Arginyl-binding sites of human plasminogen

THROMBOSIS RESEARCH 41; 689-698, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

ARGINYL-BINDINGSITES OF RUMAN PLASMINOGEN Verevka S.V., Kudinov S.A., GrinenkoT.V. Instituteof Biochemistry,Acadejnyof Sciences of the Ukrainian SSR. Kiev, USSR (Received 20.2.1985; Accepted in revised form 10.10.1985 by Editor M. Kopec) (Received in final form by the Executive Editorial Office 13.12.1985)

ABSTRACT Localizationand snecific features of lysine- and argi_ nyl-bindingsites in Lys-plasminogen,its frsgmentsand domains have been investigatedby affinitychromatography on the sorbentscontainingarginine-likeligands. Lysine-bindingsites of Lys-plasminogen,heavy chain end fragmentKl-3 interactwith the guanidyl-carboxyl pair on homoarginine-agarose. Lysine-bindingsite in domain K4, interactingwith the amine-carboxylpair on lysine-agarose,does not interactwith that of guenidyl-cerboyyl.It has been found that plasminogencontained three arginyl-bindingsites interactingwith guanidyl group in homoarginine-agerose. Two of them correspond to two bensamidine-binding sites in domain K5 end to the plasmin light chain while the third (unknownbefore) is located in fragmentK'l-3and does not interact with bensamidine-agarose.

INTRODUCTION Protein - protein interactionsbetween plasminogenand fibrin. between plasmin end dz-entiplasminplay an important role in activatingplasminogenby physiologicalactivator,specificity of the plasmin action directed in vivo only to fibrin and, consequently,in the fibrinolysismolecularmechanism (1). These interactionsare realised by special lysine-binding sites in plasminogen(plasmin)arrangedin amino-terminal. region of the molecule of the correspondingplasmin heavy chain (21. Key words: Plasminogen,plasminogenfragments,lysine-binding sites. benzemidine-binding sites. 689

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It has been found that they are responsible for the inter(4). action of plasminogen with fibrin (3) aild do-antiplasmin The sites of interaction with p-aminobe~zamidine-agarose (5) have been found in addition to the aforementioned lysinebinding ones. They do not constitute en active site for not only plasminogen but also plasmin inactivated by p-nitrophenyl-p-guanidinobenzoate over an active site (5), display an affinity for p-aminobenzamidine-agarose. They are localized in the plasmin molecule light chain and in the fifth Kringle of a heavy chain and can withstand the elution by various benzamidine concentrations (6,7). The plasmino en molecule consists of seven relatively autonomous domains ?8). Using the Kringles notations to the plasminogen molecule domain arrangement and employing the data pertaining to the protein - protein interaction sites location one may sum u that lysine-binding sites are located on domains K1 and K4 (9P , while benzsmidine-binding ones are arranged on domain K5 and on a domain of a light chain (6). It is presumed that arginine-binding sites are absent on plasminogen (10). However, benzamidine-binding sites are responsible for interaction of the plasmin light chain with immobilized D-fragment of fibrinogen (7). The role of p-aminobenzamidine in the D-fragment can be played exclusively by arginyl recidues. The functional essence of this interaction is nor clear yet. The aim of this work is to investigate the sites of intermolecular interaction of plasminogen and its fragments using the carriers containing arginine and arginyl-like ligands.

MATERIALS AND METHODS Lys-plasminogen was prepared from human plasma Cohn fraction I11Z,3 by affinity chromatography on lysine-sepharose (11). Plasmin was obtained by activating plasminogen with streptokinase (25 streptokinase units per 1 unit of the enzyme activity) in 25% of glycerol (13). The plasmin S-carboxymethyl heavy and light chain derivatives were separated according to the conditions of the plasmin reduction and csrboxymethylation (14). The low-molecular reagents excess was removed by Gel-filtration through the Sephadex G-25 column. The heavy and light chains mixture was separated through the lysine - sepharose column equilibrated by O.lM phosphate buffer pH 7.4 at 4OC. After the complete elution of the non-absorbed light chain, the plasmin heavy chain was eluted by 0.2 M 6-aminohexanoic acid in 0.1 M phosphate buffer pH 7.4. Proteins were precipitated with ammonium sulfate (0.31 g/ml solution), were dissolved in 0.05 M phosphate buffer pH 7.4 and dialyzed against the latter. The plasminogen fragments containing first three domains Kl-3 and domain K4 were obtained by spliting Lys-plasminogen with elastase isolated from the porcine pancreatic gland according to Shotton (15). The treatment by elastase was conducted at the enzyne/substrat molar ratio being 1 : 50, for 5 hours at 25OS (16). The domains X1-3, K4 and mini-plasminogen mixture

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was separated using the lysine-sepharose column (8). Fragments Kl-3 end K4 were then separated by gel-filtration on Sephadex G-75. Domain K5 was obtained by elastolysing the plasmin heavy chain with the following chromatography of the fragments mixture through the lysine-sepharose column. The obtained fragments were dialyzed against the distilled water and lyophyliaed. The preparations purity was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as was mentioned earlier (17). Iys-plasminogen, its fragments (except HI-3) as well as the plasmin heavy and light chains were homogenous as to their molecular mass. Two regions were characteristic for fragment Kl-3 which corresponded to its two forms: Tyr-79-Val-337 and tyr-79-Val-553 (18). The obtained preperations were also characterized using specific absorption coefficients and molecular mass as was described earlier (8). Affinity sorbents containing the guanidyl groups, were obtained in the following way. At the first stage, the sorbents containing lysine (or 1,6-diaminohexene end glycine) was synthesized by standart B&N-activated procedure (18). Weight ratio of the components in respect to BrCN-agarose amounted to I:1 or 0.5:0.5:1, respectively. The amount of bound lysine or 1,6-diaminohexane was determined utilizing 2,4,6_trinitrobenzenesulfonic acid (20). The amount of bound glycine was found by amino acid analyzer. The obtained sorbents incorporated 14.2 micromoles of the lysine amino groups per ml of deposited gel or 11.8 micromoles of the 1,6-dieminohexene amino groups and 8.9 micromoles of glycine per ml of deposited gel correspondingly. A part of the sorbent with immobilized 1,6-diaminohexsne end glycine was treated by ethyl diaeoacetate to esterify the carboyyl group. For this purpose, 20 ml of gel were suspended in a double volume of water on the ice bath, 2 g of ethyl diazoacetate were added with mixing and ut on a mechanical mixer for the eriod of 20 hours at rC°C* Tg en the gel was transferred on a gf ass filter end washed with water, 0.5 M NaCl end again with water. The diazoacetic acid ethyl ester was obtained according to (21). At the second stage, guaniding of amino groups by methyliso urea was carried out. 'I5ml of each sorbent were suspended in 50 ml of the Na2C0, saturated solution on the ice bath and 2.5 g of methyliso urea sulfate were added with mixing. The gel was kept at 4OC within 24 hours and washed on a glass filter by 0.1 M Na&03 , 0.1 M NeHCO,, 0.5 M NaCl and 0.05 M phosphate buffer pH 7.4. The methyliso urea sulfate was obtained according to (22). The sorbent, containing 2-amino-6-guanidyl hexanoic acid (homoarginine-agarose) immobilized over A-amino group, was obtained as a result of guaniding the lysine-agarose. The sorbents,containing I-amino-6-guanidyl-hexane end glycine with free or etherified carboxyl groups, were obtained as a result of guaniding the sorbents, incorporating 1,6-diaminohexane and glycine with free or esterified carbov1 groups. For the chromatographic experiments the next buffers were used: a. 0.05 M sodium-phosphate buffer pH 7.4; be 0.5 M NaCl in buffer a , pH 7.4; C* 0.2 M 6-aminohexanoic acid in buffer a , pH 7.4 do 9.1 M arginine monohydrochloride in buffer a , pH 7.4

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Fractions 2.5 ml monitored at 280 nm. Ail.1 columns were overloade to prevent the second sorption of the protein.

A3EiSULTS AND DISCUSSION Fig la shows that Lys-plasminogen is specifically bound to homoarginine-agarose. A part of protein can be eluted by 0.2 M solution of 6-aminohexanoic acid, and the remaining one

-CH2-C-0-CH2-C-OC2H5 C.I Y Aw.hCl

0

5

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15

20

25 30 Fraction.I".LwP

FIG. 1 Affinity chromatography of Lys-plasminogen on homoarginine-agarose and sorbent vith combined ligand: A) homoarginine-agarose (column volume 12 ml); B) sorbent containing free suanidyl and carboxyl groups (column volume 6 mly; C) sorbent containing only guanidyl groups (column volume 6 ml). - by 3.1 1J arginine solution. Approximately a half of the protein content, capable of undergoing sorption in the column in the absence of 6-aminohexanoic acid, is bound to a carrier whi-

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le applyingthe plasminogensolutionto the column equilibrated by the same buffer beforehand.Its desorptiontakes place when putting 0.1 M arginine solution through the column. Sorption does not occur in the presence of arginine.The obtaineddata are explainedin the followingway. The lysine-bindingsites of plasminogenare capable to interactwith the arginyl -carboxylic pair and sorptionof the protein part. eluted by 6-aminohexsnoic acid, takes place due to these sites. The same occurs while using arginine-sephsrose(IO). The protein part not eluted by 6-aminohexanoicacid absorbes on a carrier due to the presence of benzamidine-binding sites and is eluted with 0.1 M argininesolution. In homoarginine,used in our experimentsas a ligand, the distancebetween guauidineand carboxyl groups is enlargedby one methylene group comparedto arginine.Such an enlargement turns out to be sufficientfor efficientinteractionwith both lysine-bindingand benzamidine-binding sites of plasminogen. Arginine,immobilizedover d-amino group, does not correspond to the requirementsof the benzamidine-binding sites ligand specificity(probably,as a result of obstaclesbeing created by carboxyl group). Therefore,homoarginine-agarose simultaneously interactwith two types of the plasminogenmolecule binding sites, that is, lysine- and benzemidine-binding sites. Fot the experimentalcheck of such an assumptionwe have studied the plasminogensorption on the sorbent,containing the combined ligsnds - I-guanidyl-6-amino-hexaue and glycine immobilizedover amino groups (functionalguanidyl and carboxyl groups), as well as on the sorbentwith the esterifiedglytine carboxyl groups (functionalguanidyl groups). The data presented in Fig.lb,c shows that in both cases the specific sorption of plasminogenoccurs. Plasminogenmay undergo sorption by both lysine- and benzamidine-binding sites? and be eluted by 6-aminohexanoicacid and arginine,respectivelyon the sorbent incorporatingguanidyl and carboyylgroups, similar to the case with homoarginine-agarose (Fig. lb). Plasminogenis subjectedto sorptionby benzamidine-binding sites and elution only by arginine (Fig.'lc) on the sorbent,containingexclusively guanidinegroups. Therefore,these sites may be called guanidyl- or arginyl-bindingones. The interactionover lysinebinding sites does not take place for the carbolgl groups on sorbent'ssurface are blocked. Biospecificcapacity of these sorbentswith combined ligand is comparativelylow. This is probably explainedby the gel piercing in the course of 1,6diaminohecaneimmobilizationand by the strictnessof structural demands "made" by lysine-bindingsites to the ligsnd (IO). In the cases when guanidyl and carboxyl groups are arranged at the distancesclose to 0.68 nm the sorption over lysine-binding sites may occur. If the distancesbetween this groups are accidental the sorbent affinitycapacity is considerablylower. The nonspecificsorptionis higher than in the case of homoarginine-agarose.It is destroyedby 0.5 M NaCl. Taking into account the data pertainingto both the I&Wplasminogensorption on the sorbentswith guanidyl and carboxyl groups by two types of sites as well as the localization of these parts in the molecule frsgments,one can suggest that the plasmin light chain end domain K5 will undergo sorption on

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homoarginine-agarose by arginyl-binding sites, the plasmin hea- by two types of sites whereas fragment ICI-3and do- by lysine-binding sites. This assumption is proved by z:hE observation that domain K5 and the plasmin light chain are specifically bound to homoarginine-agarose and can not undergo desorption by 6-aminohexanoic acid (Fig. 2). These data are in a good accord with those relating to the presence of benzamidine-binding sites in the plasmin light chain and domain K5 (6,7). They explain why the protein-protein interactions between the plasmin light chain and D-fragment of fibrinogen are not destroyed by high concentration of 6-aminohexanoic acid (7). Miniplasminogen has no lysine-binding sites (7). It is possible that a great affinity of mini-plasminogen to bifrinogen (23) is due to arginyl-binding sites. The functional sighificance of these interactions is not quite cleazcyet, however, only arginyl residue is able to act as p-amino-benzamidine (or guanidyl-con-

@,2

FIG. 2 Affinity chromatography of the plasmin light chain (A) and domain K5 (B) on homoarginineagarose (column volume 12 ml). taking ligands used by us) in fragment D in the course of its interaction with the light chain or in fibrin during its interaction with miniplasminogen. The plasminogen is believed to contain no separate arginine-binding site because the interaction of the plasminogen with arginine-sepharose is easily destructed by 6-aminohexanoic acid (IO). However the arginine immobilized over d--amino group on sepharose can not serve as

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a structural analog of the arginyl residue in polypeptide chain due to the presence of free cerboxyl group. Therefore, one may suppose that beneamidine-binding (guanidyl-binding) sites are the arginyl-binding ones and possess a functional significance. The plasmin heavy chain and its fragment HI-3 underwent sorption on homoarginine-agarose and were eluted by the 6-aminohexanoic acid solution only partially with the following complete elution by 13.1M solution of arginine (Fig.3). In the presence of 0.1 M arginine these fragments, similarly to plasminogen do not interact with homoar inine-agarose. In the presence of 0.2 M 6-eminohexanoic acid 'i for the blocking of the lysine-binding sites) the sorption of plasmin heavy chain and frag-

FIG. 3 Affinity chromatography of the plasmin heavy chain (A), fragment Kl-3 (B) and domain K4 (C) on hornoar.&nine-agarose (column volume 12 ml). ment K'l-3 occurs on the given carrier. We expected such a behavior for the plasmin heavy chain, for the fifth kringle available in the chain contains argihyl-binding site. It was surprising to find incomplete elution of fragment

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RI-3 by 6-aminohexsnoic acid because it is know-n that benzamidine -binding sites are absent in this fragment (7). These results allow to assume that the site of specific affinity for the ligand guanidyl group is present not only in the plasmin light chain and domain K5 but also in fragment K?-3. This assumption is proved by the ability of fragment Kl-3 to undergo the affinity sorption on the carrier incorporating both l-amino-6-guanidyl-hexane and glycine with esterified carboql groups as a ligsnd as well as to be eluted by the 0.1 M arginine solution (Fig. 4).

FIG. 4 ent Xl-3 on sorAffinity chromatography of fr sorbent containing bents with combined ligands: A ae;m free guanidyl and carboxyl groups (column volume 6 ml); B) sorbent containing only guanidyl groups (column volume 6 ml). Proceeding from the ability of lysine-binding sites of Lys.plasminogen and plasmin heavy chain end fragment K'lto interact with the guanidyl-carboxylic pair, one could expect that tne K4 domain site would also interact with homoarginineagarose. However the isolated K4 domain did not display such an ability (Fig.3B). The absence of the domain sorption upon homoarginine-agarose may suggest different specificity of the lysine-binding sites in fragment Kl-3 and domain K4 of plasmi-

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nogen. However it is also expedient to make allowance for the fact that the isolated K4 domain lacks the natural environment and can be denaturated to some extent. RiEi-FmcES 1. 2.

3. 4.

5. 6.

Wiman, B., Collen, D. Molecular mechanism of physiological fibrinolysis. - Nature, 272, 549-550,1978. \barkus,J., De Pasquale? I., Wissler? P. Quantitative determination of the binding of E-amlnocapronic acid to nati ve plasminogen. - J. Biol. Chem., 222, 727-732, 1978. Wiman, B., Wallen, P. The specific interaction between plasminogen and fibrin. A physiological role of the lysinebinding site. - Thromb. Res., IO, p. 213-222, 1977. Wiman, B., Collen, D., Lijnen, H. On the specific interaction between plasmin and complementary in ci(z-antiplasmin and in fibrinogen. - Biochim. et bionhv S. acta, m, 1429 154, 1979. Holleman, W.H., Andreas, W.W., Weiss, L.J. The relationship between the lysine- and p-aminobensamidine-binding sites on human plasminogen. - Thromb. Res., 2, 683-693, 1975. Varodi, A., Pattby, L. Kringle 5 of human plasminogen carriers a benzamidine-binding sites. - Biochem. and Biophvs. Res. Communs, 103, 97-102, 1981.

7.

T.M. B3aHMOJJefiCTBilen~a3mmoreHa, KYJJPIHOB,C.A.,JIexea, TsmeJroB p1 Jrerxor;i. Germ mta3NIMllac ji- 18 E-& arn4eHTaPm @6pM58-60,%82, JG6. HoreHa. - ~OKJI. AH YCCP, Cep. E.,

8.

~OBOXaTHkI~, B.B., %EBem, x.B., I{pIHoB, C.A., @myo&$. AoMenHas 0 ram3arrysr ?.~oJreqvmJ.k3-rma3miHoreHa. 1 &JI., T. 17, 9k-982, 1983.

9. Trexler, M., Vali, Z., Patthy, L.

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Structure of the c-aminocarboxylic acid-binding sites of human plasminogen. Arginine 70 and aspartic !54 are essential for binding of ligand by Kringle 4. - J. Biol. Chem., a, 7401-7407, 1982. IO. Winn? E., Hu, S.-P., Hochschwender, S.M., Laursen, R.A. Studies on the lysine-binding sites of human plasminogen. Eur. J. Biochem., 104, 579-586, 1980. 11. Deutsch, D., Mertz, E.T. Plasminogen: purification from human plasma by affinity chromatography. - Science, E, 'IO%-1096, 1970. 12. Robbins, H.C., Summaria, L. Human plasminogen and plasmin. - In: Methods in Rtzvmolo~, Acad. Press, New York, London, E, 67-113, 1970. A new method of isolation and 43. Rickli, E.E., Otavsky, W. some properties of the heavy chain of human plasmin. - Eur. J. Biochem., 2, 441-447, 1975. 14. Wiman, B. Primary structure of the B-chain of human plasmin. - Eur. J. Biochem., 1977, & 129-137.

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Elastase. - In: Methods in Enz.ymology,Acad. 15. shotton, D.X. Press, New York, London, XIX, 113-140, 1970. Activation of the neoplas16. Powell, J.K., Castellino, F.J. ninogen by urokinase and strcptokinase and kinetic characterization of neo-plasmin. - J. Biol. Chen., 252, 53295335, 19~0. The subunits and biological ac17. Commins, P., Perry, S.V. tivity of polymorphic forms of tropomyosine. - Biochem. J., 1122, 765-777, 1973. 18. Sottrup-Jensen, L., Clayes, II., Ziagel, M., Peterson? T-E., The primary structure of human plasmrnogen: &gnusson, S. isolation of two lysine-binding fragments and one miniplasminogen. - In: Progress in chemical fibrinolysis and thrombolysis. Raven Press, New York, 1, 191-209, 1978. A symplified 19. March, S.C., Parikh, J., Cuatrecases, P.A. method for cyanogen bromide activation of agarose for affinity chromatography. - Anal. Biochem., 60, 149-152, 1974. The spe20. Satake, K., Okayama, T., Chashi, fit., Shinoda, T. ctrophotometric determinations of amine, amino acids andpeptide with 2,4,6-trinitrobenzene-I-sulfonic acid. - 2. Biochem., 1960, Q, 654-660. 21. Ivomack,E., Nelson, A. In: Organic Synthesis, Acad. Press, New York, Annual volumes 22-25, 512-513, 1950. The constitution of carbamides. - J. Chem. 22. ?;erner,E.A. sot., 60, 923-932, 1914. 23. Thorsen, S., Clemmensen, J., Sottrup-Jensen, L., Magnusson, S. Adsorption to fibrin of native fragments of known primary structure from human plasminogen. - Biochim. et bio7 94~s. acta, 668, 377-387, 1981.