Eur. J. Biochem. 199, 389 -393 (1991) FEBS 1991 001429569100454Z
Identification of the disulphide bonds in human platelet glycocalicin Daniel HESS
*
Johann SCHALLER ', Egon E. RICKLI and Kenneth J. CLEMETSON
Institute of Biochemistry and Theodor Kocher Institute, University of Berne, Switzerland
(Received February 13/April4, 1991) - EJB 91 0227
The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ibcc and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209 - 248 and 21 1 - 264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.
Glycoprotein Ib/IX is the major sialoglycoprotein on the platelet surface and has an essential role in primary haemostasis as the receptor for von Willebrand factor on resting platelets involved in the initial adhesion of platelets to exposed subendothelium [l - 31. Glycoprotein Ib consists of two chains a (140 kDa) and (27 kDa) joined by a disulphide bond [4]. Glycoprotein IX is tightly associated with glycoprotein Ib non-covalently in a 1 : 1 ratio [ 5 ] . The sequences of all three chains have been determined, all penetrate the membrane and contain cytoplasmic domains [6- 81. Glycocalicin consists of the extracellular part of the a-chain of glycoprotein Ib and is easily removed from the platelet surface by treatment with various proteases, in particular endogenous platelet calpain [9]. Glycoprotein contains the von Willebrand factor binding site of glycoprotein Ib/IX and also a thrombin binding site which is involved in the kinetics of the platelet response to thrombin [lo]. Glycocalicin can be considered to contain at least four recognisable domains. An N-terminal region, a region containing leucine-rich motifs similar to those found in a group of other proteins [6-8, 111, a region containing peptide sequences thought to be involved in the receptor functions [12, 131 and a region containing repeated threonine-/serine-rich motifs [6] which is highly 0-glycosylated [14, IS]. Glycocalicin contains seven cysteine residues. Two of these lie in the Nterminal region and have been shown to form a disulphidebond-linked loop while the third lies at the start of the leucinerich domain and may well have a free thiol group [16]. The four other cysteines lie in the region implicated in receptor functions. Their linkage is determined here and the structural implications for glycoprotein Ib/IX function are discussed.
a
Correspondence to K. J. Clemetson, Theodor Kocher Institute, Freiestrasse 1 , CH-3012 Berne, Switzerland
Abbreviations. Pth, phenylthiohydantoin; SBD-F, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. Enzymes. Calpain (EC 3.4.22.17); pepsin (EC 3.4.23.1); thermolysin (EC 3.4.24.4).
MATERIALS AND METHODS Pepsin was from Sigma (St. Louis, MO, USA), thermolysin was from Serva (Heidelberg, FRG) and ammonium 7fluorobenzo-2-oxa-l,3-diazolo-4-sulphonate(SBD-F) was from Dojin (Kumamoto, Japan). The reagents used for Nterminal sequence analysis were from Applied Biosystems (Foster City, CA, USA) and the reagents used for reversephase HPLC were Lichrosolv or Uvasol grade supplied by Merck (Darmstadt, FRG). All other chemicals were of analytical reagent grade, purchased either from Fluka (Buchs, Switzerland), Merck or Sigma. Glycocalicin was isolated from human platelets by a variation of an earlier method [15]. Briefly, platelets were isolated from SO buffy coats, less than 12 h after blood collection, obtained from the Central Laboratory of the Swiss Red Cross. The platelets were isolated by centrifugation and were washed twice with 12 mM sodium citrate, 30 mM glucose, 120 mM NaCl, 10 mM EDTA, pH 6.5 and once with 4.8 mM glucose, 143 mM NaCl, 6 mM Mes/NaOH pH 7.4. The platelet pellet (about 35 ml) was suspended in about 125 m14.8 mM glucose, 143 mM NaCl, 6 mM Mes/NaOH pH 7.4 and one quarter of the suspension cooled to 4°C and sonicated with a B-30 sonifier (Branson Sonic Power, Danbury, CT) for 2 min (output control 7; 50% duty cycle). The sonicated platelets were added back to the untreated platelets as a crude source of calpain, CaCl, was added to give a final concentration of 20mM, the suspension was mixed well and incubated for 30 min at 37 "C. Phenylmethylsulphonyl fluoride (end concentration 1 mM) and EDTA (end concentration 10 mM) were added to prevent further degradation. Thiol-reactive reagents, normally also added, were avoided because of the putative free thiol group in glycocalicin. After centrifugation to remove intact platelets, then cytoskeleton and fibrin (1 h, 100000 g ) , the supernatant was applied to a wheat-germ-agglutinin Sepharose 4B column (Sigma) and, after washing, the bound material was eluted with 2.5 9'0 N-acetyl-D-glucosamine, 130 mM NaC1,l mM EDTA, 20 mM Tris/HCl pH 7.6. Glycocalicin was purified and separated from the major contami-
390 nant, a soluble fragment of glycoprotein V, by chromatog- and fragments with triplets of peptides containing the remainraphy on Q-Sepharose using a 0 -0.7 NaCl gradient in 20 mM ing two disulphides (Table 1). The digest was separated by reverse-phase HPLC on a C4 column (Fig. 1b). The fluorTris/HCl pH 7.6. Cleavage with pepsin was carried out at an enzymelsub- escent fractions P,Tll, P,T119 and P,T113 were further purified strate mass ratio of 1:lOO for 24 h at 37°C in 0.01 M HCl, on a Bakerbond Diphenyl column, a Butyl Aquapore column pH 2. Subdigestion of the cysteine-containing peptic frag- or a Octyl Aquapore column, respectively. Edman degradation of the fluorescent part of fraction ments with thermolysin was performed at an enzyme/substrate mass ratio of 1 :50 for 3 h at 60°C and an additional 3 h at P,T11$8 revealed two sequences in approximately equimolar amounts. N-terminal sequence and compositional analysis 75°C in 1 mM borate, pH 6.5 containing 2 mM CaC12. Mixtures of peptides were separated first by reverse-phase indicated the presence of a tripeptide Ile3-X-Glu5 and of a HPLC on a Bakerbond C4 column (4.6 x 250 mm, wide-pore, peptide comprising Val1 5 to Asn21, thus including Cysl7. 33 nm, 5 pm; J. T. Baker Chemicals, Deventer, The Nether- Therefore Cys4 has to be connected with Cysl7. Analysis for lands) in a Hewlett Packard liquid chromatograph 1090 amino sugars by amino acid analysis revealed that Asn2l (Hewlett Packard, Waldbronn, FRG). The acetonitrile gradi- apparently carries an N-linked sugar moiety, in agreement ent systems used are described in the legends of the corre- with the results of Titani et al. [16]. The sequence of fraction P,T19@3,4 were interpreted as sponding chromatograms. For further purification a Bakerbond Diphenyl column (4.6 x 250 mm, wide-pore, 33 nm, Va1246-Gln-Cys- to Phe254, Leu208-X-Asn-Cys211 and Gly 5 pm), a Phenyl Aquapore column (2.1 x 100 mm, wide-pore, 261-Lys-Gly-Cys264-Pro-Thr. The Pth-Cysys-Pth can only 30 nm; 7 pm; Brownlee Labs, Santa Clara, CA, USA) or an be detected after both corresponding cysteines have been RP-300 Octyl Aquapore column (1 x 100 mm, wide-pore, cleaved during the Edman degradation cycle. Since in degradation steps three and four Pth-Cysys-Pth can be detected, 30 nm, 7 pm) were used in the same acetonitrile systems. Cys-containing peptides were detected according to the Cys248 has to be connected with Cys209 and Cys211 with procedure of Sueyoshi et al. [17]. The fluorescence intensities Cys264. This result could be confirmed with fraction were measured in a Perkin Elmer LS-5B spectrophotometer P,T113&3. Fraction P,Tl13@ revealed three sequences present in apwith excitation at 385 nm and emission at 515 nm. For amino acid analysis, the samples were hydrolized in proximately equimolar amounts. Their N-terminal sequence the gas phase with 6 M hydrochloric acid containing 0.1% and compositional analysis were indicative for the following (by vol.) phenol and 0.05% (by vol.) 2-mercaptoethanol for three chains: Leu208 to Cys211, Val246 to Phe254 and Val256 22 h at 115°C under vacuum. Liberated amino acids were to Thr266, c o n t a i n 3 the half-cystines 209, 211, 248 reacted with phenylisothiocyanate and the resulting phe- and 264. The Pth-Cys Cys-Pth could be detected in degradanylthiocarbomyl derivatives were analyzed by reverse-phase tion steps 3 and 9. Therefore Cys209 has to be connected with HPLC on a Nova Pak C I 8 column (4 pm, 3.9 x 150 mm; Cys248 and Cys211 with Cys264. This observation was crucial for an additional experiment. Waters, Milford, MA, USA) in a Hewlett Packard liquid chromatograph 1090 with an automatic injection system ac- Fragment P,T113@ was subjected to two degradation cycles cording to Bidlingmeyer et al. [18]; 0.14 M sodium acetate pH in the sequenator without addition of BioBrene. The se6.4 was replaced by the corresponding ammonium acetate quences Val-Gln-, Leu-X- and Val-Tyr- were detected in about equimolar amounts. The remaining fragment P,Tl13@ buffer. Disulphide-linked peptides were analyzed in an Applied (minus two positions) was recovered from the sequenator by Biosystems 477A sequenator using a programme adapted elution of the glass fiber support with aqueous trifluoroacetic from Hunkapiller et al. [19] and with a model 120A PTH acid and acetonitrile. The extract was separated on an RP300 analyzer [20]. Pth-Cys was identified after its release in the Octyl Aquapore column into fragments P,T113$8,1 and corresponding cycle as previously reported by Lu et al. [21] P,T1134,2. Analysis of P,Tll3@3,1 indicated the presence of a chain and Marti et al. [22]. Since glycocalicin had to be analyzed with intact disulphide bonds, the first cysteine of a disulphide- Asn-210-X and a second chain extending from Lys258 to linked pair is not released in its degradation cycle and is Thr266, thus including Cys264. Therefore, Cys2ll has to be therefore denoted by X. Thus, this symbol stands for a cysteine connected to Cys264. Analysis of P,Tl13@,2 indicated the presence of Cys209 residue (half-cystine) expected from the known sequence but which is not detected in the sequence analysis. Not until the and a chain from Cys248 to Phe254. Therefore, Cys209 has cysteine to which it is linked is cleaved can the corresponding to be connected to Cys248. product, Pth-Cysys-Pth, be identified in the sequenator. RESULTS Limited cleavage of glycocalicin with pepsin The peptic digest of unreduced glycocalicin yielded 18 fractions by reverse-phase HPLC on a C4 column (Fig. 1 a). Aliquots of 50 - 200 pmol of each fraction were treated with SBD-F to identify cysteine-containing peptides. The fluorescent peptic fractions P11- PI6 were pooled and further digested with thermolysin. Cleavage with thermolysin Thermolysin lysis of the pooled peptic fractions P11 -PI6 generated one pair of peptides, containing only one disulphide
DISCUSSION The results obtained here confirm the presence of a disulphide bond between cysteines 4 and 17 in the N-terminal region of glycocalicin and also that cysteine 65 in all likelihood exists as a free thiol [16]. In addition, the linkage between the remaining four cysteines has been determined to be 209 to 248 and 211 to 264, thus forming two interlinked loops one containing a total of 20 amino acids and the other a total of 40 amino acids (including the overlap). The structure formed by these loops and the loops and the sequences involved are shown in Fig. 2. In recent years there have been several studies directed at identifying the binding sites for von Willebrand factor and
391
10
20
30
40
60
50
Elution Time (min)
0.10
1 PE
3-P
P29
25
0 05
0 I
I
1
15
30
45
.
Elution Time (min)
Fig. 1. Separation of ( a ) peptic digest of unreducedglycocalicin and ( b ) thermolytic digest ofpeptic fractions PI 1 - P16 ofglycocalicin. Reversephase HPLC on Bakerbond C4 (wide-pore, 33 nrn, 5 prn, 4.6 x 250 mm), using a linear acetonitrile gradient. Solution A : 0.1% (by vol.) trifluoroacetic acid in distilled water. Solution B: 0.1 % (by vol.) trifluoroacetic acid, 80% (by vol.) acetonitrile in distilled water. Flow rate: 1 ml/min. Detection: 210 nm. Fractions pooled are indicated by horizontal bars
thrombin on glycoprotein Ib. These have been based either on cross-linked methods [23] or on inhibition studies using synthetic peptides from the sequence of glycoprotein Ibcl [12, 131. Studies on the interaction between von Willebrand factor and glycoprotein Ib are complicated by the fact that these two molecules normally do not interact and require the presence of material in subendothelial tissue (probably either collagen or proteoglycan) to which von Willebrand factor binds and thereby undergoes a conformational change allowing it to bind glycoprotein Ib. The interaction between von Willebrand factor and glycoprotein Ib can be simulated in vitro by using the antibiotic ristocetin [24] or the snake venom peptide botrocetin [25] or by using animal von Willebrand factor such as bovine or porcine or asialo human von Willebrand factor [26, 271. It is nevertheless still not clear how far these approaches really approximate to the natural interaction and, indeed, differences in potential binding sites have been observed [28]. Using 26 overlapping synthetic short (mostly 15-residue) peptides from the N-terminal 45-kDa fragment of glycoprotein Iba the region Ser251- Glu285 was identified by Vicente et al. [12] as inhibiting both ristocetin- and botrocetindependent von Willebrand factor binding to platelets. Two peptides, Ser251 -Tyr279 (29 residues) and Gly271- Glu285 (I 5 residues) were most effective. Nevertheless, the linear peptides were considerably poorer inhibitors than the native 45-kDa N-terminal tryptic fragment, particularly with
botrocetin. In addition, they showed that the negative charges a t 281 -283 and 285 were not critical. The peptide Lys231Ser245 was also inhibitory as was peptide Gln71 -Ser80 though somewhat less. Katagiri et al. [I31 examined the effect of short peptides (24 - 28 residues) on von Willebrand factor and thrombin binding and found the strongest inhibition of ristocetin-induced platelet aggregation with a peptide As11235 - Lys262. A group of overlapping peptides from Phe216-Asp274 with maximum activity in the sequence Phe216 -Thr240 inhibited platelet aggregation by thrombin. All this work points to the von Willebrand factor and thrombin receptors on glycoprotein Ib lying on and/or in the vicinity of these two disulphide-bond-formed loops. There is, however, no direct evidence that the binding sites overlap or directly interact. Many monoclonal antibodies have been described that block only the von Willebrand factor binding site but not thrombin binding. Only a few monoclonal antibodies have been described that affect both [29, 301. An additional piece of evidence that these disulphide loops are involved in von Willebrand factor binding comes from studies on pseudo-von-Willebrand's disease. In this disease the platelet glycoprotein Ib is abnormal and reacts spontaneously with normal von Willebrand factor in plasma, implying that the binding site is expressed even in the absence of ristocetin or botrocetin. The gene for glycoprotein Ibcl from a patient with pseudo-von-Willebrand's disease has been sequenced and
392 Table 1. Edman degradation data and identification of disulphide bonds in peptic, thermolytic,frugments Sequences in capital letters indicate the results of Edman degradation of unreduced peptides; X denotes a Cys residue expected from the known sequence but not observed. Asn21 could not be identified by Edman degradation due to its carbohydrate moiety. C40 indicates carbohydrate Peptide
Disulphide bond c y s -c y s
Structural data
'D \
3 IXE ~
V N C D K R (N) 17 I CHO
248 VQCDNSDK F 211
209 - 248
I
211-264
L2x: GKGCPT 264 248 VQCDNSDKF 211 LXNX
I
I
209 - 248 21 1 - 264
209 VYKYPGKGCPT 264
21 I NX
21 1 - 264
I
KYPGKGCPT 264 P,T11348,2
209 C
Fig. 2. Schematic drawing of the Ala200 - Pro280 disulphide-loop region of glycocalicin. The amino acid sequence within the double disulphide-loop region is shown. The thin superscript lines indicate peptides which have been reported to inhibit von Willebrand factor/ glycoprotein Ib binding [12,13], while the thick subscript line indicates peptides which have been reported to inhibit thrombin/glycoprotein Ib binding [13]. The large arrow indicates Gly233. Mutation of this residue to valine has been reported to be associated with spontaneous binding of glycoprotein Ib to von Willebrand factor in pseudo or 'platelet-type' von Willebrand's disease [31]
209 - 248
CDNSDKF 248
The determination of the disulphide bond linkages in this region of glycoprotein Ibcl should now enable strategies to be developed based either upon molecular biology approaches (site-directed mutagenesis, or deletions), or protein chemistry (synthesis of circular or fixed-conformation peptides) to establish definitively the binding sites of von Willebrand factor and thrombin within the disulphide-linked loop structures. The authors thank Ms Susanne Weber for technical assistance and the Swiss Red Cross Central Laboratory for providing buffy coats. This work was supported by the Swiss National Science Foundation (grants 31.26264.89 and 31.25633.88).
REFERENCES 1. Wicki, A. N. & Clemetson, K. J . (1987) Eur. J . Biochem. 163,
a single point mutation was found leading to the putative change Gly233 -+ Val [31]. A further indication that the conformation maintained by disulphide bonds is critical for von Willebrand factor comes from the fact that their reduction leads to the loss of binding between von Willebrand factor and glycoprotein Ib in asialo von Willebrand factor or botrocetin-dependent assays [ 121, though this seems to be less important for ristocetin-dependent assays or for thrombin binding [l].Putting all this information together leads to the conclusion that both loops have an important role in von Willebrand factor and thrombin binding and that they may indeed interact; also, that perhaps the smaller loop is more involved in von Willebrand factor binding, while the larger loop may be more involved in thrombin binding.
43 - 50. 2. Booth, W. J., Andrews, R. K., Castaldi, P. A,, Berndt, M. C. (1990) Platelets 1, 169-176. 3. Meyer, D. & Baumgartner, H. R. (1983) Br. J . Haematol. 54, 1-9. 4. Phillips, D. R. & Poh-Agin, P. (1977) J . Biol. Chem. 252, 2121 2126. 5. Du, X., Beutler, L., Ruan, C.-H., Castaldi, P. A. & Berndt, M. C. (1987) Blood 69, 1524-1527. 6. Lopez, J . A,, Chung, D. W., Fujikawa, K., Hagen, F. S., Papayannopoulou, T. & Roth, G. J. (1987) Proc. Nut1 Acad. Sci. U S A 84, 561 5 - 5619. 7. Lopez, J. A., Chung, D. W., Fujikawa, K., Hagen, F. S., Davie, E. W. & Roth, G. J. (1988) Proc. Nut1 Acad. Sci. U S A 85, 2135 -2139. 8. Hickey, M. J., Williams, S. A. & Roth, G. J. (1989) Proc. Nutl Acud. Sci. U S A 86, 6773 - 6777. 9. Clemetson, K. J . , Naim, H. Y. & Liischer, E. F. (1981) Proc. Nutl Acad. Sci. U S A 78,2712-2716. 10. Okumura, T. & Jamieson, G. A. (1978) Thromh. Res. 8, 701 706.
393 11. Takahashi, N., Takahashi, Y. & Putnam, F. W. (1985) Proc. N a f l Accld. Sci. U S A 82, 1906-1910. 12. Vicente, V., Houghten, R. A. & Ruggeri, Z. M. (1990) J . Biol. Chem. 265, 274-280. 13. Katagiri, Y., Hayashi, Y., Yamamoto, K., Tanoue, K., Kosaki, G. & Yamazaki, H. (1990) Thromb. Haemost. 63, 122-126. 14. Tsuji, T., Tsunehisa, S., Watanabe, Y., Yamamoto, K., Tohyama, H. & Osawa, T. (1983) J . Biol. Chern. 258, 6335-6339. 15. Korrel, S. A. M., Clemetson, K. J., van Halbeek, H., Kamerling, J . P., Sixma, J. J. &Wegenthart, J. F. G. (1984) Eur. J . Biochem. 140, 571 -576. 16. Titani, K., Takio, K., Handa, M. & Ruggeri, Z. M. (1987) Proc. Natl Acud. Sci. U S A 84, 5610-5614. 17. Sueyoshi, T., Miyata, T., Iwanaga, S., Toyo'oka, T. & Imai, K. (1985) J . Biochem. ( T o k y o ) 97,1811 -2813. 18. Bidlingmeyer, B. A , , Cohcn, S. A. & Tarvin, T. L. (1984) J . Chromatogr. 336, 93 - 104. 19. Hunkapiller, M. W., Hewick, R. M., Dreyer, W. J. & Hood, L. E. (1983) Methods Enzymol. 91, 399-413. 20. Hunkapiller, M. W. (1987) in Proteins structure and ,function (L'Italien, J., ed.) pp. 363-381, Plenum Press, New York. 21. Lu, H. S., Klein, M. L., Everett, R. R. & Lai, P.-H (1987) in Proteins structure andfunction (L'Italien, J., ed.) pp. 493-501, Plenum Press, New York.
22. Marti, T., Rosselet, S. J., Titani, K. & Walsh, K . A. (1987) Biochemistry 26, 8099 - 8109. 23. Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A. & Berndt, M. C. (1989) Biochemistry 28, 83268336. 24. Howard, M. A. & Firkin, B. G. (1971) Thrornh. Diath. Haemorrh. 25, 362-369. 25. Read, M. S., Smith, S. V., Lamb, M. A. & Brinkhous, K . M. (1989) Blood 74, 1031 -1035. 26. De Marco, L. & Shapiro, S. S. (1981) J . Clin. Invest. 68, 321 328. 27. Kirby, E. P. (1982) J . Lab. Clin. Med. 100, 963-976. 28. Andrews, R. K., Booth, W. J., Gorman, J . J., Castaldi, P. A., Berndt, M. C. (1989) Biochemistry 28, 8317-8326. 29. Kunicki, T. J., Montgomery, R. R. & Pidard, D. (1983) Blood62, 260a (abstr.) 30. Yamamoto, N., Kitagawa, H., Tanoue, K. Yamazaki, H. (1985) Thrornb. Res. 39, 751 -759. 31. Miller, J . L., Cunningham, D., Lyle, V. A,, Finch, C. N. & Basurto-Garcia, M. A. (1990) Circulation 82, 595 (abstr.)
Identification of the disulphide bonds in human platelet glycocalicin Daniel HESS
*
Johann SCHALLER ', Egon E. RICKLI and Kenneth J. CLEMETSON
Institute of Biochemistry and Theodor Kocher Institute, University of Berne, Switzerland
(Received February 13/April4, 1991) - EJB 91 0227
The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ibcc and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209 - 248 and 21 1 - 264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.
Glycoprotein Ib/IX is the major sialoglycoprotein on the platelet surface and has an essential role in primary haemostasis as the receptor for von Willebrand factor on resting platelets involved in the initial adhesion of platelets to exposed subendothelium [l - 31. Glycoprotein Ib consists of two chains a (140 kDa) and (27 kDa) joined by a disulphide bond [4]. Glycoprotein IX is tightly associated with glycoprotein Ib non-covalently in a 1 : 1 ratio [ 5 ] . The sequences of all three chains have been determined, all penetrate the membrane and contain cytoplasmic domains [6- 81. Glycocalicin consists of the extracellular part of the a-chain of glycoprotein Ib and is easily removed from the platelet surface by treatment with various proteases, in particular endogenous platelet calpain [9]. Glycoprotein contains the von Willebrand factor binding site of glycoprotein Ib/IX and also a thrombin binding site which is involved in the kinetics of the platelet response to thrombin [lo]. Glycocalicin can be considered to contain at least four recognisable domains. An N-terminal region, a region containing leucine-rich motifs similar to those found in a group of other proteins [6-8, 111, a region containing peptide sequences thought to be involved in the receptor functions [12, 131 and a region containing repeated threonine-/serine-rich motifs [6] which is highly 0-glycosylated [14, IS]. Glycocalicin contains seven cysteine residues. Two of these lie in the Nterminal region and have been shown to form a disulphidebond-linked loop while the third lies at the start of the leucinerich domain and may well have a free thiol group [16]. The four other cysteines lie in the region implicated in receptor functions. Their linkage is determined here and the structural implications for glycoprotein Ib/IX function are discussed.
a
Correspondence to K. J. Clemetson, Theodor Kocher Institute, Freiestrasse 1 , CH-3012 Berne, Switzerland
Abbreviations. Pth, phenylthiohydantoin; SBD-F, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. Enzymes. Calpain (EC 3.4.22.17); pepsin (EC 3.4.23.1); thermolysin (EC 3.4.24.4).
MATERIALS AND METHODS Pepsin was from Sigma (St. Louis, MO, USA), thermolysin was from Serva (Heidelberg, FRG) and ammonium 7fluorobenzo-2-oxa-l,3-diazolo-4-sulphonate(SBD-F) was from Dojin (Kumamoto, Japan). The reagents used for Nterminal sequence analysis were from Applied Biosystems (Foster City, CA, USA) and the reagents used for reversephase HPLC were Lichrosolv or Uvasol grade supplied by Merck (Darmstadt, FRG). All other chemicals were of analytical reagent grade, purchased either from Fluka (Buchs, Switzerland), Merck or Sigma. Glycocalicin was isolated from human platelets by a variation of an earlier method [15]. Briefly, platelets were isolated from SO buffy coats, less than 12 h after blood collection, obtained from the Central Laboratory of the Swiss Red Cross. The platelets were isolated by centrifugation and were washed twice with 12 mM sodium citrate, 30 mM glucose, 120 mM NaCl, 10 mM EDTA, pH 6.5 and once with 4.8 mM glucose, 143 mM NaCl, 6 mM Mes/NaOH pH 7.4. The platelet pellet (about 35 ml) was suspended in about 125 m14.8 mM glucose, 143 mM NaCl, 6 mM Mes/NaOH pH 7.4 and one quarter of the suspension cooled to 4°C and sonicated with a B-30 sonifier (Branson Sonic Power, Danbury, CT) for 2 min (output control 7; 50% duty cycle). The sonicated platelets were added back to the untreated platelets as a crude source of calpain, CaCl, was added to give a final concentration of 20mM, the suspension was mixed well and incubated for 30 min at 37 "C. Phenylmethylsulphonyl fluoride (end concentration 1 mM) and EDTA (end concentration 10 mM) were added to prevent further degradation. Thiol-reactive reagents, normally also added, were avoided because of the putative free thiol group in glycocalicin. After centrifugation to remove intact platelets, then cytoskeleton and fibrin (1 h, 100000 g ) , the supernatant was applied to a wheat-germ-agglutinin Sepharose 4B column (Sigma) and, after washing, the bound material was eluted with 2.5 9'0 N-acetyl-D-glucosamine, 130 mM NaC1,l mM EDTA, 20 mM Tris/HCl pH 7.6. Glycocalicin was purified and separated from the major contami-
390 nant, a soluble fragment of glycoprotein V, by chromatog- and fragments with triplets of peptides containing the remainraphy on Q-Sepharose using a 0 -0.7 NaCl gradient in 20 mM ing two disulphides (Table 1). The digest was separated by reverse-phase HPLC on a C4 column (Fig. 1b). The fluorTris/HCl pH 7.6. Cleavage with pepsin was carried out at an enzymelsub- escent fractions P,Tll, P,T119 and P,T113 were further purified strate mass ratio of 1:lOO for 24 h at 37°C in 0.01 M HCl, on a Bakerbond Diphenyl column, a Butyl Aquapore column pH 2. Subdigestion of the cysteine-containing peptic frag- or a Octyl Aquapore column, respectively. Edman degradation of the fluorescent part of fraction ments with thermolysin was performed at an enzyme/substrate mass ratio of 1 :50 for 3 h at 60°C and an additional 3 h at P,T11$8 revealed two sequences in approximately equimolar amounts. N-terminal sequence and compositional analysis 75°C in 1 mM borate, pH 6.5 containing 2 mM CaC12. Mixtures of peptides were separated first by reverse-phase indicated the presence of a tripeptide Ile3-X-Glu5 and of a HPLC on a Bakerbond C4 column (4.6 x 250 mm, wide-pore, peptide comprising Val1 5 to Asn21, thus including Cysl7. 33 nm, 5 pm; J. T. Baker Chemicals, Deventer, The Nether- Therefore Cys4 has to be connected with Cysl7. Analysis for lands) in a Hewlett Packard liquid chromatograph 1090 amino sugars by amino acid analysis revealed that Asn2l (Hewlett Packard, Waldbronn, FRG). The acetonitrile gradi- apparently carries an N-linked sugar moiety, in agreement ent systems used are described in the legends of the corre- with the results of Titani et al. [16]. The sequence of fraction P,T19@3,4 were interpreted as sponding chromatograms. For further purification a Bakerbond Diphenyl column (4.6 x 250 mm, wide-pore, 33 nm, Va1246-Gln-Cys- to Phe254, Leu208-X-Asn-Cys211 and Gly 5 pm), a Phenyl Aquapore column (2.1 x 100 mm, wide-pore, 261-Lys-Gly-Cys264-Pro-Thr. The Pth-Cysys-Pth can only 30 nm; 7 pm; Brownlee Labs, Santa Clara, CA, USA) or an be detected after both corresponding cysteines have been RP-300 Octyl Aquapore column (1 x 100 mm, wide-pore, cleaved during the Edman degradation cycle. Since in degradation steps three and four Pth-Cysys-Pth can be detected, 30 nm, 7 pm) were used in the same acetonitrile systems. Cys-containing peptides were detected according to the Cys248 has to be connected with Cys209 and Cys211 with procedure of Sueyoshi et al. [17]. The fluorescence intensities Cys264. This result could be confirmed with fraction were measured in a Perkin Elmer LS-5B spectrophotometer P,T113&3. Fraction P,Tl13@ revealed three sequences present in apwith excitation at 385 nm and emission at 515 nm. For amino acid analysis, the samples were hydrolized in proximately equimolar amounts. Their N-terminal sequence the gas phase with 6 M hydrochloric acid containing 0.1% and compositional analysis were indicative for the following (by vol.) phenol and 0.05% (by vol.) 2-mercaptoethanol for three chains: Leu208 to Cys211, Val246 to Phe254 and Val256 22 h at 115°C under vacuum. Liberated amino acids were to Thr266, c o n t a i n 3 the half-cystines 209, 211, 248 reacted with phenylisothiocyanate and the resulting phe- and 264. The Pth-Cys Cys-Pth could be detected in degradanylthiocarbomyl derivatives were analyzed by reverse-phase tion steps 3 and 9. Therefore Cys209 has to be connected with HPLC on a Nova Pak C I 8 column (4 pm, 3.9 x 150 mm; Cys248 and Cys211 with Cys264. This observation was crucial for an additional experiment. Waters, Milford, MA, USA) in a Hewlett Packard liquid chromatograph 1090 with an automatic injection system ac- Fragment P,T113@ was subjected to two degradation cycles cording to Bidlingmeyer et al. [18]; 0.14 M sodium acetate pH in the sequenator without addition of BioBrene. The se6.4 was replaced by the corresponding ammonium acetate quences Val-Gln-, Leu-X- and Val-Tyr- were detected in about equimolar amounts. The remaining fragment P,Tl13@ buffer. Disulphide-linked peptides were analyzed in an Applied (minus two positions) was recovered from the sequenator by Biosystems 477A sequenator using a programme adapted elution of the glass fiber support with aqueous trifluoroacetic from Hunkapiller et al. [19] and with a model 120A PTH acid and acetonitrile. The extract was separated on an RP300 analyzer [20]. Pth-Cys was identified after its release in the Octyl Aquapore column into fragments P,T113$8,1 and corresponding cycle as previously reported by Lu et al. [21] P,T1134,2. Analysis of P,Tll3@3,1 indicated the presence of a chain and Marti et al. [22]. Since glycocalicin had to be analyzed with intact disulphide bonds, the first cysteine of a disulphide- Asn-210-X and a second chain extending from Lys258 to linked pair is not released in its degradation cycle and is Thr266, thus including Cys264. Therefore, Cys2ll has to be therefore denoted by X. Thus, this symbol stands for a cysteine connected to Cys264. Analysis of P,Tl13@,2 indicated the presence of Cys209 residue (half-cystine) expected from the known sequence but which is not detected in the sequence analysis. Not until the and a chain from Cys248 to Phe254. Therefore, Cys209 has cysteine to which it is linked is cleaved can the corresponding to be connected to Cys248. product, Pth-Cysys-Pth, be identified in the sequenator. RESULTS Limited cleavage of glycocalicin with pepsin The peptic digest of unreduced glycocalicin yielded 18 fractions by reverse-phase HPLC on a C4 column (Fig. 1 a). Aliquots of 50 - 200 pmol of each fraction were treated with SBD-F to identify cysteine-containing peptides. The fluorescent peptic fractions P11- PI6 were pooled and further digested with thermolysin. Cleavage with thermolysin Thermolysin lysis of the pooled peptic fractions P11 -PI6 generated one pair of peptides, containing only one disulphide
DISCUSSION The results obtained here confirm the presence of a disulphide bond between cysteines 4 and 17 in the N-terminal region of glycocalicin and also that cysteine 65 in all likelihood exists as a free thiol [16]. In addition, the linkage between the remaining four cysteines has been determined to be 209 to 248 and 211 to 264, thus forming two interlinked loops one containing a total of 20 amino acids and the other a total of 40 amino acids (including the overlap). The structure formed by these loops and the loops and the sequences involved are shown in Fig. 2. In recent years there have been several studies directed at identifying the binding sites for von Willebrand factor and
391
10
20
30
40
60
50
Elution Time (min)
0.10
1 PE
3-P
P29
25
0 05
0 I
I
1
15
30
45
.
Elution Time (min)
Fig. 1. Separation of ( a ) peptic digest of unreducedglycocalicin and ( b ) thermolytic digest ofpeptic fractions PI 1 - P16 ofglycocalicin. Reversephase HPLC on Bakerbond C4 (wide-pore, 33 nrn, 5 prn, 4.6 x 250 mm), using a linear acetonitrile gradient. Solution A : 0.1% (by vol.) trifluoroacetic acid in distilled water. Solution B: 0.1 % (by vol.) trifluoroacetic acid, 80% (by vol.) acetonitrile in distilled water. Flow rate: 1 ml/min. Detection: 210 nm. Fractions pooled are indicated by horizontal bars
thrombin on glycoprotein Ib. These have been based either on cross-linked methods [23] or on inhibition studies using synthetic peptides from the sequence of glycoprotein Ibcl [12, 131. Studies on the interaction between von Willebrand factor and glycoprotein Ib are complicated by the fact that these two molecules normally do not interact and require the presence of material in subendothelial tissue (probably either collagen or proteoglycan) to which von Willebrand factor binds and thereby undergoes a conformational change allowing it to bind glycoprotein Ib. The interaction between von Willebrand factor and glycoprotein Ib can be simulated in vitro by using the antibiotic ristocetin [24] or the snake venom peptide botrocetin [25] or by using animal von Willebrand factor such as bovine or porcine or asialo human von Willebrand factor [26, 271. It is nevertheless still not clear how far these approaches really approximate to the natural interaction and, indeed, differences in potential binding sites have been observed [28]. Using 26 overlapping synthetic short (mostly 15-residue) peptides from the N-terminal 45-kDa fragment of glycoprotein Iba the region Ser251- Glu285 was identified by Vicente et al. [12] as inhibiting both ristocetin- and botrocetindependent von Willebrand factor binding to platelets. Two peptides, Ser251 -Tyr279 (29 residues) and Gly271- Glu285 (I 5 residues) were most effective. Nevertheless, the linear peptides were considerably poorer inhibitors than the native 45-kDa N-terminal tryptic fragment, particularly with
botrocetin. In addition, they showed that the negative charges a t 281 -283 and 285 were not critical. The peptide Lys231Ser245 was also inhibitory as was peptide Gln71 -Ser80 though somewhat less. Katagiri et al. [I31 examined the effect of short peptides (24 - 28 residues) on von Willebrand factor and thrombin binding and found the strongest inhibition of ristocetin-induced platelet aggregation with a peptide As11235 - Lys262. A group of overlapping peptides from Phe216-Asp274 with maximum activity in the sequence Phe216 -Thr240 inhibited platelet aggregation by thrombin. All this work points to the von Willebrand factor and thrombin receptors on glycoprotein Ib lying on and/or in the vicinity of these two disulphide-bond-formed loops. There is, however, no direct evidence that the binding sites overlap or directly interact. Many monoclonal antibodies have been described that block only the von Willebrand factor binding site but not thrombin binding. Only a few monoclonal antibodies have been described that affect both [29, 301. An additional piece of evidence that these disulphide loops are involved in von Willebrand factor binding comes from studies on pseudo-von-Willebrand's disease. In this disease the platelet glycoprotein Ib is abnormal and reacts spontaneously with normal von Willebrand factor in plasma, implying that the binding site is expressed even in the absence of ristocetin or botrocetin. The gene for glycoprotein Ibcl from a patient with pseudo-von-Willebrand's disease has been sequenced and
392 Table 1. Edman degradation data and identification of disulphide bonds in peptic, thermolytic,frugments Sequences in capital letters indicate the results of Edman degradation of unreduced peptides; X denotes a Cys residue expected from the known sequence but not observed. Asn21 could not be identified by Edman degradation due to its carbohydrate moiety. C40 indicates carbohydrate Peptide
Disulphide bond c y s -c y s
Structural data
'D \
3 IXE ~
V N C D K R (N) 17 I CHO
248 VQCDNSDK F 211
209 - 248
I
211-264
L2x: GKGCPT 264 248 VQCDNSDKF 211 LXNX
I
I
209 - 248 21 1 - 264
209 VYKYPGKGCPT 264
21 I NX
21 1 - 264
I
KYPGKGCPT 264 P,T11348,2
209 C
Fig. 2. Schematic drawing of the Ala200 - Pro280 disulphide-loop region of glycocalicin. The amino acid sequence within the double disulphide-loop region is shown. The thin superscript lines indicate peptides which have been reported to inhibit von Willebrand factor/ glycoprotein Ib binding [12,13], while the thick subscript line indicates peptides which have been reported to inhibit thrombin/glycoprotein Ib binding [13]. The large arrow indicates Gly233. Mutation of this residue to valine has been reported to be associated with spontaneous binding of glycoprotein Ib to von Willebrand factor in pseudo or 'platelet-type' von Willebrand's disease [31]
209 - 248
CDNSDKF 248
The determination of the disulphide bond linkages in this region of glycoprotein Ibcl should now enable strategies to be developed based either upon molecular biology approaches (site-directed mutagenesis, or deletions), or protein chemistry (synthesis of circular or fixed-conformation peptides) to establish definitively the binding sites of von Willebrand factor and thrombin within the disulphide-linked loop structures. The authors thank Ms Susanne Weber for technical assistance and the Swiss Red Cross Central Laboratory for providing buffy coats. This work was supported by the Swiss National Science Foundation (grants 31.26264.89 and 31.25633.88).
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a single point mutation was found leading to the putative change Gly233 -+ Val [31]. A further indication that the conformation maintained by disulphide bonds is critical for von Willebrand factor comes from the fact that their reduction leads to the loss of binding between von Willebrand factor and glycoprotein Ib in asialo von Willebrand factor or botrocetin-dependent assays [ 121, though this seems to be less important for ristocetin-dependent assays or for thrombin binding [l].Putting all this information together leads to the conclusion that both loops have an important role in von Willebrand factor and thrombin binding and that they may indeed interact; also, that perhaps the smaller loop is more involved in von Willebrand factor binding, while the larger loop may be more involved in thrombin binding.
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