210

Biochimica et Biophysica Acta, 580 (1979) 210--215 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 31283

PARTIAL AMINO ACID SEQUENCE OF GLYCOPHORIN FROM PORCINE ERYTHROCYTE MEMBRANES

KIKUKO HONMA, MOTOWO TOMITA* and A K I RA HAMADA

School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo (Japan) (Received June 6th, 1979)

Key words: Glycophorin; Amino acid sequence; (Erythrocyte membrane)

Summary Glycophorin from porcine erythrocyte membranes was digested with trypsin and chymotrypsin. Some of the peptides were isolated by conventional techniques. The amino acid sequence was determined for two isolated peptides: a chymotryptic glycopeptide of 19 residues and a tryptic peptide of 36 residues which represented the carboxy terminal of the glycophorin.

Prior reports have noted marked similarity of major protein components of animal erythrocyte membranes when analyzed by SDS-gel electrophoresis. There was also evidence that glycophorin, the major sialoglycoprotein of erythrocyte membranes, varied greatly in apparent molecular weight, depending on species [1--3]. We were interested in structural differences in glycophorins from various animals. Glycophorin is one of the few membrane glycoproteins which are readily available and easily purified. Elucidation of amino acid sequences of several glycophorins seemed to be reasonably accessible. Indeed, the complete sequence of glycophorin A, a glycophorin of human erythrocyte membranes, has been determined [4]. In this paper we describe preliminary results of the amino acid sequence of porcine glycophorin. Porcine blood was procured at a local slaughterhouse, and anticoagulated with citrate. Erythrocyte membranes were prepared from washed cells by the method of Dodge et al. [5]. Porcine glycophorin was readily prepared by the method of Marchesi and Andrews [6], which was initially developed for the preparation of glycophorin A. Approximately 30 mg of the glycophorin was obtained from 1 g of lyophilized membrane. The glycophorin mi*To

whom correspondence should

be addressed.

211 grated as a single band having an apparent molecular weight of 53 000 when analyzed by SDS-gel electrophoresis. Chemical and physical properties of our preparations were consistent with those of porcine glycophorin prepared from Yorkshire and Duroc breeds by Fujita and Cleve [7]. Porcine glycophorin (313 mg) was digested with 3.0 mg of ~-chymotrypsin in 40 ml of 0.05 M Tris-HC1 buffer, pH 8.2, at 37°C for 24 h. The incubation mixture was directly lyophilized, dissolved in 5 ml of 0.1 M ammonium bicarbonate, and applied to a Sephacryl $200 column (2.5 X 150 cm) previously equilibrated with 0.1 M ammonium bicarbonate. Fig. 1A shows a typical elution profile. The material was collected in five major fractions, labeled as shown in the figure. Fractions A (77 mg), B (99 mg), C (63 mg) and D (24 mg) were obtained from 313 mg of glycophorin. The weight of the four fractions totaled 263 mg (84% recovery). Fraction E was eliminated from the recovery calculation, because it contained large amounts of salts such as Tris-HC1. Fraction A contained hydrophobic peptides derived from the hydrophobic segment of the glycophorin, and fraction Bcontained a major glycopeptide derived from the amino terminal segment. The amino

""

i

I.C

A O.

CH2

H2B 0.4

40

160

80

0.2 ~

120

140

B

g

T21~ T2C

--r z

I,

I,C

~.4

0.2

,

60 80 I00 120 FRACTION NUMBER

,

140

,

,

160

20 40 60 80 FRACTION NUMBER

I00

120

Fig. 1. F r a c t i o n a t i o n o f c h y m o t r y p t i c ( A ) a n d t r y p t i c (B) p e p t i d e s f r o m p o r c i n e g l y c o p h o r i n . P e p t i d e s w e r e a p p l i e d t o a S e p h a c r y l $ 2 0 0 c o l u m n ( 2 . 5 × 1 5 0 c m ) w h i c h w a s p r e v i o u s l y equilibrated w i t h 0.1 M a m m o n i u m b i c a r b o n a t e . T h e c o l u m n w a s e l u t e d w i t h t h e s a m e b u f f e r a t a f l o w r a t e o f 12 m l / h , and 5 . 0 - m l f r a c t i o n s w e r e c o l l e c t e d . F r a c t i o n s w e r e a n a l y z e d b y a b s o r b a n c e a t 2 2 6 rim. ( A ) C h y m o t r y p t i c digest o f 3 1 3 m g o f g l y c o p h o r i n w a s l o a d e d . (B) T r y p t i c digest o f 3 5 0 m g o f g l y c o p h o r i n w a s l o a d e d . Fig. 2 P u r i f i c a t i o n o f c h y m o t r y p t i c and t r y p t i c p e p t i d e s b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y . ( A ) F r a c t i o n C ( 2 0 r a g ) o f Fig. 1 A w a s a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n ( 1 . 0 X 2 0 c m ) e q u i l i b r a t e d with 0.01 M a m m o n i u m bicarbonate. The peptides were eluted with the same b u f f e r at a flow rate of 3 0 m l / h , and 6 . 0 - m l f r a c t i o n s w e r e c o l l e c t e d . A linear g r a d i e n t o f a m m o n i u m b i c a r b o n a t e ( 0 . 0 1 - - 0 . 5 M) w a s u s e d . (B) F r a c t i o n C ( 4 5 m g ) o f Fig. 1B w a s a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n ( 1 . 0 X 3 0 cm) equilibrated with 0.01 M ammonium bicarbonate. The peptides were eluted with the same buffer a t a f l o w r a t e o f 3 0 m l / h , a n d 6 . 0 - m l f r a c t i o n s w e r e c o l l e c t e d . A linear gradient o f a m m o n i u m b i c a r b o n a t e ( 0 . 0 1 - - 0 . 5 M) w a s u s e d .

212 acid sequence of the two peptides has not been determined. Three peptides were purified from fraction C (Fig. 2A). Two glycopeptides were labeled CH2A and CH2B, and a peptide was labeled CH3 as shown in the figure. Their amino acid compositions are shown in Table I. The two peptides CH2A and CH2B were identical in amino acid composition and contained a large amount of carbohydrate. The micmheterogeneity was responsible for the separation of the two glycopeptides. Peptide CH3 had no carbohydrate and was rich in Asp and Pro (Table I). The amino acid sequence of CH2B was determined for the first 17 residues (Table II) by a manual Edman procedure [8]. Thin-layer chromatography [9], gas-liquid chromatography [10] and amino acid analysis after HI hydrolysis [ 11 ] were used to identify amino acid phenylthiohydantoins. No amino acid phenylthiohydantoin was detected in the 4th, 6th or 13 th residue. The dansyl-Edman technique [12] was used to identify two of the three residues: the 4th (Asp), and the 6th (Thr), but was unsuccessful for the 13th. Simultaneous application of the direct Edman and the dansyl-Edman procedures has been used for the determination of glycosylated amino acid residues [4]. These results indicate that both residues may be glycosylated; the 4th residue N-glycosylated, and the 6th residue O-glycosylated. To determine the 13th residue and the sequence of the carboxy terminal of CH2B the peptide was digested with thermolysin after desialylation by 0.1 M sulfuric acid treatment. Two peptides were obtained from the thermolytic digest by gel filtration on a Sephadex G50 column, and labeled DCH2TH1 and DCH2TH2. Their amino acid compositions are shown in Table I. Thermolysin cleaved at just one position of CH2B, the Val-Val linkage (Table II). Since the 5th residue of DCH2TH2, Thr, could be identified only by the dansylation technique, it is probably O-glycosylated. The carboxy terminal of CH2B was determined to be Ser-Tyr by carboxypeptidase treatment [ 13]. The peptide DCH2TH2 also had Ser-Tyr as the carboxy terminal sequence. The comTABLE I AMINO ACID COMPOSITION OF PEPTIDES USED FOR ANALYSIS OF AMINO ACID SEQUENCE Numbers in parenthesis indicate mol/mol peptide assumed from sequence, and a dash r e s i d u e o r less

denotes

Residues per I 0 0 amino acids

CH2B Asp Thr Ser Glu Pro GIy

19.5 5.4 15.5 6.2 5.9

Ala 1/2 C y s

.

Val Met Ile

13.3

(4) (I) (3) (2) (I)

13.2 .

14.1 (3) .

3.2 (0)

i0.0 8.8 26.5 10.2 8.5

(1) .

13.4 (1) .

DCH2TH2

(1)

33.2 (3) -----

5.8 (1)

(i) (1) (3) (1) (I)

2.0 (0) .

CH3

.

T2BAI

(3)

18.4

(6)

13.4 10.9 19.9 15.1 3.0

(4) (3) (4.5) (4.5) (1)

11.8 9.9 16.4 20.1 2.6

(5) (4) (6.5) (6.5) (I)

.

14.9 (2)

.

T2B

13.7

.

7.6 (1)

4.3 ( I )

2.4 ( i )

7.1 ( I ) 7.0 (1)

.

12.4 (1)

--

3.5 (1)

4.4 (2)

--

8.3 (2)

5.0 (2)

5.9 (1)

Phe

. 10.3 . . .

. (2)

14.5 . . .

. (1) . . .

6.9 (1) . 9.1 (1) . . .

-.

--

7.1 (1) --

. --

. . .

(2) (2) (2) (3) (0)

8.3 (2)

---

--

2.6 (0)

13.9 14.1 16.5 23.6 0.6

7.9 (2) .

5.6 ( i )

Leu Tyr Lys His Arg Trp

DCH2THI

5.7 (1)

0.8 . . .

(0)

--

0.1

213 TABLE

II

AMINO ACID SEQUENCE OF PEPTIDES CH2B AND T2B

--~denotes residues identified as amino acid phenylthiohydantoins; (--7) denotes residues identified only by dansyl-Edmanprocedure; ~-- denotes residues identified by caxboxypeptidasetreatment. Proposed sequence of CH2B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Sequence Thr-Ile-Lys- Asn-Thr- Thr -Ala-Val-Val-Gln-Lys-Glu- Thr -Gly-Val-Pro-Glu-Ser-Ty~ (~HO (~HO (~HO CH2B --~ ~ - 7 (--~) ~ (----~) . . . . . . (--~) . . . . . . DCH2TH1 . . . . . . . . DCH2TH2 . . . . . . . . . ~-~ Proposed sequence of T2B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15' 16 17 18 Sequence Pro-Gln-Asp-Ser-Pro-Asp-Ile-Gly-Thr-Glu-Asn-Thr-Ala-Asp-Pro-Ser-Glu-LeuT2B . . . . . . . . . . . . . CH3

.

.

.

.

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Sequence Prl~-Asp-Thr-Glu-Asp-Pro-Pro-Leu-Thr-Ser-Val-Glu-Ile-Glu-Thr-Pro-Ala-Ser T2B CH3 T2BA1

~ .

.

.

.

.

.

. .

. .

.

.

.

.

.

.

.

.

.

-~-

~--~ ~ -

p l e t e s e q u e n c e o f CH2B, d e t e r m i n e d f r o m t h e a b o v e results, is s h o w n in Table II. P e p t i d e C H 2 A had the same s e q u e n c e as p e p t i d e CH2B. It is n o w evid e n t t h a t t h e d i f f e r e n c e in t h e sialic acid c o n t e n t o f t h e oligosaccharide chains caused t h e s e p a r a t i o n o f C H 2 A f r o m C H 2 B in ion e x c h a n g e c h r o m a t o g r a p h y using DEAE-cellulose (Fig. 2A). T h e sialic acid c o n t e n t o f C H 2 A was less t h a n t h a t o f CH2B (data n o t shown). T h e s e q u e n c e o f these glycop e p t i d e s is c o n s i s t e n t with the suggestion t h a t N - g l y c o s y l a t i o n occurs o n l y in t h e s e q u e n c e Asn-X-Thr (or Ser) [ 1 4 ] , b u t this is t h e first evidence t h a t Thr, in t h e N-glycosylated Asn-X-Thr, is also O-glycosylated. T h e a m i n o acid s e q u e n c e o f CH3 was d e t e r m i n e d f o r t h e first 15 residues (Table II). T h e a m i n o acid c o m p o s i t i o n and s e q u e n c e o f CH3 are similar t o t h o s e o f t h e c a r b o x y terminal s e g m e n t o f g l y c o p h o r i n A, indicating t h a t the p e p t i d e CH3 is also derived f r o m the c a r b o x y terminal s e g m e n t o f p o r c i n e glycophorin. T r y p t i c p e p t i d e s o f p o r c i n e g l y c o p h o r i n were p r e p a r e d as follows: porcine g l y c o p h o r i n ( 3 5 0 mg) was digested with 5 mg o f T P C K - t r y p s i n in 40 m l o f 0.05 M Tris-HC1 b u f f e r , p H 8.2, at 37°C f o r 24 h. T h e i n c u b a t i o n m i x t u r e was acidified t o p H 2.8 w i t h 1 N HC1. T h e p r e c i p i t a t e c o n t a i n i n g h y d r o p h obic peptides, which was derived f r o m t h e h y d r o p h o b i c s e g m e n t o f t h e glyc o p h o r i n , was r e m o v e d b y c e n t r i f u g a t i o n at 15 000 r e v . / m i n f o r 40 min. T h e soluble p e p t i d e s were initially s e p a r a t e d b y gel f i l t r a t i o n o n a S e p h a c r y l $ 2 0 0 c o l u m n (2.5 × 150 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.1 M a m m o n i u m bic a r b o n a t e . Fig. 1B shows a t y p i c a l e l u t i o n profile. Five m a j o r fractions, A--E,

214 were taken as shown in the figure. Fractions A (64 mg), B (84 mg), C (45 mg), D (31 mg) and the precipitate (87 mg) were obtained from 350 mg of glycophorin. The five fractions totaled 311 mg (89% recovery). Fraction E, containing salts such as Tris-HC1, was eliminated from the recovery calculation. Fraction A, eluted in the excluded volume of the column, contained the incomplete digests which included hydrophobic segments of the molecules. Fraction B was a mixture of glycopeptides originating in the amino terminal segment. From fraction C, three peptides were purified by ion exchange chromatography using DEAE-cellulose and labeled T2A, T2B and T2C (Fig. 2B). They were similar in amino acid composition and devoid of carbohydrate. Table I shows the composition of the largest peak, T2B. The amino acid sequence of T2B was determined for the first 17 residues (Table II). The sequence starting from 12th residue of this peptide overlaps with the amino terminal sequence of CH3. To determine the sequence of the carboxy terminal half of T2B, the peptide was treated with 70% HCOOH at 37 ° C for 44 h; this treatment selectively cleaves the Asp-Pro linkage [ 15]. A peptide labeled T2BA1 was purified from the cleavage mixture by DEAE-cellulose chromatography (elution profile n o t shown). From the amino acid composition (Table I) this peptide was estimated to comprise 13 amino acid residues. The complete sequence of T2BA1 was determined by a manual Edman procedure and by carboxypeptidase Y treatment (Table II). The amino terminal sequence of T2BA1 is identical with the sequence starting from the 13th residue of CH3 (Table II). When T2B was treated with carboxypeptidase Y, Ser was first released and followed by Ala and Pro, indicating that the peptide T2BA1 was derived from the carboxy terminal end of T2B. Thus, we can n o w construct the complete sequence of T2B by using the sequences of CH3 and T2BA1. The amino acid composition of T2B almost agrees with the complete sequence, b u t a small discrepancy between the amino acid composition obtained by amino acid analysis (Table I) and that estimated from the amino acid sequence was observed for some amino acids: Asp, Glu, Pro and Ala. The sequence heterogeneity of porcine glycophorin might explain this discrepancy since the two amino acids, Gln and Pro, were both found in the 19th position of T2B. The sequence heterogeneity may also explain w h y the three peptides, T2A, T2B and T2C, were obtained from the same segment of the glycophorin. The amino terminal sequence of T2B was also observed for peptide T2C. Since the carboxy terminal sequence of intact glycophorin was determined to be Ala-Ser, by carboxypeptidase Y treatment, peptide T2B should represent the carboxy terminal end of porcine glycophorin. We have determined, as described above, 55 residues of amino acid sequence of porcine glycophorin; 36 residues of the carboxy terminal segment and 19 residues of an internal glycosylated segment. The most interesting fact a b o u t this sequence is that the amino acid sequence of porcine glycophorin differs from that of human glycophorin (glycophorin A) more than might be expected. It is unlikely that the gene origin of porcine glycophorin does not relate to that of human glycophorin, since the sequence, Ser-Val-

215 Glu-Ile-Glu is found near the carboxy terminal of both glycophorins. A possible explanation of the difference is that the rate of sequence evolution in glycophorin molecules is faster than in other protein molecules. More information on the structure of animal glycophorins is needed to judge the plausibility of this explanation. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Hamaguchi, H. and Cleve, H. (1972) Biochem. Biophys. Res. C ommun. 47, 459--464 Tillack, T.W., Scott, E.R. and Marchesi, V.T. (1972) J. Exp. Med. 135, 1209--1227 Ko bylka, D., Khettry, A., Shin, B.C. and Carraway, K.L. (1972) Arch. Biochem. Biophys. 148, 475--487 Tomita, M., F u r t h m a y r , H. and Marchesi, V.T. (1978) Biochemistry 17, 4 7 5 6 - - 4 7 7 0 Dodge, J.T., Mitchell, C. and Hanahan, D.J. (1963) Arch. Biochem. Biophys. 100, 119--130 Marchesi, V.T. and Andrews, E.P. (1971) Science 174, 1247--1248 Fujita, S. and Cleve, H. (1975) Biochim. Biophys. Acta 406, 206--213 Peterson, J.D., Nerrlich, S., Oyer, P.E. and Steiner, D.F. (1972) J. Biol. Chem. 247, 4866--4871 Summers, M.R., Smythers, G.W. and Oroszlan, S. (1973) Anal. Biochem. 53, 624--628 Pisano, J.J., Bronzert, T. and Brewer, H.B. (1972) Anal. Biochem. 45, 43--59 Smithies, O., Gibson, D., Fanning, E.M., Goodfries, R.M., Gilman, J.G. and Ballantyne, D.H. (1971) Biochemistry 10, 4912--4921 Gray, W.R. (1972) in Methods in E n z y m o l o g y (Hirs, C.H.W. and Timasheff, S.N., eds.), Vol. 25, pp. 326--332, Academic Press, New York Hayashi, H. (1977) in Methods in E n z y m o l o g y (Hits, C.H.W. and Timasheff, S.N., eds.), Vol. 47, pp. 84--96, Academic Press, New York Spiro, R.G. (1966) in Methods in E n z y m o l o g y (Neufeld, E.F. and Ginsburg, V., eds.), Vol. 8, pp. 3--25, Academic Press, New York Omenn, G.S., Fontana, A. and Anfinsen, C.B. (1970) J. Biol. Chem. 245, 1895--1902