Staffan Magnusson

A thiol‐ester in α2‐macroglobulin cleaved during proteinase complex formation

The glycoprotein crs-macroglobulin ((YAM), M, 725 000, is unique among plasma proteins in being able to form complexes with proteinases from all 4 classes (EC 3.4 2 1-24) [ l-31. The function of olM is not well understood. However, the rapid clearance of osM-proteinase complexes from the blood by the reticuloendothelial system [4] and the uptake of a,M or r+M-proteinase complexes via receptors by several types of cell in culture [5-81 indicates that cuzM plays a role in the transport of proteinases and possibly other proteins into cells. The binding of proteinases to crzM is initiated by cleavage of one or more of the Mr 180 000 subunits of crzM in the ‘bait region’ 193, producing two Mr 85 000 fragments [ 1 ,I O-121. There appears to be a close-fitting binding site in this area since even auhydrotrypsin binds to crIZM [131. The exact position of cleavage in the ‘bait region’ reflects the known substrate specificity of the particular proteinase being complexed (trypsin, plasmin, thrombin’, elastase). Following cleavage, the initial complex, involving the proteinase active site and the cvzM ‘bait region’, is rearranged to form a final complex, involving a second site in the proteinase and probably also in cr2 M (L. S.-J., T. E. P., S. M., 11. Jdrnvall, in preparation). In the final complex cvzM may well be covalently linked to the proteinase [9 ,141. The subunits of ol,M can be cleaved specifically by heating, into two fragments of Mr 120 000 and Mr 60 000 [15-l 71. This polypeptide

Primary structure of the vitamin K-dependent part of prothrombin

The biosynthesis of normal prothrombin is vitamin K-dependent [ 11. During dicoumarol treatment a prothrombin is produced [2,3] which can neither be adsorbed to barium citrate nor bind calcium ions [4] and therefore is not activated in the normal Ca’+, phospholipid systems. One reason for determining the primary structure of prothrombin has been to find the structural modification caused by vitamin K. Analyses of carbohydrate and amino acid compositions as well as N-terminal sequences of normal [5-71 and dicoumarol prothrombin [8-lo] indicated no clearcut difference between the two proteins and no unusual constituents. The first clue to the identity of the structural modification was the isolation [ 1 l] of the tryptic peptides Gly-Phe-Leu-Glx-Glx-Val-Arg and Gly-Phe-Leu-Glx-Glx-Val-Arg-Lys from positions 410 and 411 in normal prothrombin. Both peptides were free of carbohydrate and carried two negative charges more at neutral pH than could be attributed to normal glutamic acid residues [ 12, 131. The adsorption of a different tryptic peptide to barium citrate [ 141 indicated that this peptide had also been modified by vitamin K. Stenflo [ 151 has recently confirmed that peptides 410 and 411 from normal prothrombin carry the extra negative charges, and found that the corresponding sequences in dicoumarol prothrombin contain normal Glu-

Adsorption to fibrin of native fragments of known primary structure from human plasminogen

Limited proteolysis of native Glu-plasminogen with pancreatic elastase produced three major fragments, K1+2#3, K4, K5-light chain (miniplasminogen). Fibrin-binding was determined by clotting fibrinogen in the presence of 125I-labelled fragments and measuring 125I in the washed fibrin and in the supernatant. Of the fragments miniplasminogen showed the highest fibrin-binding, the strength of which was intermediate between those of Glu-plasminogen and Lys-plasminogen. The fibrin-binding of all three fragments was decreased by 6-aminohexanoic acid or tranexamic acid. This decrease was most pronounced with K1+2+3. The fibrin-binding of K1+2+3, but not that of K4 and miniplasminogen was decreased by alpha 2-antiplasmin. The fibrin-binding of K1+2+3 and mini-plasminogen was lower in a plasma clot than in a purified fibrin clot. Our results indicate that each of the three fragments can bind to fibrin. They confirm that an alpha 2-antiplasmin-binding site is located on K1+2+3. Furthermore two of the fragments, namely K4 and K1+2+3 contain lysine-binding site(s).

Amino acid sequence of the factor XIIIa acceptor site in bovine plasma fibronectin

Blood coagulation factor XIII is a proenzyme which can be activated by thrombin [I] to the transglutaminase factor XIII, [2%4]. Factor XIII, catalyzes the formation of e(y-glutamyl)lysyl amide bonds between pairs of y-chains in aggregated fibrin, resulting in its transformation to a highly stable and insoluble covalently cross-linked clot (reviewed in [S-7]). Two other plasma proteins cYz-macroglobulin and fibronectin contain acceptor sites for factor XIII, as shown by incorporation of dansylcadaverine [8]. Only fibronectin, but not aa-macroglobulin, was shown to be crosslinked to fibrin [8]. Cross-linking of fibronectin to collagen [9 ,I 0] and to StaphyZoccocus aureus cells [ 1 l] has been demonstrated. It has also been suggested that rYa-antiplasmin could be covalently linked to. fibrin in a Ca’+dependent reaction probably catalyzed by factor XIII, [ 121. Here we report the ammo acid sequence in bovine fibronectin which contains the glutamine residue labelled with radioactive putrescine by factor XIII,. This glutamine is located at position 3 from the N-terminus of fibronectin.

Primary structure of the ‘bait’ region for proteinases in α2-macroglobulin: Nature of the complex

Most proteinase inhibitors in blood plasma, e.g., antithromb~.III, Q i-antitrypsin, inter+trypsin inhibitor, are specific towards serine proteinases and form 1 :l complexes engaging the active site and thus completely inhibiting the proteinase activity. a,-Macroglobulin is a tetrameric glycoprote~,~r 725 000, consisting of four app~ently identical chains. ‘Halfmolecules’,Mr 360 000 can be obtained under nonreductive denaturing conditions [ 1,2]. Unlike the other inhibitors (YAM can form complexes with proteinases from different classes having different substrate specificities [3-51. furthermore, the active site of the complexed proteinase is accessible to smaller molecules as shown by the facts that the cr,M-trypsin can cleave synthetic substrates [6] and be inhibited by the bovine pancreatic trypsin inhibitor (Kunitz) (M, 6514) [7]. It. IS not accessible to larger protein substrates or ~hibitors, e.g., soy bean trypsin inhibitor, Mr 20 095 [?I. However, neither zymogens such

Trypsin‐induced activation of the thiol esters in α2‐macroglobulin generates a short‐lived intermediate (‘nascent’ α2M) that can react rapidly to incorporate not only methylamine or putrescine but also proteins lacking proteinase activity

The Glx-residue is located 469 residues from the C-terminus of at,M [3]. Complement component C3 also contains a reactive thiol ester [4,5], located in an identical heptapeptide sequence in itsor’chain [6]_ Fo~owinglimited proteolysis in the ‘bait’ region of cwzM [2,3] or at that site in C3 which generates C3a and C3b [4-61 the thiol esters are rapidly cleaved resulting in the appearance of free sul~yd~l groups 12-61. Reaction of azM and C3 with methylamine leads to relatively slow cleavage of their thiol esters [2,4-61. During this process a stoichiometric amount of CHsNH2 is incorporated into the reactive Glx-residue to form y-glutamylmethylamide [l-4,6,7]. As a result of this reaction the proteinase binding capacity of cu,M [ 1,2,8] and the hemolytic activity of C3

Sequence location of the reactive thiol ester in human α2-macroglobulin

It was shown recently that native az-macroglobulin (cQM), Af, 725 000 contains one reactive, labile thiol ester in each of its 4 identical subunits of Iw, 180 000, which is formed by a particular Glx-residue, whose y-carboxyl group is esterified to the sulfhydryl group of a cysteinyl residue [ 13. Complex formation with proteinases involves not only limited proteolysis in the ‘bait’ region but also the rapid cleavage of this thiol ester as evidenced by the concurrent appearance of the SH-groups (max. 4 mol/mol azM) [ 11. Denaturation of CY,M or treatment with CH3NH2 also leads to cleavage of this thiol ester [I]. During ‘inactivation’ of the proteinase binding capacity with CHaNH2 the latter,is incorporated covalently to form a y-glutamyl methylamide [ 1,2] indicating that this site in ozM may be a site for covalent binding of proteinase. Complement component C3 also reacts covalently with CH3NH2 again leading to the formation of y-glutamyl methylamide f3-51. The recent demonstration that the a-chain of C3 probably also contains a thiol ester [4,5] later shown [6] to involve the Glx-residue and the Cys-residue’in the sequence: -Gly-Cys-Gly~lu~lx-Asn-Met which is identical to the sequence around the CHaNHa-reactive Glx-residue of oZM [ 1,7,8] shows that cuzM and C3 are structurally and functionally

Primary and secondary cleavage sites in the bait region of α2-macroglobulin

The plasma glycoprotein cuzM forms complexes with proteinases from all 4 classes (serine, thiol, carboxy1 and metalloproteinases, EC 3.4.21-24) [l-3]. Complex formation is initiated by specific limited proteolysis in the bait region, located near the middle of the 4 identical Mr 180 000 subunits [ l-31. Following cleavage in the bait region ar?M undergoes a change in conformation resulting in entrapment of the proteinase [2] and cleavage of the internal y-glutamyl+3cysteinyl thiol ester [4] in each subunit of a,M [4-6]. A fraction of the proteinase molecules becomes covalently bound to (Y*M [7,8]. The site of cleavage in the bait region of azM has been identified for trypsin [9,10], plasmin [9] and thrombin [9], respectively. In each case the same Arg-Leu bond was cleaved. Elastase was found to cleave at a ValGly bond (major cleavage) and at the adjacent GlyPhe bond (minor cleavage) [9]. The new N-terminal sequence generated by cleavage of CQM with Stuphylococcus aureus strain V8 proteinase (SP) was also determined [lo]. From these data and the sequence of the bait region [9] it is evident that cleavage in the bait region of CQM by these different proteinases reflects their known substrate specificities [9,10]. The common cleavage site for trypsin, plasmin, thrombin and the SP cleavage site (Glu-Ser) are located 15 and 5 residues, respectively, after the main elastase cleavage site [9,10]. Here, we report the identification of the cleavage sites in the bait region of 02M for a variety of other proteinases with known primary structures from all 4 classes. We show that proteolytic cleavage can occur at each residue in the hexapeptide sequence: -Arg-Val-Gly-Phe-TyrGlu-. This sequence most likely contains the sites in the bait region at which initial proteolytic cleavage occurs. Additional probably secondary cleavage can occur in two other regions situated -15 and 27 residues, respectively, from the hexapeptide region.

Multiple gene duplication in the evolution of plasminogen. Five regions of sequence homology with the two internally homologous structures in prothrombin

In order to investigate the substrate specificity of urokinase (EC 3.4.99.26) and other plasminogen activators we isolated and sequenced a chymotryptic fragment of 38 residues from the activation cleavage site region of plasminogen [ 1 ] . The fragment overlapped the last 28 residues in the heavy chain of plasmin (EC 3.4.21.7) (derived from the N-terminal 3/4 of the single chain plasminogen molecule) to the previously known sequence [2] of the first 10 residues in the light chain (derived from the C-terminal l/4 of the plasminogen). The fragment thus contained the Argapprox. 600Val bond which is cleaved on activation of plasminogen to plasmin [3]. In the N-terminal region of this chymotryptic fragment residues 2-l 1 showed 70% sequence identity with two sequences in prothrombin (residues 138-147 and 243-252) [l], which constitute the C-terminal parts of the two internally homologous kringle

Analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography using gradient elution with ethanol

region (residues 62-144 and 167-249) [4] in the ‘pro’ part (residues l-274) of the prothrombin single chain (582 residues) [5]. These two 83-residue kringle regions in