydrolyzed in 2 N HCl for 4 h at 1 "C in a final volume of.2 ml. The acid was removed by evaporation. When mannose-labeled glycopeptides were used, the dried hydrolysate was resuspended in water and spotted directly for paper chromatography. When glucosamine-labeled glycopeptides were hydrolyzed, the released sugars were reacetylated with acetic anhydride and then desalted by passage over a column of Amberlite mixed bed resin (2). The column was washed with 5 ml of water, the eluate containing the acetylated sugars was dried by evaporation under reduced pressure, and the sugars were suspended in water and spotted for paper chromatography. Conditions for Release of Oligosaccharides from Glycopeptides by Mild Alkaline-Borohydride Treatment-Glycopeptides were treated with 1 M NaB& in 5 mm NaOH for 16 h at 45 "C as described (2). Sodium was removed by passing the incubation mixture over a column of Dowex 5-H+. The borate was removed by repeatedly evaporating the sample with methanol containing.1 M acetic acid. The treated amino sugar (2). The standards, N-a~etyl-[~H]glucosamine and N- a~etyl-['~c]neuraminic acid were prepared by hydrolysis of UDP-Na~etyl-[~H]glucosamine and CMP-N-a~etyl-['~C]neuraminic acid. sample was applied directly to a Bio-Gel P-1 column. QAE-Sephadex The sugar alcohols of N-a~etyl-['~C]galactosamine and N-a~etyl-[~H] column chromatography was performed in 1-ml columns of resin as glucosamine were prepared by reduction with NaBH4 (21). Standard described previously (3). [3H]mannose-labeled high mannose-type oligosaccharides were pre- Glycosidase Treatments-Radiolabeled glycopeptides were treated pared as described previously (22). with 1 milliunits of endo-p-n-acetylglucosaminidase H in 5 pl of Antibody Preparations-Antiserum to the EGF receptor was pre-.1 M citrate-phosphate buffer at 37 "C for 48 h in a toluene atmospared by immunizing a rabbit with EGF receptor purified by affinity phere. Glycopeptides were treated with 1 milliunits of E, freundii chromatography on EGF-agarose (23). endo-@-galactosidase in 4 pl of.1 M sodium acetate buffer at ph Cell Culture, Metabolic Labeling of EGF Receptor, and Immunopre- 5.6 containing 1 mg/ml bovine serum albumin at 37 "C for 48 h in a cipitation-a-431 cells were grown for either 1 h (to label the receptor toluene atmosphere. Exoglycosidase treatments were conducted for precursor) or 24 h (to label the mature receptor) in complete Dulbec- 48 h in 4 pl of.1 M citrate, ph 4.6, in a toluene atmosphere, as co's modified Eagle's medium containing 1% calf serum, garamycin described previously (26). The amounts of activity employed were (5 pg/ml) and either [35S]methionine, 2-[3H]mannose, or 6-[3H] 15 milliunits of a-mannosidase, 5 milliunits of @-galactosidase, 1 glucosamine as described previously (14). After incubation, cells were milliunits of P-N-acetylglucosaminidase, 1 milliunits of neuraminisolubilized at 2 X lo6 cells/ml for 2 min in 1% Triton X-1, 2 mm HEPES, ph 7.4, 1% glycerol, and 1 mm phenylmethylsulfonyl dase, 5 milliunits of a-l-fucosidase, and 1 milliunits of cu-n-acetylfluoride. To each 1 pl of cell extract, 1 rl of antireceptor antiserum galactosaminidase. To determine the release of radioactive sugars was added and incubated 15 min at room temperature. After incuba- after exoglycosidase treatment, the treated glycopeptides were anation, 1 pl of a 1% suspension of fixed Staphylococcus aureus cells lyzed by descending paper chromatography in either solvent B or C, were added and incubated 15 min at room temperature. The mixture as described previously (26). was centrifuged and the pellet was washed in buffer. The immuno- Desialylation of Glycopeptides by Mild Acid Treatment-Radiolaprecipitates were heated in Laemmli buffer (24) and electrophoresed beled glycopepkkjes were desialylated by treatment with either 2 N in 7.5% sodium dodecyl sulfate-polyacrylamide gels. The dried gels acetic acid at 1 "@$g 1 h or.1 M HC1 at 8 "C for 1 h. The acids were then fluorographed to locate the radiolabeled receptor bands as were removed by evapo&a$on under reduced pressure. The released described previously (14). Preparation of Radiolabeled Glycopeptides by Pronase Digestion of SDS-Polyacrylamide Gel Electrophoresis Slices Containing the Purisugar was analyzed by desckding paper chromatography in solvent A. In other cases the treated glycopeptides were analyzed directly by chromatography on QAE-Sephadex.
11946 Glycosylution EGF Receptor RESULTS Radiolabeling of the EGF Receptor with f5s]methionine, 2- PHIMannose and 6-fH]Glucosamine-Numerous studies have shown that Asn-linked oligosaccharides, but not the typical Ser/Thr-linked oligosaccharides, in animal cell glycoproteins contain mannose residues and thathey can, therefore, be biosynthetically radiolabeled with 2- [3H]mannose (22). In contrast, the amino sugar residues in both Asn- and Ser/Thr-linked oligosaccharides can be biosynthetically radiolabeled by culturing cells in complete medium containing 6-[3H]gluc~~amine (25). We have already shown that when A-431 cells are grown for 24 h in the presence of glucose and 6-[3H]glucosamine, the primary radiolabeled sugar residues in glycoproteins are N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and that these residues appear to be labeled to similar specific activities (25). To radiolabel the sugar chains of the EGF receptor, A-431 cells were grown for 24 h in media containing either 2-[3H] mannose or 6-[3H]glucosamine. In a similar experiment cells were grown in the presence of [35S]methionine. The mature EGF receptors in the cell extracts were precipitated with anti- EGF receptor antibody and the immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. A single major protein of apparent M, = 17, was radiolabeled by all three precursors, as shown in our recent report (14). ConA-Sepharose Column Chromatography of Radiolabeled Glycopeptides-The sections of gel containing the [3H]mannose- or [3H]glucosamine-labeled receptors were removed and glycopeptides were prepared by digesting the samples directly with Pronase, as described under Experimental Procedures. Approximately 55 cpm of [3H]mannose-labeled and 7 cpm of [3H]glucosamine-labeled glycopeptides were obtained after this treatment with an estimated recovery of at least 8%. The radiolabeled glycopeptides were applied directly to columns of ConA-Sepharose. Previous experiments have shown that ConA-Sepharose does not interact with high affinity with glycopeptides containing either Ser/Thr-linked oligosaccharides or tri- or tetraantennary complex-type Asnlinked oligosaccharides (26, 31, 32). However, biantennary complex-type Asn-linked oligosaccharides bind to the immobilized lectin and can be eluted with 1 mm a-methylglucoside. High mannose- and hybrid-type Asn-linked oligosaccharides also bind to ConA-Sepharose, and elution requires high concentrations of a-methylmannoside. When the [3H]mannoselabeled glycopeptides from the receptor were fractionated by chromatography on ConA-Sepharose, 44% of the radioactivity did not bind (peak I), whereas 12% was bound and eluted with 1 mm a-methylglucoside (peak 11) and 43% was bound and eluted with 1 mm a-methylmannoside (peak 111) (Fig. 1A). In contrast, 74% of the [3H]glucosamine-labeled glycopeptides from the receptor did not bind to ConA-Sepharose (peak I), 15% was bound and eluted with 1 mm a-methylglucoside (peak 11), and 11% was bound and eluted with 1 mm a-methylmannoside (peak 111) (Fig. 1B). In agreement with the data from Endo H treatment of the mature receptor (13, 14), these results suggest that the M, = 17, receptor contains either high mannose- or hybrid- type as well as complex-type Asn-linked oligosaccharides. These data do not, however, give any direct indication about the possible presence of -linked sugar chains. The oligosaccharideswere, therefore, further characterized in the following experiments, which examine the structures of the oligosaccharides in glycopeptide peaks I and I11 derived by ConA- Sepharose chromatography. Due to limited material, the radiolabeled glycopeptides in peak I1 were not further charac- z I O LL \ 2ooor It I11 4, 9 2 - Peak % of Number Radioactivity I 44 I1 12 43 A. 5 IO 15 FRACTION Peak Number I I1 I11 % of Radioactivitv 74 15 II IO mm a-m-man I FRACTION FIG. 1. Chromatography on ConA-Sepharose of [ Hlmannose- and [SH]glucosamine-labeled glycopeptides from the EGF receptor. The Pronase digests of the [3H]mannose- (A) and [3H]glucosamine-labeled (B) mature EGF receptors purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied directly to columns of Cod-Sepharose. Bound glycopeptides were eluted sequentially with 1 mm a-methylglucoside and 1 mm a-methylmannoside as described under Experimental Procedures. The fractions contained in peaks I, 11, and I11 were pooled and analyzed separately. terized. However, since they were bound by ConA-Sepharose and were eluted with 1 mm a-methylglucoside, they probably contain biantennary complex-type Asn-linked oligosaccharides. Analysis of the PHIMannose- and fh]glucosamine-labeled Glycopeptides in Peak III-An aliquot of the [3H]glucosaminelabeled glycopeptides that were bound with high affinity by ConA-Sepharose (peak 111, Fig. 1B) was hydrolyzed in strong acid. The released monosaccharides were reacetylated and analyzed by descending paper chromatography (Fig. 2A). All of the radioactivity recovered co-migrated with authentic N- acetylglucosamine. To examine whether these glycopeptides contained high mannose- or hybrid-type Asn-linked oligosaccharides, both the [3H]glucosamine- and [3H]mannose-labeled glycopeptides in peak I11 were treated with Endo H. This enzyme cleaves between the two N-acetylglucosamine residues in the N,N - di-n-acetylchitobiose core of certain high mannose- and hybrid-type Asn-linked oligosaccharides, thereby releasing oligosaccharides having a residue of N-acetylglucosamine at the reducing termini from GlcNAc-Asn-R (33, 34). ConA-Sepharose should bind the released oligosaccharide, but not the GlcNAc-Asn-R. Successful digestion by Endo H of a [3H] glucosamine-labeled high mannose-type Asn-linked oligosaccharide should release 5% of the radioactivity as oligosaccharide and 5% as GlcNAc-Asn-R. Greater than 5% of the B.
Gal,NAc Receptor EGF Glycosylation 11947 GlcNAc A. =A a A B. 62% 5 1 15 CM FIG. 2. Descending paper chromatography of the strong acid hydrolysates of [3H]glucosamine-labeled glycopeptides from the EGF receptor. Aliquots of (A) the [3H]glucosaminelabeled glycopeptides from peak I11 (Fig. 1B) and (B) the [3H] glucosamine-labeled glycopeptides from peak I (Fig. 1B) were hydrolyzed in 2 N HC1 for 4 h at 1 C. The released sugars were reacetylated and analyzed by descending paper chromatography as described under Experimental Procedures. The migration of standards is indicated. 5 2 25 n 49 % 6%, i ill i\ IO 2 3 4 CM FIG. 3. Separation by descending paper chromatography of [3H]mannose-labeled oligosaccharides released by Endo H treatment of glycopeptides. An aliquot of the [3H]mannose-labeled glycopeptides in peak I11 (Fig. la) was treated with Endo H, and the released oligosaccharides were separated by descending paper chromatography as described under Experimental Procedures. The migration of standards is shown. radioactivity would appear in the oligosaccharide fraction if hybrid-type chains were digested with Endo H, since hybrid oligosaccharides have additional GlcNAc residues in their Man-a1,3 branch. After Endo H treatment of [3H]glucosamine-labeled glycopeptides in peak III,49% of the radioactivity was no longer bound by ConA-Sepharose and 51% was bound and eluted with 1 mm a-methylmannoside (data not shown). This result indicates that the glycopeptides in peak I11 have high mannose-type rather than hybrid-type oligosaccharides. The size of the high mannose-type oligosaccharides released by Endo H was determined by descending paper chromatography (Fig. 3). [3H]Mannose-labeled oligosaccharides released by Endo H were passed through a column of Amberlite mixed bed ion exchange resin to which they did not bind. The oligosaccharides were then separated by descending paper chromatography into 4 major species, corresponding in migration to Man9GlcNAcl (6%), MansGlcNAcl (49%), Man7GlcNAcl (25%), and Man6GlcNAcl (2%). These experiments indicate that the mature EGF receptor contains neutral, high mannose-type Asn-linked oligosaccharides. Analysis of the ~H]Glucosamine-labeled Glycopeptides in Peak I-The radiolabeled glycopeptides not bound by ConA- Sepharose (peak I, Fig. 1, A and B) could contain either certain complex-type Asn-linked oligosaccharides, Ser/Thrlinked oligosaccharides, or a mixture of both types. To inves- tigate these possibilities, the presence and proportion of N- acetylglucosamine, N-acetylgalactosamine, and sialic acid in the peak I glycopeptides was first determined. The presence of sialic acid was investigated by treating an aliquot of the [3H]glucosamine-labeled glycopeptides with.1 M HC1 at 8 C for 1 h. The treated material was then analyzed by descending paper chromatography (Fig. 4). Twelve per cent of the radioactivity was released and co-migrated with authentic N-acetylneuraminic acid. An additional aliquot of [3H] glucosamine-labeled glycopeptides in peak I was hydrolyzed in strong acid and the released monosaccharides were reacetylated and analyzed by descending paper chromatography (Fig. 2B). Thirty-eight per cent of the radioactivity was recovered as N-acetylgalactosamine and 62% was recovered as N-acetylglucosamine. Since sialic acid is destroyed by this treatment, N-acetylgalactosamine actually accounts for 33% and N-acetylglucosamine accounts for 55% of the total radioactivity in [3H]glucosamine-labeled glycopeptides in peak I. These data demonstrate that the glycopeptides from the ma- ture receptor that are not bound by ConA-Sepharose contain significant quantities of N-acetylgalactosamine, which is commonly found in -linked oligosaccharides, as well as N-acetylglucosamine and sialic acid, which are found in both N- and O-linked oligosaccharides. To analyze whether the N-acetylgalactosamine residues were in -linked or N-linked oligosaccharides, an aliquot of the [3H]glucosamine-labeled glycopeptides in peak I was applied to a column of Bio-Gel P-1 before and after treatment with.5 M NaOH containing 1 M Na13Ha, as described under Experimental Procedures. The untreated glycopeptides were slightly included in the column and eluted as a single symmetrical peak. Treatment of glycopeptides containing - linked oligosaccharides with NaOH/NaBH4 causes the cleavage of the -linked oligosaccharides from peptide via,8- elimination, with concomitant reduction and protection of the reducing terminus (35). Thus a glycopeptide containing - linked oligosaccharides should appear larger in apparent molecular weight than the base-released oligosaccharide, as was observed in studies on the LDL receptor (25). However, base- treatment of the glycopeptides from the EGF receptor did not cause any apparent shift in the elution position of the radiolabeled glycopeptides on a column of Bio-Gel P-1 (Fig. 5). These results suggest that the EGF receptor, while containing high amounts of N-acetylgalactosamine, does not contain - linked oligosaccharides. However, these results could also be..k E NeuAc IO 2 crn FIG. 4. Descending paper chromatography of the mild acidtreated [3H]glucosamine-labeled glycopeptides. An aliquot of the [3H]glucosamine-labeled glycopeptides inpeak I (Fig. 1B) was treated at ph 2. for 1 h at 1 C. The acid was removed by evaporation under reduced pressure and the treated sample was analyzed directly by descending paper chromatography. The migration position of N-acetylneuraminic acid is shown.
11948 EGF Receptor Glycosylation Fetuin Complex vo Glycopeptide Mon9G cnacl ve loor 1 z c l -BEFORE NoOH/BHi -4 AFTER NaOH/BHi IO n 2 7 14 2 25 1 mg NaCl /L A. RACTION FIG. 5. Bio-Gel P-1 column chromatography of [3H]glucosamine-labeled glycopeptides from the EGF receptor before and after treatment with NaOH/NaBHd. An aliquot of the [3H] glucosamine-labeled glycopeptides in peak I (Fig. 1B) was applied to a column of Bio-Gel P-1. An identical aliquot of material was treated with.5 M NaOH containing 1 M NaBH4 for 16 h at 45 C before chromatography on the same column, as described under Experimental Procedures. The sample was prepared and the V, and V, of the column are shown and were determined by the elution positions of bovine serum albumin and galactose, respectively. The elution positions of the complex Asn-linked oligosaccharides from fetuin and MansGlcNAcl are indicated. B. explained by the presence of very large size -linked oligosaccharides, which, even after release from peptide, still appear large in apparent molecular weight. Alternatively, the presence of a free a-carboxyl group on the serine or threonine residues containing -linked chains may prevent the P-dimination reaction (36). These possibilities, however, are ruled out in the following experiments. To assess the average degree of sialylation of the glycopeptides derived from the EGF receptor, the [3H]glucosaminelabeled glycopeptides in peak I (Fig. 1B) were applied to columns of QAE-Sephadex before and after treatment with mild acid to remove sialic acid. Bound radioactivity was eluted by increasing concentrations of salt, as described by Varki and Kornfeld (3). While uncharged glycopeptides do not bind to the resin, those glycopeptides containing one negative charge are bound and can be eluted with 2 mm NaC1. Those containing two, three, or four negative charges can be eluted with 7, 14, and 2 mm NaC1, respectively. As shown in Fig. 6, only 3% of the untreated glycopeptides did not bind to QAE-Sephadex. The amounts of bound radioactivity eluted at each salt concentration were 2 mm NaCl (29%), 7 mm NaCl (48%), and 14 mm NaCl (16%). After treatment with mild acid, the profile of unbound and eluted radioactivity on QAE-Sephadex was slightly altered (Fig. 6B). Twelve per cent of the radioactivity did not bind to the column and the amounts of bound radioactivity eluted at each salt concentration were 2 mm NaCl (56%), 7 mm NaCl (22%), and 14 mm NaCl (1%). These data demonstrate that the glycopep- tides derived from the receptor and not bound by ConA- Sepharose are not highly sialylated, consistent with the result that sialic acid constituted a small percentage of the t3h] glucosamine-derived radioactivity in these glycopeptides, as discussed above. The shift in apparent charge of the glycopeptides after mild acid treatment indicates that only about one-third to one-half of the glycopeptides contain sialic acid, and that these have no more than 1 to 2 sialic acid residues each. To determine whether all [3H]glucosamine-derived radioactivity, including that in N-acetylgalactosamine, might be present in Asn-linked oligosaccharides, an aliquot of the [3H] mannose-labeled glycopeptides in peak I (Fig. 1A) was also applied to a QAE-Sephadex column (Fig. 6C). The elution profile of the [3H]mannose-labeled glycopeptides was similar 1 2 3 4 FRACTION FIG. 6. QAE-Sephadex chromatography of [3H]glucosamine-labeled glycopeptides before and after mild acid treatment and of untreated [3H]mannose-labeled glycopeptides. A, an aliquot of the [3H]glucosamine-labeled glycopeptides in peak I (Fig. 1B) was applied to a column of QAE-Sephadex and the bound material eluted with increasing concentrations of NaCl as described under Experimental Procedures. B, a second aliquot was treated at ph 2. at 1 C for 1 h and the solvent was removed by evaporation under reduced pressure before analysis by QAE-Sephadex chromatography. C, an aliquot of the [3H]mannose-labeled glycopeptides in peak I (Fig. 1A) was applied to a column of QAE-Sephadex. to that of the untreated [3H]glucosamine-labeled glycopeptides in Fig. 6A. These data suggest that the [3H]glu~~saminederived radioactivity is contained in oligosaccharides that could also be labeled with 2-[3H]mannose, as would be expected of Asn-linked oligosaccharides. Analysis of the PHIMannose-labeled Glycopeptides in Peak I-The [3H]mannose-labeled glycopeptides not bound by ConA-Sepharose (peak I, Fig. la) were hydrolyzed in strong acid and the released monosaccharides were separated by descending paper chromatography (Fig. 7). It should be recalled that 2-[3H]mannose selectively labels only mannose and fucose residues in glycoproteins, since conversion of the precursor to any sugar other than fucose requires epimerization at C-2 with subsequent loss of radiolabel. Fifty-two per cent of the radioactivity was recovered as fucose and 48% as mannose, suggesting that the complex-type Asn-linked oligosaccharides in the EGF receptor contain large numbers of fucose residues. Since typical complex-type Asn-linked chains contain 3 mannose residues in the core structure (37, 38), these results suggest that there are about equal numbers, or 3 residues, of fucose on average per Asn-linked chain. This interpretation assumes that the fucose and mannose residues have been radiolabeled to similar specific activities, which has been shown to occur in other types of cells labeled under similar conditions (26, 27). Previous reports have shown that certain complex-type bi- and triantennary Asn-linked oligosaccharides that contain fucose residues linked a1,6 to core N-acetylglucosamine are bound by lentil lectin-sepharose (27). Tetraantennary Asn-
25r Man Fuc A ir CM FIG. 7. Descendingpaperchromatography of thestrong acid hydrolysate of [3H]mannose-labeled glycopeptides. An aliquot of the [3H]mannose-labeled glycopeptides in peak I (Fig. la) was treated with 2 N HC1 at 1 C for 4 h and the released monosaccharides were separated by descending paper chromatography. The migration positions of standard mannose and fucose are indicated. linked oligosaccharides are not bound by this immobilized lectin regardless of their fucose content (26, 27). To examine the fucose linkages in complex-type oligosaccharides of the EGF receptor, an aliquot of [3H]mannose-labeled glycopeptides in peak I (Fig. L4) was applied to a column of lentil lectin-sepharose. Approximately 7% of the radioactivity was not bound by the immobilized lectin, while the remaining 3% was bound and could be eluted with 5 mm a-methylmannoside (data not shown). These results indicate that approximately one-third of the complex-type Asn-linked oligosaccharides are triantennary-type that contain a fucose residue in the core. Exoglycosidme Treatment of PHIMannose and ph]glucosamine-labeled Glycopeptides in Peak I-Complex-type Asnlinked oligosaccharides in animal cell glycoproteins contain a common core unit composed of 3 mannose residues. Two of these residues are a-linked to a mannose residue that is 8- linked to the N,N -di-n-acetylchitobiose core (37, 38). Thus, a-mannosidase should release two-thirds of the mannose residues from [3H]mannose-labeled glycopeptides containing terminal mannose residues. However, if the a-linked mannose residues are substituted by N-acetyllactosamine, which occurs commonly in animal cell glycoproteins, then 6-N-acetylglucosaminidase and @-galactosidase in addition to a-mannosidase would be required for complete release of the a-linked mannose residues. Additional outer sugar residues, e.g. sialic acid or N-acetylgalactosamine, would require additional appropriate enzymes for complete digestion. To examine the outer sugar residues of the complex-type oligosaccharides of the receptor, this rationale was followed in the experiments below. The results are shown in Table I. No radioactivity was released as a result of a-mannosidase treatment of the [3H]mannose-labeled glycopeptides in peak I from Fig. la. The combination of @-N-acetylglucosaminidase, @-galactosidase, and a-mannosidase caused the release of only 4% of the radioactivity. The inclusion of neuraminidase in this mixture enhanced the release of radioactivity to 21%. These results suggest that some of the glycopeptides may contain the common sequence sialic acid-ga1actose-nacetylglucosamine-mannose. a-fucosidase treatment alone released 19% of the radioactivity as fucose, and the combi- nation of all of the exoglycosidases above allowed the release of 4% of the radioactivity. However, when a-n-acetylgalactosaminidase was included in the mixture of exoglycosidases, 86% of the radioacivity in the glycopeptides was released as mannose and fucose. This release of radioactivity is close to the maximal amount expected if all fucose residues and two- Glycosylation EGF Receptor 11949 TABLE I Exoglycosidase digestions of [3H]manmse-labeled EGF receptor glycopeptides not bound by ConA-Sephurose No treatment a-mannosidase Enzyme treatment a-mannosidase + @-N-acetylglucosaminidase + p-galactosidase m-mannosidase + 8-N-acetylglucosaminidase +,&galactosidase + neuraminidase a-fucosidase a-mannosidase + @-N-acetylglucosaminidase + @-galactosidase + neuraminidase + a-fucosidase a-mannosidase + P-N-acetylglucosaminidase + 8-galactosidase + neuraminidase + a-fucosi- dase + a-n-acetylgalactosaminidase z n u 4 3 Ot \ LC&* GalNAc 4 % Radioactivity released as mannose and/ or fucose 4 6.?\ A.I IO 2 3 4 5 cm FIG. 8. Descending paper chromatography of [SH]glucosamine-labeled glycopeptides treated with either P-N-acetylglucosaminidase or a-n-acetylgalactosaminidase. Aliquots of the [3H]glucosamine-labeled glycopeptides in peak I (Fig. 1B) were A. B. 21 19 4 treated for 48 h with either (A) @-N-acetylglucosaminidase or (B) a- N-acetylgalactosaminidase and then analyzed by descending paper chromatography in solvent A, as described under Experimental Procedures. The migration position of N-acetylgalactosamine is indicated. thirds of the mannose residues were released. These results suggest that N-acetylgalactosamine residues are present in complex-type Asn-linked oligosaccharides of the EGF receptor. To determine if the N-acetylgalactosamine residues are present at the nonreducing termini of the Asn-linked chains, aliquots of the [3H]glucosamine-labeled glycopeptides in peak I (Fig. 1B) were treated with a-n-acetylgalactosaminidase, and the amount of radioactivity released was determined by descending paper chromatography. Another sample was treated with jack bean 6-N-acetylglucosaminidase and analyzed similarly. As shown in Fig. 8B, a-n-acetylgalactosaminidase released about 3% of the radioactivity from the glycopeptides, and the released material co-migrated with N-acetylgalactosamine. In contrast, no radioactivity was released from glycopeptides treated with @-N-acetylglucosaminidase (Fig. 8A). The amount of radioactivity released by a-nacetylgalactosaminidase corresponds well with the total percentage of radioactivity contained in N-acetylgalactosamine (33%) and indicates that all residues of N-acetylgalactosa- mine are a-linked and occur at the terminal positions in these oligosaccharides. These results also indicate that there are no terminal N-acetylglucosamine residues in these glycopeptides. 86
1195 EGF Receptor Glycosylation Additionally, since the jack bean @-N-acetylglucosaminidase is also effective against @-linked N-acetylgalactosamine residues (19), these results support the conclusion that all N- acetylgalactosamine residues are a-linked. Endo-&galactosidase Treatment of ph]glucosamine-la- beled Glycopeptides in Peak I-To assess whether the complex-type Asn-linked oligosaccharides in the mature receptor contain the repeating type 1 (Galpl,3GlcNAcpl,3) or type 2 (Gal@l,4GlcNAcpl,3) polylactosamine sequences, an aliquot of the [3H]glucosamine-labeled glycopeptides from ConA- Sepharose peak I (Fig. 1B) was treated with E. freundii endo- @-galactosidase. This enzyme cleaves these types of sequences and releases small molecular weight di- to hexasaccharides (39-41). The treated glycopeptide sample was passed over a column of Sephadex G-25 to allow recovery of any released material. However, as shown in Fig. 9, less than 5% of the radioactivity appeared to be released by this enzyme. To ensure that the enzyme was active, 6-[3H]galactose-labeled complex-type Asn-linked oligosaccharides from the mouse lymphoma cell line BW5147 were prepared and also treated with this enzyme and analyzed under identical conditions. These glycopeptides from BW5147 cells contain the type 2 polylactosamine sequence as shown previously and are sensitive to endo--galactosidase (41). Approximately 3% of the radioactivity from the BW5147 cell glycopeptides was released by endo-@-galactosidase treatment, demonstrating that the enzyme was active toward susceptible sequences (data not shown). These results indicate that the complex-type Asnlinked oligosaccharides from the mature EGF receptor in A- 431 cells contain low amounts, if any, of the polylactosamine sequences efficiently cleaved by endo-p-galactosidase. Analysis of ph]mannose-labeled Glycopeptides from the M, = 16, EGF Receptor Precursor-A-431 cells were labeled for 1 h in medium containing 2-[3H]mannose, and the radiolabeled EGF receptors were purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis, as described under Experimental Procedures. The major band detected by fluorography had an apparent M, = 16,, which corresponds to the molecular weight of the [35S]methionine-labeled 2 a 24 16 8 VO Ve 1 1 L 41 2 3 FRACTION NUMBER FIG. 9. Sephadex G-25 column chromatography of [3H]glucosamine-labeled glycopeptides from the mature EGF receptor after treatment with E. freundii endo-&galactosidase. [3H] Glucosamine-labeled glycopeptides in peak I (Fig. 1B) were treated for 48 h with endo-p-galactosidase as described under Experimental Procedures and then applied to a column of Sephadex G-25. The V, and V, were determined as described in the legend to Fig. 5. precursor (14). This band was excised, the material was digested with Pronase, and was then analyzed by chromatography on ConA-Sepharose (Fig. 1, inset). In contrast to the results obtained with the mature receptor (Fig. la), all of the [3H]mannose-labeled glycopeptides from the receptor precursor were bound by ConA-Sepharose and required 1 mm a- methylmannoside for elution. The bound glycopeptides were desalted and treated with endo-@-n-acetylglucosaminidase H and the released oligosaccharides (>go% released) were separated by HPLC. AS shown in Fig. 1, most of the released material (approximately 7%) corresponded in size to Man8GlcNAcl. DISCUSSION The current experiments indicate that the EGF receptor in A-431 cells contains both complex-type and high mannosetype Asn-linked oligosaccharides but does not contain - linked oligosaccharides. The high mannose-type oligosaccharides in the mature receptor are neutral species and are of the size ManG9GlcNAc2. Interestingly, the complex chains in the receptor contain low amounts of sialic acid, high amounts of fucose, and significant quantities of N-acetylgalactosamine in a-linkage at the nonreducing termini. Sequence analysis of cdna for the EGF receptor from A- 431 cells indicates that there are 1-12 potential glycosylation sites on what is expected to be the extracellular domain of I a-m-glc 4 z 2 loomm 5 1 15 FRACTION NUMBER 1 1 1 1 1 1 A LJ IO 2 3 4 5 6 FRACTION NUMBER FIG. 1. Separation by HPLC of [3H]mannose-labeled oligosaccharides released by Endo H treatment of the high mannose-type Asn-linked oligosaccharides from the EGF receptor precursor. A-431 cells were grown in 2-[3H]mannose for 1 h. The M, = 16, EGF receptor precursor was purified from these cells by immunoprecipitation and SDS-polyacrylamide gel electrophoresis, as described under Experimental Procedures. After Pronase digestion of the precursor, the [3H]mannose-labeled glycopeptides were applied directly to a ConA-Sepharose column, and were eluted as shown in the inset. The glycopeptides bound and eluted with a- methylmannoside were treated with Endo H and the released oligosaccharides were separated by HPLC. The elution of standards is shown.
EGF Receptor Glycosylation 11951 the receptor (42,43). Limited Endo digestion H of the receptor precursor and the overall decrease in its apparent size after exhaustive Endo H digestion, indicate that the precursor may contain approximately 11 Asn-linked oligosaccharides (13, 14). We have shown that the oligosaccharides of the receptor precursor are high mannose-type Asn-linked chains. Endo H treatment of the mature receptor causes a decrease of only 8-1 kda in its apparent size (13). Thus, if each chain is assumed to be 2 Da in size, the mature receptor would contain 4-5 high mannose-type chains. Our results from ConA-Sepharose chromatography of the receptor glycopeptides (Fig. 1A) suggest that the mature receptor contains approximately twice as many complex as high mannose-type chains. Therefore, we would expect about 7-8 complex-type Asn-linked oligosaccharides in the mature EGF receptor. Several recent reports have suggested that the oligosaccharide moieties of the EGF receptor in A-431 cells contain blood group-related sequences. Fredman et al. (15) described a monoclonal antibody prepared against A-431 cells that precipitated receptor from A-431 cells but not other cell types. They found that binding of the antibody to A-431 cell membranes was inhibited by oligosaccharides containing the sequence of H type 1 antigens. Whether binding to A-431 membranes was also inhibited by oligosaccharides containing other blood group antigens, however, was not investigated. Parker et al. (16) examined the specificity of a number of monoclonal antibodies prepared against A-431 cells. All of the antibodies were capable of precipitating the EGF receptor from A-431 cells, but not from several other types of cells, suggesting, as do the similar results of Fredman et al. (15), that the blood group antigens are not required for receptor function. Parker et al. (16) also demonstrated that anti-egf receptor antibodies agglutinated type A human erythrocytes but not type B erythrocytes. These experiments of Fredman et al. (15) and Parker et al. (16) suggest that the oligosaccharides in the mature EGF receptor in A-431 cells are structurally related to human blood types A and H. Human blood types A and H are specified by terminal a-linked N-acetylgalactosamine residues and terminal a-linked fucose residues, respectively. Our demonstration that the complex-type chains in the mature EGF receptor contain terminal a-linked N- acetylgalactosamine and high amounts of fucose residues is consistent with and may partly explain these observations. The data from Fredman et al. (15) and Parker et al. (16) do not give any insight into whether the blood group-related sequences of the receptor are in N- or -linked oligosaccharides. However, Childs et al. (17) studied the binding of a series of previously characterized monoclonal antibodies to purified EGF receptor transferred to nitrocellulose. The receptor appeared to contain sequences related to blood type A, type 1 chain ALeb and Lea, as well as, type 2 chain unsubstituted, mono- and difucosylated antigens. Based on an examination of [3H]glucosamine- and [3H]fucose-labeled receptor glycopeptides, Childs et al. (17) concluded that the EGF receptor lacks -linked oligosaccharides, suggesting that the blood group-related sequences occurred on N-linked chains, as we have shown directly. Recent studies have demonstrated that blood group-related sugar residues can occur on Asn-linked oligosaccharides (38, 41,44-47). For example, Tsuji et al. (47) performed structural studies on the Asn-linked oligosaccharides from type A human erythrocyte band 3 glycoprotein and found that they contained polylactosamine sequences terminating in N-acetylgalactosamine and fucose residues. Similar results have been reported by Krusius et al. (48) and Jiirnefelt et al. (49) in studies on the polylactosamine-containing oligosaccharides derived from type A, B, and AB erythrocytes. Additionally, Cummings and Kornfeld (41) recently reported the presence of polylactosamine sequences containing terminal a-linked galactose residues in complex-type Asn-linked oligosaccharides from a mouse lymphoma cell line. Moreover, they observed that these polylactosamine-containing sequences were present primarily in the tri- and tetraantennary complex-type chains and not in the biantennary chains. An interesting finding in our study was that only a small percentage of the [3H]glucosamine-labeled complex-type Asnlinked oligosaccharides from the mature EGF receptor was sensitive to endo-p-galactosidase treatment. This result suggests that either a majority of the glycopeptideslack the polylactosamine sequences or that they contain unusual sugar substitutions or branching on a polylactosamine chain which prohibits cleavage by this enzyme. The refractoriness of these oligosaccharides to endo-p-galactosidase is similar to the results obtained by Childs et al. (17). However, they found that the binding of certain monoclonal antibodies directed against carbohydrate determinants was reduced after treatment of the receptor with endo-@galactosidase. Altogether, these data may indicate that only a small percentage of the carbohydrate chains in the receptor or a small percentage of receptors contain the polylactosamine sequence. The occurrence of the blood group related sequences in Asn-linked, but not -linked oligosaccharides could be due to many factors. For example, it is possible that the relative activities of the sialyltransferases and galactosyltransferases that add the terminal sugars to -linked chains influence the degree of chain elongation and complexity of these chains. This phenomenon has been observed in studies on the porcine and ovine submaxillary mucins (5-53). Ovine submaxillary glands contain high levels of CMP-sialic acidgalnac-mucin a2,6-sialyltransferase and synthesize the disaccharide sialic acid a2,6-galnac-ser(thr). However, porcine submaxillary glands contain a higher level of UDP-Ga1:GalNAc-mucin ~l,3-galactosyltransferase relative to sialyltransferase. Thus, a higher percentage of the chains in porcine mucins are galactosylated, which contributes to the eventual elongation of the chains to blood type A-related structures. Other studies have demonstrated that there are preferred reaction pathways for - and N-linked oligosaccharide biosynthesis that are dependent on the relative activities of many different glycosyltransferases (54, 55). Additionally, it is possible that some of the enzymes in A-431 cells involved in synthesizing the blood group related structures in N-linked oligosaccharides may not efficiently utilize -linked oligosaccharides as acceptors. In this regard, it is interesting to note that the low density lipoprotein (LDL) receptor in A-431 cells has 9-18 -linked oligosaccharides. All of these were found to be mono- and disialylated derivatives of the simple sequence Gal-GalNAc- Ser(Thr) (25). Thus, there are no blood group-related sequences in -linked oligosaccharides of the LDL receptor. It has not been determined whether blood group-related sequences are present in the 1-2 Asn-linked oligosaccharides of the LDL receptor in A-431 cells. However, the absence of these sequences in the -linked chains of the LDL receptor, together with the results of the present study on the EGF receptor in the same cell type, raises the possibility that blood group-related sequences in A-431 cells are found only in Asnlinked oligosaccharides. Our laboratory is currently conducting studies on A-431 cells to address this possibility. Acknowledgments-We thank Connie Moore and Lynn Shaver for
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