Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

Lijnen, H. Roger; Nelles, Luc; Hoef, Berthe Van; Demarsin, E.; Collen, D.

doi:10.1111/j.1432-1033.1988.tb14409.x

Cited by 34 publications

(8 citation statements)

References 48 publications

(16 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant inactive human plasminogen with Ser 741 mutagenized to Ala (r-Pg-Ala 741 ) and recombinant scuPA with Ile 159 mutagenized to Gly (r-scuPA-Gly 159 ) were obtained and characterized as described. 28,29 uPA and tPA (both greater than 99% single-chain form), a sheep antibody directed against uPA, and an immunoglobulin G 1 mAb 34D3 were obtained as described. 20,30 The mAb 34D3 reacts with plasminogen fragment K1 ϩ 2 ϩ3, blocks the LBS function of K1, and shows no cross-reaction with plasminogen K4.…”

Section: Reagentsmentioning

confidence: 99%

Fibrinolytic cross-talk: a new mechanism for plasmin formation

Dejouvencel

Doeuvre

Lacroix

et al. 2010

Blood

Self Cite

View full text Add to dashboard Cite

Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by ⑀-aminocaproic acid (⑀-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles. (Blood. 2010;115:2048-2056) IntroductionFibrinolysis and pericellular proteolysis depend on plasminogen activation at fibrin and cell surfaces, respectively. Plasminogen is a 92-kDa protein composed of 791 amino acids (Glu 1 -Asn 791 ; Glu-plasminogen) arranged in a serine protease region, 5 tripleloop structures (approximately 80 amino acid residues constrained by 3 disulfide bridges) called kringle (K) domains and an aminoterminal sequence (Glu 1 -Lys 77 /Val 79 ). 1 Domains K1 and K4 contain lysine-binding sites (LBSs) 2 that allow binding of plasminogen to cells, 3 fibrin, 4 and extracellular matrix (ECM) components 5 with a moderate affinity (overall dissociation constant, K d ϭ 0.5-1M). This interaction is inhibited by carboxypeptidase B (CpB), an exopeptidase that targets the activation surface by cleaving exposed carboxy-terminal lysines (C-ter Lys), and by ⑀-aminocaproic acid (⑀-ACA), a lysine analog that targets the LBS of plasminogen. Domain K5 of plasminogen contains a LBS of weak affinity that interacts with the amino-terminal peptide of Glu-plasminogen and favors a predominant compact "closed" conformation 6-8 over a very short-lived extended "open" form. 8,9 Proteolytic removal of the amino-terminal peptide by plasmin (cleavage at either Lys 62 , Arg 68 , or Lys 77 ) yields a stable truncated open form (Lysplasminogen), unable to adopt the compact conformation. [10][11][12] In a similar manner, saturation of the weak LBS in Glu-plasminogen with ⑀-ACA causes a transition from the compact closed form to the open extended conformation 8,9 characteristic of Lys-plasminogen. [12][13]...

show abstract

Section: Reagentsmentioning

confidence: 99%

Fibrinolytic cross-talk: a new mechanism for plasmin formation

Dejouvencel

Doeuvre

Lacroix

et al. 2010

Blood

Self Cite

View full text Add to dashboard Cite

show abstract

“…Construction and functional evaluation of recombinant mutants of scu-PA, in which the plasmin cleavage site was destroyed by site-specific mutagenesis of Lys158 or Ile159, has suggested that the enzymic properties of scu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). The structure of the NH2-terminal region of the B-chain of tcu-PA thus appears to be an important determinant for its enzyme activity, whereas the COOH-terminal region of the A-chain is less critical [16]. In order to further investigate the structure/function relations underlying the enzyme activity of both scu-PA and tcu-PA, we have in the present study prepared and characterized recombinant mutants of scu-PA with deletion of the NH2-terminal polypeptide chain and/or the Cys148 -Cys279 interchain disulphide bond in u-PA.…”

Section: Discussionmentioning

confidence: 99%

“…Conversion of scu-PA to tcu-PA in the vicinity of a fibrin clot apparently constitutes a primary positive-feedback mechanism for clot lysis 16, 141. In order to investigate the structure/function relationship underlying the enzymic properties of scu-PA and tcu-PA, recombinant mutants of scu-PA have previously been produced in which the plasmin cleavage site was destroyed by site-directed mutagenesis [I5 -181. Studies with such mutants have suggested that the enzymatic properties of scu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and in position 159 (requirement for Ile) [16]. Exposure of Ile159 apparently plays an important role in the increased enzyme activity of tcu-PA, probably as a result of refolding of Ile159 into the binding pocket of the enzyme.…”

mentioning

confidence: 99%

Biochemical properties of recombinant single‐chain urokinase‐type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala

Lijnen

Nelles

et al. 1992

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The contribution of the NH,-terminal polypeptide chain and of the Cys148 -Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2 -Phel57)rscu-PA (rscu-PA with deletion of Asn2 -Phel57), [Ala279]rscu-PA (rscu-PA with Cys279jAla mutation) or des(Asn2 -Phel57)[Ala279]rscu-PA [des(Asn2 -Phel57)rscu-PA with Cys279jAla mutation].Des(Asn2 -Phel57)rscu-PA, [Ala279]rscu-PA and des(Asn2 -Phel57)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with M , approximately 30000, 54000 and 30000, and specific fibrinolytic activities on fibrin plates of (mean A SD; n = 3) 860 & 150 IU/mg, 43.0 f 2.5 IU/pg and 240 rt_ 20 IU/mg, respectively, compared to 69.0 & 4.3 IU/pg for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ald279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 pg/ml [Ala279]rscu-PA or 3.4 pg/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 pg/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 1U/pg for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/pg to 75.0 IU/pg for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2 -Phel57)rscu-PA or des(Asn2 -Phel57)[Ala279]rscu-PA with plasmin.The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7 -1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants.These results indicate that (a) deletion of residues Asn2 -Phel57 results in abolition of the enzyme activity of rscu-PA, (b) the interchain disulphide bond in u-PA is not required for the enzymic activity of scu-PA, (c) all the determinants required for the enzymic activity of two-chain u-PA are contained within the B-chain, and (d) the region comprising residues Asn2 -Phel57 of u-PA is not required for the rapid in vivo clearance. Abbrc.viution.7. u-PA, urokinasc-type plasminogen activator; scu-PA, single-chain u-PA ; rscu-PA, recombinant scu-PA, obtained by expression of cDNA cncoding u-PA in Chinese hamster ovary cells; rtcu-PA, recombinant two-chain u-P...

show abstract

“…In order to obtain additional information on the structure/function relationships underlying the enzymatic properties of single chain and two chain u-PA moieties, the properties of scu-PA mutants produced by site-directed mutagenesis of Lysl58, He 159 or He 160 were studied (173). scu-PA K158R appeared to be indistinguishable from wild-type scu-PA with respect to plasminogen activating potential, conversion to active two chain urokinase by plasmin, specific activity and fibrinolytic potential in a plasma milieu.…”

Section: Gene Structure Of Scu-pamentioning

confidence: 99%

“…Because clot lysis with scu-PA is associated with a higher degree of fibrin-specificity as compared to tcu-PA, attempts were made, using recombinant DNA technology, to prevent its conversion to a two chain molecule. Several investigators have constructed mutants of scu-PA with the aim to destroy the plasmin cleavage site by site-specific mutagenesis of Lysl58 or Ilel59 (167,173,(188)(189)(190). Alternatively, scu-PA has been rendered less sensitive to cleavage by both thrombin and plasmin by substitution of both Argl56 and Lysl58 by Thr (190), or by introduction of a nega tive charge near the cleavage sites (substitution of Phel57 by Asp) (191).…”

Section: Plasmin-resistant Mutants Of Scu-pamentioning

confidence: 99%