Complete Amino Acid Sequence of a Membrane Receptor for Glycoproteins SEQUENCE OF THE CHICKEN HEPATIC LECTIN*

(Received for publication, September 30, 1980, and in revised form, January 8, 1981)

Kurt DrickamerS From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11 724

The chicken hepatic lectin is involved in the clearance of glycoproteins from circulation (Kawasaki, T., and Ashwell, G . (1977) J Biol. Chem 252,6536-6543). The complete amino acid sequence of chicken hepatic lectin has been established by analysis of peptides generated by chemical cleavage at methionine or tryptophan residues. Larger BrCN fragments were further digested with trypsin, chymotrypsin, and clostripain. All sequences were determined by automated sequential Edman degradation. Extensive use was made of high performance liquid chromatography in the purification of peptides and identification of phenylthiohydantoin derivatives of amino acids. The complete sequence is:

10 20 A c - M e t - A s p - G l u - G l u - A r g - L e u - S e r - A s p - A s n Val-Arg-Leu-~-Lys-Gly-Gly-Ser-Ile-Arg-Gln-Gly-Leu-Arg-Ser-Phe-Ala-Ala-Val-~r-

30 40 50 Val-Leu-Leu-Ala-Leu-Ser-Phe-Leu-Leu-Leu-Thr-Leu-Leu-Ser-Ser-Val-Ser-Leu-Ala-Arg-lle-Ala-Ala-Leu-Ser-Ser-Lys-Leu-Ser-Thr-

60 Cho 70 Leu - Gln - Ser ~ Glu - Pro - Lys - His - Asn - Phe - Ser - Ser - Arg- Asp - Ser - Leu - Leu - Phe - Pro - Cys - Gly - Ala - Gln - Ser ~ Arg ~ Gln - Trp - Glu - Tyr - Phe - Glu - 90 100 110 Gly-Arg-Cys-~r-Tyr-Phe-Ser-Leu-Ser-Arg-Met-Ser-Trp-His-Lys-Ala-Lys-Ala-Glu-Cys-Glu-Glu-Met-His-Ser-His-Leu-Ile - Ile - Ile. 120 130 Asp - Ser - Tyr - Ala - Lys - Gln - Asn - Phe - Val - Met - Phe - Arg ~ Thr - Arg - Asn - Glu - Arg- Phe - Trp - Ile - Gly - Leu - Thr . Asp - Glu - Asn - Gln - Glu - Gly - Glu - 150 160 Trp - Gln - Tq - Val - Asp - Gly - Thr - Asp - T h r - Arg - Ser - Ser - Phe - T h r - Phe - Trp - Lys - Glu - Gly - Glu - Pro - Asn - Asn - Arg - Gly - Phe - Asn - Glu - Asp - cys - 180 190 A l a - H i s - V a l - T q - T h r - S e r - C l y - G l n - T q - A s n - A s p - V a l - ~ - C y s - T h r - T y r - G l u - C y s - T y r - T y r - V a l - C y s - G l u . L y ~ - ~ o - ~ ~ ~ p r o - ~ ~ ~ -

80

140

170

200

The stretch of uncharged amino acids from residue 25 to 48 is a possible membrane-interaction region. Carbo- hydrate is attached to residue 67.

Rapid transfer of partially desialated rat serum glycopro- teins from plasma to liver has been extensively studied by Ashwell and Morrell (1) and colleagues. These investigators have isolated a carbohydrate-binding protein from mamma- lian liver (2). This “lectin” appears to mediate the specific binding phase of the clearance phenomenon (3). It recognizes the terminal galactose residues exposed upon removal of sialic acid from glycoproteins.

Although this carbohydrate recognition system is quite widely distributed in mammalian species (2 ,4, 5 ) , some differ- ences are found in birds (6). The plasma of birds contains significant levels of glycoproteins whose carbohydrate moie- ties are abbreviated by the absence of sialic acid. These glycoproteins (with exposed terminal galactose residues) are not rapidly cleared from the avian circulation unless N-ace- tylglucosamine residues are uncovered by removal of galac- tose. Correspondingly, a chicken hepatic lectin, which recog-

* Indicated portions of this work were supported by Research Grants HL-06400 and GM-2747 from the National Institutes of Health and Grant GB-29334 from the National Science Foundation to Robert L. Hill, Duke University; the remainder of the study was supported by the Cancer Research Center Grant CA13106 from the National Cancer Institute to Cold Spring Harbor Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Postdoctoral Fellow of the Helen Hay Whitney Foundation.

nizes terminal N-acetylglucosamine (instead of galactose), has been identified and isolated by Kawasaki and Ashwell ( 7 ) . The size of this protein (Mr = 26,000) and its ready availability from a plentiful source made it an attractive object for struc- tural studies.

Chicken hepatic lectin is an example of that class of mem- brane receptors responsible for selective uptake of macromol- ecules by cells. Receptors in this group are specific for low density lipoproteins (€9, transferrin (9), and immunoglobulins (lo), among others. It is hoped that structural studies on chicken hepatic lectin will increase our understanding of the general process of receptor-mediated endocytosis (11).

The evidence presented in this paper documents the com- plete amino acid sequence of chicken hepatic lectin. This sequence is the first step toward elucidating the role of chicken hepatic lectin as a receptor for selective endocytosis.

EXPERIMENTAL PROCEDURES’

’ Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 3 to 21, Tables I to XXXI, and additional refer- ences) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20014. Request Document No. 80M-2044, cite author(s), and include a check or money order for $25.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

5827

5828 Sequence of Membrane Receptor

A c - M E T - A S P - G L U - G L U - A R G - L E U - S E R - A S P - A S F t V - S E R - I L E - A R G - G L N - G L Y - L E U - A R G - S E R - P H E - A L A - A L A - V A L - T Y R - IO 20

TRP-GLN-TRP-VAL-ASP-GLY-THR-ASP-THR-ARG-SER-SER-PHE-THR-PHE-~P-LYS-GLU-GLY-GLU -PRO-ASN-ASN-ARG-GLY-PHE-ASN-GLU-ASP-CYS- I50 160 I70

""22" CB-ll cI-c-I"""cI-B,,,,,, ::""n

~~~p-~x~~,""Bp-Vu""- ::"""BP,Iv,"""

I80 ALA-HIS-VAL-TRP-THR-SER-GLY-GLN.TRP-ASN-ASP-VAL-TYR-CYS-THR-TYR-GLU-CYS-TYR-TYR-VAL-CYS-GLU-LYS-PRO-LEU-PRO.L~S

190 200

CB-ll I

FIG. 1. Complete amino acid sequence of chicken hepatic lectin. -, extent of the various fragments used to construct the sequence; rzghtward arrows (+), assignment of residues by sequential Edman degradation; leftward arrows ( 4 , results of carboxypeptidase digestion.

RESULTS

Overall Sequencing Strategy

The strategy employed in the determination of the complete amino acid sequence of chicken hepatic lectin is summarized in Fig. 1. Two complete sets of fragments were isolated from the total protein: (a) those derived by BrCN cleavage at the four methionine residues, and ( b ) those resulting from BPNS2- mediated breaking of peptide bonds at the eight tryptophan residues. The purification of these fragments and subfrag- ments produced from them is summarized in Fig. 2.

BrCN Fragments-The five BrCN fragments were used as the starting point for the sequence determination. Conse- quently, the following description is divided into three parts. The Fist is devoted to the sequence of the NHp-terminal BrCN fragments CB-I and CB-V. The second concerns the two small fragments CB-I11 and CB-IV which derive from the middle of the molecule. The final section presents results for the COOH-terminal peptide, CB-11.

The abbreviations used are: BPNS, 2-(2 nitrophenylsulfenyl)-3- methyl-3"bromoindole; RCM, reduced and carboxymethylated; DNS, 5-dimethylaminonaphthalene-1-sulfonyl; CHL, chicken hepatic lec- tin.

The BrCN fragments were separated by gel fitration chro- matography on a Sephadex G-50 column (see Fig. 4). Aggre- gated fragments eluted in the void volume, followed closely by the two large fragments CB-I and CB-11. Identification of these peptides and determination of which fractions to com- bine was based on the results of dodecyl sulfate/urea poly- acrylamide gel electrophoresis (see Fig. 5). Peptides CB-111 and CB-IV were detected by the ninhydrin method. The large amount of 280-nm absorbing material which elutes with and following CB-IV is ninhydrin-negative and contains no amino acids by analysis. It is probably residual Triton X-100 in the RCM chicken hepatic lectin preparation. Peptide CB-V elutes in the included volume of the column. The compositions of these fragments are summarized in Table 1.

BPNS Fragments-The nine fragments produced by cleav- age at tryptophan residues were used to provide overlaps for the BrCN fragments, overlaps of subfragments within the larger BrCN fragments, and confirmatory sequence in certain regions. The use of fragments produced by treatment of polypeptides with BPNS has heretofore not been extensive. One reason is the fact that cleavage yields have not generally been very high. In the current work it was found advantageous to maintain the fragments in 50% acetic acid where cleavage

Sequence of Membrane Receptor RCM-CHL

1 I CB-Ill CB-IV CB-v

CHYMOTRYPSIN CHYMOTRYPSIN I I I

TRYPSIN CLOSTRIPAIN I

G-,50 , G j 2 5 , I I G - f 5 I I , CHY CHY CHY HPLC HPLC T-A T-B HPLC HPLC T-M CI-A Ins - A -B I 1

CHY.C CHY-D CI-B CI-C CI-D CI-E CI-F T.K T-L

5829

B RCM-CHL I

BPNS I

BP-1 6.50 6-25

I , BP4 BP.III/IV 6P.V

I HP:C H P : C CLOSTRIPAIN BP-VI BP-VI1 BP-VI11 BP-IX

I 2?l

BP-Ill BP-IV BP-IV CI-A CI-B

FIG. 2. Summary of strategy for preparing and purifying fragments and subfragments from RCM chicken hepatic lectin. CHL, chicken hepatic lectin; HPLC, high performance liquid chromatography.

continues to near completion during the purification proce- dures. Thus, for example, when preliminary fractionation was performed on a Sephadex G-75 column (Fig. 6A) , fragment BP-I1 was contaminated with a slightly larger fragment (pep- tide BP-11’ in Fig. 7). When the mixture was rechromato- graphed on a G-50 column several days later, most of the peptide BP-11’ had disappeared and the yield of BP-I1 in- creased. Peptide BP-11’ was probably peptides BP-I1 and BP- V still connected and containing a tryptophan residue which was derivatized but had not yet cyclized to cleave the adjacent peptide bond (12). Thus, considerable care was required to ensure that peptides isolated were ultimate cleavage products. Incompletely cleaved peptides were detectable by their grad- ual decomposition with increasing time in acid solution. This necessitated repeated chromatography of some fractions over the same column (Fig. 6, B and C ) .

After the fragments were fractionated on the series of gel fiitration columns (concluding with the Sephadex G-50 col- umn shown in Fig. 6 D ) , the smaller fragments were further purified by reversed phase chromatography (Fig. 8). Finally, peptides BP-I11 and BP-IV were separated by treatment of the mixture with clostripain followed by rechromatography on a Sephadex G-25 column (Fig. 9). The rationale for this step is discussed below (“Sequence of Residues 130 to 207”). The compositions of the BPNS fragments are given in Table 2.

Sequence of Residues 1 to 102

Generation of Subfragments-The NH2-terminal half of the chicken hepatic lectin molecule proved to be the most difficult to sequence. Of the several proteases employed to digest CB-I, only trypsin provided a complete set of peptides which could be readily purified. These fragments were sepa- rated into five pools by chromatography on a Sephadex G-25

column (Fig. 10). Three of these pools contained pure frag- ments (CB-I-T-A and CB-I-T-B and CB-I-T-M). The remain- ing two pools were each resolved by reversed phase chroma- tography (Fig. 11). The compositions of all of the fragments are given in Table 3. An extra peptide, CB-I-T-X, was also obtained, due to limited trypsin action after the arginine in position 5 in spite of the adjacent negatively charged residues (see below and Fig. 1).

Some overlaps for the trypsin cleavage sites were estab- lished using the BPNS fragments discussed above, but the majority of the alignment was based on fragments produced by chymotrypsin digestion of CB-I and intact RCM chicken hepatic lectin. Initial fractionation of the CB-I chymotrypsin peptides was on a Sephadex G-25 column (Fig. 12). One peptide (CB-I-CHY-A) was obtained directly by this method. Others were further purified by reversed phase chromatogra- phy of lower molecular weight pools (Fig. 13). The composi- tion of the peptides obtained are given in Table 4. Other chymotryptic peptides, from the COOH-terminal end of CB- I, were difficult to obtain. However, an additional peptide covering this region was produced by digestion of intact RCM chicken hepatic lectin directly with chymotrypsin. The pep- tide CHY-Ins was purified by precipitation from 20% acetic acid followed by chromatography on Sephadex G-50 in 50% acetic acid (Fig. 14). The purification was monitored by do- decyl sulfate urea polyacrylamide gel electrophoresis (Fig. 15). The composition of CHY-Ins is also given in Table 4.

Finally, CB-I was also digested with clostripain. The digest was applied to a Sephadex G-25 column (Fig. 16) and one pure fragment was obtained (CB-I-C1-A). Its composition is given in Table 5. Purification of other clostripain peptides was not pursued.

Identification of Blocked N H , Terminus-DNS end-group analysis and Edman degradation of RCM chicken hepatic

5830 Sequence of Membrane Receptor

lectin failed to produce any results, suggesting that the ter- minal amino group is blocked. A blocked peptide was isolated as follows. RCM chicken hepatic lectin was digested with chymotrypsin and the resulting ethanol-soluble peptides were passed over a Sephadex G-25 column (Fig. 17) to remove glycopeptides which otherwise would contaminate the acidic peptide fraction. Peptides eluting after the void volume were pooled and redigested with S. aureus V-8 protease. This digest was then passed over a sulfonated polystyrene column under acidic conditions to remove all peptides with free amino groups. The peptide which passed through the ion exchange column (CHY/V-I) gave no result by DNS-end group analysis and had the composition given in Table 5.

Further information about the NH2-terminal structure was obtained by analysis of peptide CB-V. This peptide yielded no peaks when applied directly to the amino acid analyzer; after hydrolysis, homoserine was the sole amino acid present. Acetate was detected by the DNS-hydrazide method. This suggests that the structure of peptide CB-V is AcHsr. Com- bined with the composition of peptide CHY/V-I, the results indicate that NHn-terminal structure of RCM chicken hepatic lectin is AcMet-(AspGlus).

Sequence of Residues I to 13-Edman degradation of intact CB-I (Table 7) and of CB-I-T-C (Table 8) provided the sequence Asp-Glu-Glu-Arg-Leu-Ser-Asp-Asn-Val-Arg-Leu- Tyr. The composition of peptide CB-I-CHY-C is exactly ac- counted for by this sequence. Further, the sequence Asp-Glu- Glu at the beginning of CB-I is consistent with the alignment of CB-I immediately following CB-V since this would account for the structure of peptide CHY/V-I discussed above. These sequence analyses therefore establish positions 2 to 13 in the overall sequence. The low initial yields in these runs indicates that peptide CB-I and its derivatives CB-I-T-C and CB-I- CHY-C are still largely blocked; the presence of methionine in these peptides (Tables 1, 3, and 4) confirms that cleavage of the NH2-terminal acetyl methionine was inefficient.

The sequence of peptide CB-I-T-X (Table 9) corresponds to the COOH-terminal portion of CB-I-T-C (residues 6 to 11). It is presumably produced by a low level of tryptic cleavage after the preceding arginine (position 5) in spite of a cluster of negative charges nearby (13).

Sequence of Residues 12 to 53-The interval from residue 24 to 48 contains no charged and very few polar residues; as a result, peptides containing this region have poor solubility characteristics. It was difficult to subfragment this region, SO

the sequence was determined by extended Edman degrada- tion. Peptide CB-I-T-A produced a very low yield of serine in the first cycle of automated Edman degradation and no readily identifiable residues in subsequent cycles. The larger frag- ments CB-I-CHY-A and CB-I-C1-A were much more readily analyzed. The results for these two peptides are given in Tables 10 and 11. The sequence Leu-Tyr at the beginning of peptide CB-I-C1-A provides overlap with the sequence dis- cussed above. This indicates that peptides CB-I-C1-A and CB-I-CHY-A begin at residues 12 and 14, respectively.

Residues 12 to 14 and 15 to 19 were confirmed by sequencing tryptic peptides CB-I-T-H and CB-I-T-G (Tables 12 and 13). Peptide CB-I-T-J, however, gave no NH2-terminal residue by DNS-end-group or Edman analysis; its composition matches residues 20 to 23. Since residue 20 is glutamine, the negative NHp-terminal result is presumably due to cyclization of this residue to form a 5-pyrrolidone-2-carboxyl residue. Also, al- though peptide CB-I-T-A was difficult to sequence directly (as discussed above), its composition matches the predicted tryptic peptide running from residue 24 to 49.

Sequence of Residues 50 to 83-The sequence in this region was established by aligning a series of short tryptic peptides

(CB-I-T-D, CB-I-T-E, CB-I-T-B, CB-I-T-F) with overlapping chymotryptic peptides (CB-I-CHY-D, CB-I-CHY-B, and CB- I-CHY-E). The results of sequence analyses of these peptides are given in Tables 14 to 20. First it was necessary to investi- gate the site of carbohydrate attachment which falls in this region. This site was localized by isolating a very short gly- copeptide from a trypsin digest of RCM chicken hepatic lectin which was redigested with thermolysin. The isolation of pep- tide T/TH-I is shown in Fig. 18. The composition of the isolated fragment (Table 5) indicates that only histidine and aspartic acid are present; DNS-end-group analysis indicates that the sequence must be His-Asx. By analogy with many other carbohydrate attachment sites (14), it was concluded that the carbohydrate must be of the “N-linked” type, bound to the asparagine residue in the sequence His-Asn.

The sequence Ile-Ala-Ala at residues 50 to 52 indicates that the next tryptic peptide after CB-I-T-A must be CB-I-T-D, as analyzed in the Edman degradation reported in Table 14. This peptide, therefore, represents residues 50 to 56. The results for peptide CB-I-CHY-D (Table 15; residues 54 to 57) extend this sequence by one residue to Leu at position 57. This means that the tryptic peptide following CB-I-T-D must begin with leucine. Only one such peptide (CB-I-T-E; Table 16) remains, so it was concluded that this peptide follows next in the sequence, running from residues 57 to 65. This is the shortest overlap (one amino acid) employed in the present structural study.

The two glycopeptides CB-I-T-B and CB-I-CHY-B follow next in the sequence. The sequence of peptide CB-I-T-B is reported in Table 17. The chymotryptic glycopeptide yielded no results on direct Edman degradation, so it was digested with 5-pyrrolidone-2-carboxylic acid amino peptidase and reanalyzed. The results of this sequence analysis (Table 18) indicate that the sequence of the original peptide must have been Gln-Ser-Glu-Pro-Lys-His-Asn-Phe. This sequence (po- sitions 61 to 68) overlaps peptides CB-I-T-E and CB-I-T-B, extending the sequence to residue 71. In the sequence of both glycopeptides the absence of an identifiable residue at the cycle corresponding to residue 67 was interpreted to indicate that this residue is the Asn-carbohydrate site.

Finally, the sequence of peptide CB-I-CHY-E (Table 19) provides the information that the next three residues (72 to 74) are Asp-Ser-Leu. This indicates that peptide CB-I-T-F (Table 21) is the next tryptic peptide in the sequence, com- pleting the identification of residues to position 83.

Sequence of Residues 75 to 202-The sequence of the remainder of CB-I plus one additional residue was established by analysis of CHY-Ins and BP-V. Although chymotryptic peptides from the COOH-terminal end of peptide CB-I were difficult to isolate, the large insoluble peptide CHY-Ins was readily purified from chymotryptic digests of intact RCM chicken hepatic lectin. The results of the sequence analysis of this peptide (Table 21) were complicated by heterogeneity in the NHn-terminal residue. Roughly one-third of the peptide has NH2-terminal leucine (corresponding to leucine at position 75) and the remainder is one amino acid shorter, beginning with phenylalanine (residue 76). In spite of this, it was possible to interpret the sequence for at least 12 residues, which bridges the gap between peptide CB-I-T-F ending at residue 83 and the beginning of peptide BP-V.

The sequence of BP-V was determined by Edman degra- dation (Table 22). The NH2 terminal sequence Glu-Tyr-Phe provides the necessary overlap with CHY-Ins (residues 86 to 88). The COOH-terminal end provides the remainder of the sequence of CB-I, plus two residues which allow alignment of the next BrCN fragment (see below).

The sequence determined for residues 84 to 99 also predicts

Sequence of Membrane Receptor 5831

three additional tryptic peptides. The compositions of these peptides exactly match the remaining tryptic peptides: CB-I- T-L (residues 84 to 91), CB-I-T-K (residues 92 through 99), and CB-I-T-M (homoserine derived from methionine at po- sition 100). Neither of the first two of these peptides gave any results by DNS-end group or Edman analysis. This is presum- ably due to cyclization of a glutamyl to 5-pyrrolidone-2-car- boxyl residue in the case of peptide CB-I-T-L. It is possible that a similar cyclization of carboxymethylcysteine at the NHz-terminal end of CB-I-T-K accounts for the negative results in this case (15).

Finally, the sequence of residues 1 to 85 accounts for the properties of peptide BP-I. The composition of the peptide matches the sequence of these residues, and the failure to obtain sequence results is explained by the presence of an acetylated methionine residue at the NHz-terminal.

Sequence of Residues 86 to 138 The sequence of residues 86 to 138 was determined by

Edman degradation of two BrCN peptides and two BPNS peptides. Almost the complete sequence of peptide CB-IV was determined in a single sequenator analysis (Table 23), while only the first 14 residues of peptide CB-I11 were readily identified (Table 24).

The alignment of these peptides was established by analysis of BPNS fragments. First, the previously discussed sequence of peptide BP-V indicates that the residue following peptide CB-I must be serine (position 101). The sequence Ser-Trp-His found at the NH2 terminus of peptide CB-IV indicates that this BrCN peptide follows CB-I. The presence of tryptophan in the second position indicates that peptide BP-V terminates at residue 102 in the complete sequence. This is consistent with the lack of an identifiable residue in cycle 17 after serine in cycle 16 of the sequenator run on peptide BP-V (Table 22).

The remaining overlaps for the BrCN fragments are pro- vided by analysis of peptide BP-11. Sequential Edman degra- dation of this peptide (Table 25) indicates that it includes the COOH-terminal 10 residues of peptide CB-IV (residues 103 to 112), all of peptide CB-I11 following immediately thereafter (residues 113 to 129), and the fist eight residues of the next BrCN peptide, CB-I1 (see below). This completes the se- quence analysis of the central part of chicken hepatic lectin.

Sequence of Residues 130 to 207

The sequence of the remainder of the protein was estab- lished by analysis of peptide CB-I1 and a complete set of subfragments generated by digestion of this peptide with clostripain. Overlaps and c o n f i a t o r y sequence were pro- vided by several BPNS fragments. The clostripain fragments were separated by gel filtration on Sephadex G-25 (Fig. 19), reversed phase chromatography (Fig. 20), and paper chro- matography (Fig. 21). The compositions of the resultant pep- tides are summarized in Table 6.

Sequence of Residues 130 to 162-Direct sequence analysis of intact CB-I1 (Table 26) allowed identification of the first 28 residues. The sequence of the fist eight residues is identical with the sequence at the COOH-terminal end of peptide BP- I1 (residues 130 to 137) which confirms that peptide CB-11, the only remaining BrCN peptide, follows CB-I11 in the se- quence. This is also consistent with the lack of homoserine in CB-I1 which means that it must be at the COOH-terminal end of chicken hepatic lectin. This sequence also accounts for several clostripain and BPNS peptides: CB-11-C1-F (residues 130 and 131), CB-11-C1-D (residues 132 and 133), CB-I-C1-E (residues 134 to 136), BP-VI (residues 139 to 150), and BP-IX (residues 151 and 152).

The sequence was extended by analysis of peptide BP-VII.

Sequential Edman degradation of this peptide (Table 27) revealed that it overlaps the sequence so far obtained, begin- ning at residue 153. In addition, it extends the sequence by several residues (Thr-Arg-Ser-Ser-Phe; positions 158 to 162).

Sequence of Residues 160 to 182-The sequence just estab- lished accounts for an additional clostripain peptide, CB-II- C1-C (residues 137 to 159). In addition, the sequence Ser-Ser- Phe at residues 160 to 162 is identical with the NHz-terminal sequence of peptide CB-11-C1-B (results of Edman degrada- tion given in Table 28). This indicates that peptide CB-11-C1- B must occupy positions 160 to 173.

The sequence was continued to residue 182 by analysis of peptide BP-IV. This analysis was complicated by the difficulty of resolving peptides BP-I11 and BP-IV. The difficulty was overcome in the following way. First the mixture was se- quenced directly, with two residues identified at each position (Table 29). Next it was observed that only one of the two peptides contains arginine, located at position 8 in the com- bined sequence. The mixture was therefore digested with clostripain and rechromatographed on Sephadex G-25 (Fig. 9). This procedure yielded three peptides: BP-111, BP-IV-C1- A, and BP-IV-C1-B. The compositions of the two clostripain subfragments are given in Table 5. The purified peptide BP- I11 was submitted to Edman degradation (Table 30), which allowed interpretation of the previous degradation of the mixture as given in Table 29. The deduced sequence of peptide BP-IV overlaps the COOH-terminal end of peptide CB-11-C1- B and, thus, extends the sequence to residue 182.

Sequence of Residues 174 to 207-Most of the remainder of the sequence was determined by Edman degradation of pep- tide CB-11-C1-A (Table 31). The COOH-terminal residues were identified by analysis of BPNS peptides and carboxy- peptidase digestion. The sequence of peptide BP-I11 (dis- cussed above; Table 30) is identical to the final 17 residues of the larger clostripain peptide plus two additional residues (Pro-Lys; residues 206 and 207). Also, the sequence of residues 184 to 188 accounts for peptide BP-VIII, the sole remaining BPNS peptide.

The COOH-terminal sequence of the lectin was confirmed by carboxypeptidase digestion. Digestion of either intact lectin or BP-I11 with carboxypeptidases B or mixed carboxypepti- dases A and B released only lysine (0.7 mol/mol of peptide after 3 h). Digestion with carboxypeptidase Y released lysine, proline, and leucine in equal amounts (0.8, 0.9, and 0.9 mol/ mol of peptides, respectively, after 3 h) in either case. These results are consistent with the proposed sequence if one as- sumes that release of proline preceeded by lysine (positions 203 and 204) is very slow with carboxypeptidase Y. This is consistent with the known activity of this enzyme (16).

DISCUSSION

Sequencing Methods-The present experiments on the pri- mary structure of chicken hepatic lectin follow standard pro- tein-sequencing strategy. However, several relatively unusual methods of generating fragments aided considerably in the sequencing work. For example, although the use of BrCN fragments in sequencing is now common, other large, chemi- cally generated fragments have not been widely employed. In the study of chicken hepatic lectins, the peptides created by specific cleavage at tryptophan residues with BPNS were an important asset. Use of this reagent allowed construction of complete sets of fragments produced by two different chemical methods, which helps to ensure that all parts of the poly- peptide are included in the final structure. The precise mech- anism of peptide cleavage by BPNS is not known and, al- though a structure has been proposed for the new COOH- terminal amino acid residue produced, based on model studies,

5832 Sequence of Membrane Receptor

the homogeneity of this product has not been demonstrated under the conditions required for protein cleavage. Hetero- geneity in this group may account for the elution of certain BPNS peptides as several peaks on reversed phase columns. Nevertheless, the peptides are very useful for automated Edman degradation, because they are of a convenient size (owing to the low frequency of tryptophan residues) and are retained well in the cup in the 0.1 M Quadrol-Polybrene method.

On the basis of the current work it would also seem that clostripain cleavage of peptides a t arginine residues is a useful alternative to trypsin cleavage after lysine modification.

An additional novel aspect of the present work is the use of high performance liquid chromatography with reversed phase columns to separate peptides. For the purification of small peptides (less than 25 amino acid residues) the technique is very powerful. The combination of preliminary gel filtration steps followed by reversed phase chromatography make pu- rification of tryptic peptides quite rapid. Peptides which have been exposed to more drastic conditions, such as those re- quired to effect chemical cleavage, tend to elute in several discrete peaks; this phenomenon is generally seen with pep- tides containing residues which may have been altered (oxi- dized or deamidated) by the harsh chemical treatment. Fi- nally, Polybrene carrier employed with the 0.1 M Quadrol program makes the spinning cup sequencer an effective tool for sequencing peptides of all sizes.

Based on the amino acid sequence just presented, the molecular weight of the peptide portion of chicken hepatic lectin is calculated to be 24,259. With the addition of carbo- hydrate of molecular weight approximately 2,000 (7), the total molecular weight of the lectin is 26,259. This is in good agreement with the value of 26,000 estimated by electropho- resis in dodecyl sulfate (7).

Chicken Hepatic Lectin and Receptor Structure-Apart from the present results on chicken hepatic lectin there is only limited information about the molecular structure of eukar- yotic membrane receptors. Some other receptors involved in endocytosis have been isolated (such as the immunoglobulin F, receptor (17) and the carbohydrate-binding receptor from mammalian liver (2)). These have not been as amenable as chicken hepatic lectin to structural studies because of their larger size or limited availability. Other receptors, including that for low density lipoproteins are well defined by binding and uptake experiments but have, so far, resisted purification.

In spite of the lack of molecular information in many systems, much has been learned about the movement of certain receptors within the cell. For example, low density lipoproteins are initially bound to a plasma membrane recep- tor. They then move to coated vesicles and eventually to lysosomes (11). A similar pathway for asialoglycoproteins has been found in mammalian liver (18). In the case of low density lipoproteins, direct evidence for the movement of the receptor itself to the membrane of coated vesicles has been obtained ( 19).

It is believed that many membrane receptors are transmem- brane molecules. This view stems from observations of recep- tor organization and movement at the cell surface which appears to be correlated with the arrangement and rearrange- ment of proteins in the underlying cytoplasm. Of particular interest is the positioning of receptors in coated pits. These regions of the plasma membrane are associated with the protein clatherin at the cytoplasmic surface of the membrane. Thus, for example, the location of low density lipoprotein receptors at the cell surface reflects organization on the cyto- plasmic side of the membrane. This strongly suggests trans- membrane linkage. Variant forms of the low density lipopro-

tein receptor have been detected which seem to be unable to conform to the usual arrangement in coated pits (20). It has been suggested that a cytoplasmic portion of this receptor is altered in such a way that it fails to interact normally with an organizing protein such as clatherin.

In another system, some evidence indicates that the recep- tor for epidermal growth factor moves to form patches on the plasma membrane only after binding ligand. This is taken to indicate that transmembrane signalling of receptor occupancy results in cytoplasmically directed movement (21).

In light of these observations, it is interesting to examine the sequence of chicken hepatic lectin given in Fig. 1 for structural features which might reflect this proposed trans- membrane orientation. A suggestion about the arrangement of the chicken hepatic lectin polypeptide in the hepatocyte membrane can be made based on the following features. (a) The sequence from residues 24 to 48 contains no amino acid residues with charged groups in their side chains; in fact, the preponderance of residues are hydrophobic. This region is thus likely to be involved in membrane interaction. ( b ) The site of carbohydrate attachment is located at residue 67; this site is presumably external to the cell since the carbohydrate is believed to face the extracellular medium. These two facts suggest that if the receptor is a transmembrane protein, the first 23 residues would likely be located in the cytoplasm. The presence of an acetylated NH2-terminal residue is consistent with a cytoplasmic location for this end of the polypeptide since most cytoplasmic proteins are acetylated at the NH:! terminus (22).

It is interesting to note that this proposed orientation of chicken hepatic lectin (NH, terminus in cytoplasm and COOH terminus ,extracellular) would be quite distinct from the ori- entation of some other membrane proteins such as erythrocyte glycophorin (23) and lymphocyte HL-A antigens (24). These proteins appear to be arranged in the reverse direction, with NHz terminal ends extracellular. On the other hand, there are other transmembrane proteins whose NH2 termini are cyto- plasmic; an example is the erythrocyte anion transport protein (25). Current suggestions about the synthesis of transmem- brane proteins based on the “signal hypothesis” (26) would not appear to provide any ready explanation for how a chicken hepatic lectin polypeptide comes to be inserted in the mem- brane.

Further Structural Studies-It is clear that additional evi- dence must be obtained to substantiate this proposal. Vectoral labeling studies with membrane-impermeable reagents such as those employed to study erythrocyte membrane proteins (25) may well provide direct information about the disposition of chicken hepatic lectin in the membrane. Other areas of chicken hepatic lectin structure which must be investigated include the assignment of disulfide bonds and the location of the carbohydrate (ligand) binding site.

Acknowledgments-The purification of chicken hepatic lectin, iso-

performed in the laboratory of Robert L. Hill, Duke University lation of many of the peptides, and preliminary sequence studies were

Medical Center; I wish to thank him for his support and guidance. I also acknowledge James D. Watson for his support of the completion of the project at Cold Spring Harbor Laboratory. Georgia Binns was an enthusiastic technical assistant. The expert secretarial work of Madelyn Nathanson is also appreciated.

REFERENCES

1. Ashwell, G., and Morell, A. G. (1974) in Aduances in Enzymology (Meister, A,, ed) Vol. 41, pp. 99-128, John Wiley & Sons, New York

2. Hudgin, R. L., Pricer, W. E., Jr., Ashwell, G., Stockert, R. J. , and Morell, A. G. (1974) J. Biol. Chern. 249,5536-5543

Sequence of Membrane Receptor 5833

3. Pricer, W. E., Jr., and Ashwell, G. (1976) J. Biol. Chem. 251,

4. Tanabe, T., Pricer, W. E., Jr., and Ashwell, G. (1979) J. Biol.

5. Baenziger, J. U., and Maynard, Y. (1980) J. Biol. Chem. 255,

6. Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A.

7. Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536-

8. Goldstein, J. L., and Brown, M. S. (1976) in Curr. Top. Cell.

9. Hemmaplardh, D., and Morgan, E. H. (1976) Biochim. Biophys

7539-7544

Chem. 254, 1038-1043

4607-4613

73, 341-343

6543

Regul. 11, 147-181

Acta 426, 385-398 10. Linden, C. D., and Roth, T. F. (1978) J. Cell Sci. 33, 317-328 11. Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1979)

12. Fontana, A. (1972) Methods Enzymol. 25, 419-423 13. Smyth, D. G. (1967) Methods Enzymol. 11, 214-231 14. Spiro, R. G. (1973) Adu. Protein Chem. 27, 349-467

Nature 279, 679-685

15. Doolittle, R. F. (1972) Methods Enzymol. 25, 231-244 16. Hayashi, R. (1977) Methods Enzymol. 4 7 , 8 4 9 3 17. Thoenes, J., and Stein, H. (1979) J. Exp. Med. 150, 1049-1066 18. Wall, D. A,, Wilson, G., and Hubbard, A. L. (1980) Cell 21, 79-93 19. Mello, R. J., Brown, M. S., Goldstein, J . L., and Anderson, R. G.

20. Anderson, R. G. W., Goldstein, J . L., and Brown, M. S. (1977)

21. Maxfield, F. R., Schlessinger, J., Schechter, Y., Pastan, I., and

22. Brown, J. L., and Roberts, W. K. (1976) J. Biol. Chem. 251,1009-

23. Tomita, M., and Marchesi, V. T. (1975) Proc. Natl. Acad. Sci. U.

24. Pober, J . S., Guild, B. C., and Strominger, J . S. (1978) Proc. Natl.

25. Drickamer, K. (1980) Ann. N. Y. Acad. Sci. 341, 419-432 26. Rothman, J . E., and Lenard, J. (1977) Science 195, 743-754 Additional references are found on p. 5834.

W. (1980) Cell 20,829-837

Nature 270,695-699

Willingham, M. C. (1978) Cell 14,805-810

1014

S. A. 72,2964-2968

Acad. Sci. U. S. A. 75,6002-6006

5834

SUPPLFIC?NTARY MATERIAL TO

COMPLETE AMINO AClO SEQUENCE OP A MEMBRANE RECEPTOR POR GLYCOPROTEINS

SEQUENCE OF THE CHICKEN HEPATIC LECTIN Kurt Dnckmcr

1 6 12)

1.n (21

2 5 (31

0.2

0.1

0 1

Sequence of Membrane Receptor

w I 2

3

5 6

5836 Sequence of Membrane Receptor

a I

2

3

a 1 2 3

I

w 1 z 3 4

5 6

2

4

2

6

8

9

9 1

2 0 I 8

I ? 5.2

3 0 2.8

1 2 1 7

0 7

0 5

0 8 1 1

HPLC IUI ' lkld

20.8 24.4 14.6

25.1 8.1

13.6 24.6

20.8

1.1

28.9 10.7

29 a 8.4 21.1

15 5 10.9 4 . 5 11.8

12 6

3 L 8.1

Sequence of Membrane Receptor

s r w m ULW a scsnm ~ Q - I I I

I Y L C 10

I s l e : 90 ml

vleld * I n ASP

53.7

16.6 V N 45.1 W I 19.2 cvs 18.9 ml 6.4 T * I GLU

27.3 25.2

cvs 9.9 I*@ WI)

19.3

V N 20.0 21.0

("I GLU 14.2

9.5

LIS' 12.0 van' LC" 4.6

1.7

QRO' 2.3

WC s9.5 m 29.1 mn 9.)

m 16.1

GLU

W . 6 m.a

m mc 67.6

13.1

IRP ILC GL" LC"

f i n

m.* 51.5 59. I

mt 69.4

ASP 25.6 15.8

GL" 38.7 AS* 36.1

un 21.0 01" GI"

32.1

GI" 50.9 16.1

mv 11.4 tl. I R V

43.9 , 5

V N 30.5 ASV

G(" 33.3 7.8

mn 15.6 LIP 9.6

IW.7 4.9

10.0

8.1

59.2

7.4

18.4 9.0

I 2 3 . 5 6 7 8 9 IO I I I2 I 3 I4 15 16 I 7 I8 I 9 20 21 22 21 21 25 26 21 28

C d t

I 2 3 . 5 6 7 8 9

I O II I 2

I 2 3 4 5 6 7 8 9 IO II I 2 I1 I 4 I 5 16 I7 I8

I 2 3 . 5 6 1 8 9

I O I I I2 I1 I 6

I 5 I 6 I 7 18 19 20 21 22 23 24

25 26 27 28 29 30 31 12

56.1

U . 6 27.2

01.7

19.2

31.0 11.2

25.9 10.0

a.0 1.7 9.6

21.6 12.6

10.6 8.8

10.1 11.5 10.4

9.0

13.0 a . 4

11.3

4 . 1

4.6

9.5 3.9

4.9 1.3

0.9 6.9

I .4

23.2 8.0

23.9 28.0 16.4

5.9

10.6

7.1

6.1

4.7 ULb I 2 1 4

5 6 7 8 9

I O II I 2 I1

c- I 2 1 1 5 6 7 8 9

I O I1 I 2 I1 II I 5 I 6

I 7 I8 I 9

t 20

t Y K+F

"vTiT6 "U 11.2

54.8 37.6

%.I 21.4

8.6 30.a 17.2 34.2 14.5

41.1 w . 9

47.9 19.8 1S.5 24.1 16.8 38.3 11.4 9.a 2.7 5.6

8P.N l l t l d =RPL(W

WU 51.8 L I S 59.0

GI" 71.8 GIU 72.8 PW 19.2 AS1 71.2 38.2 ASK 57.5 Is 11.5 G L I 25.4 mc a .1 E n 52.7 mu 57.2 36.a L I P 24.7 CIS 16.1 19.6 1u 11.1 81.6 111s 16.1 17.1 v u 25.2 10.7

IO ELUTION TIME lnln)

5838 Sequence of Membrane Receptor

c

r P E

5 IC

a

+

I 0 Do 200 300

ELUTION VOLUME lnll

I - REDIOEST

0- ELUTION VOLUME in11

100 200 300 E L U T I O N V O L U M E l m l l

Sequence of Membrane ,II.

Receptor 5839