Enhancement through Mutagenesis of the Binding of the Isolated Kringle 2 Domain of Human Plasminogen to ω-Amino Acid Ligands and to an Internal Sequence of aStreptococcalSurface Protein

In order to produce plasminogen activators (PA) more specific and more active than their natural counterparts, we designed recombinant genes encoding mutant forms of urokinase (u-PA) and chimaeric molecules combining fragments of tissue type plasminogen activator (t-PA) and of u-PA. The following constructs have been realized : 1°) u-PA where amino acids Arg156 and Lys158 have been replaced by Thr. The purpose of this approach was to obtain a prourokinase molecule displaying similar properties as the natural single chain urokinase (scu-PA) but resistant to the cleavage by plasmin ; 2°) u-PA where the second cleavage site, Lys135-Lys136, was also eliminated either by replacing amino acid 132 to amino acid 147 by a shorter link (Ser-Thr) as found in t-PA, or by replacing the two lysines by glutamine residues. The resulting molecules correspond thus to completely uncleavable scu-PA forms ; 3°) an hybrid composed of the finger domain of t-PA and of the B-chain of u-PA ; 4°) an hybrid made of the A-chain of t-PA and of the B-chain of u-PA ; 5°) an hybrid where the kringle 2 of t-PA has been inserted between the kringle domain and the B-chain of u-PA. The last three constructs have been made to confer the fibrin binding specificity of t-PA to the B-chain of u-PA.All recombinant DNAs were introduced, via an expression vector, into R1610 and CosI cells. Secretion of the recombinant products was monitored by ELISA and activities were assayed in an immobilized system involving a monoclonal antibody (AAU2) raised against 33K u-PA, plasminogen and the specific chromogenic substrate S2251. In this assay, all recombinant products, except the plasmin resistant (156-158) scu-PA, showed apparent specific activities comparable to the activity of natural two-chain u-PA. Potential interest of these new plasminogen activators in therapy will be discussed and further characterization of the new molecules will-be presented.

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Amino acid sequence analysis of the asparagine-288 region of the carbohydrate variants of human plasminogen

Biochemistry ◽

10.1021/bi00273a033 ◽

1983 ◽

Vol 22 (4) ◽

pp. 923-927 ◽

Cited By ~ 20

Author(s):

James R. Powell ◽

Francis J. Castellino

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Amino Acid Sequence ◽

Amino Acid Sequence Analysis ◽

Human Plasminogen

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Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein 1 1Edited by R. Huber

Journal of Molecular Biology ◽

10.1006/jmbi.2001.4646 ◽

2001 ◽

Vol 308 (4) ◽

pp. 705-719 ◽

Cited By ~ 41

Author(s):

Jorge L. Rios-Steiner ◽

Mónica Schenone ◽

Igor Mochalkin ◽

Alexander Tulinsky ◽

Francis J. Castellino

Keyword(s):

Surface Protein ◽

Group A ◽

Human Plasminogen ◽

Internal Peptide ◽

Kringle 2 ◽

Binding Determinants

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1 Human plasminogen kringle 2: Ligand binding properties and NMR solution structure

Fibrinolysis and Proteolysis ◽

10.1016/s0268-9499(98)80030-1 ◽

1998 ◽

Vol 12 ◽

pp. 3

Keyword(s):

Ligand Binding ◽

Solution Structure ◽

Binding Properties ◽

Nmr Solution Structure ◽

Human Plasminogen ◽

Kringle 2

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Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.2999-3006.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 2999-3006 ◽

Cited By ~ 17

Author(s):

Emi Sakuno ◽

Ying Wen ◽

Hidemi Hatabayashi ◽

Hatsue Arai ◽

Chiemi Aoki ◽

...

Keyword(s):

Amino Acid ◽

Biosynthetic Pathway ◽

Aspergillus Parasiticus ◽

Enzyme Reaction ◽

Cyclase Activity ◽

Aflatoxin Biosynthesis ◽

High Concentration ◽

Cytosol Fraction ◽

Internal Sequence ◽

Its Gene

ABSTRACT In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.

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Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers

10.22541/au.163257642.25497842/v1 ◽

2021 ◽

Author(s):

Olawole Ayinuola ◽

Yetunde Ayinuola ◽

Cunjia Qiu ◽

Shaun Lee ◽

Victoria Ploplis ◽

...

Keyword(s):

Group A Streptococcus ◽

Tight Binding ◽

Coiled Coil ◽

M Protein ◽

Temperature Dependent ◽

Functional Monomers ◽

Molecular Weights ◽

Group A ◽

Human Plasminogen ◽

Kringle 2

M-protein (PAM) largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C - 25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.

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Molecular Conversions of Recombinant Staphylokinase During Plasminogen Activation in Purified Systems and in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649612 ◽

1993 ◽

Vol 70 (03) ◽

pp. 495-499 ◽

Cited By ~ 7

Author(s):

S Ueshima ◽

K Silence ◽

D Collen ◽

H R Lijnen

Keyword(s):

Amino Acid ◽

Human Plasma ◽

Catalytic Amount ◽

Clot Lysis ◽

Lag Phase ◽

Plasminogen Activation ◽

Plasma Clot ◽

Acid Protein ◽

Human Plasminogen

SummaryRecombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-Δ6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-Δ10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma.In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-Δ10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K m = 3.6 μM and k 2 = 0.38 s−1. The specific clot lysis activities of HMW-STAR (122,000 ± 8,000 units/mg) and LMW-STAR (129,000 ± 8,000 units/mg) were indistinguishable.In an in vitro system consisting of a 60 μl plasma clot submerged in 250 μl plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR. This was associated with fibrinogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Addition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown (≥ 90% residual fibrinogen after 2 h), and was not associated with significant coversion to LMW-STAR (≤10% within 2 h). With 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytically active concentrations which do not cause fibrinogen breakdown, no significant conversion of HMW-STAR to LMW-STAR occurs in human plasma in the absence of a clot.These findings indicate that the conversion of HMW-STAR to LMW-STAR may occur in association with clot lysis, but is not required to induce it.

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Fibrinolytic Activity of Two Polypeptide Chains from Human Plasminogen#

Current Proteomics ◽

10.2174/1570164616666190112120215 ◽

2019 ◽

Vol 16 (4) ◽

pp. 277-281

Author(s):

Agustín Joison ◽

Gustavo Baiardi ◽

Rocío Donalisio ◽

Federico Gallo

Keyword(s):

Amino Acid ◽

Fibrinolytic Activity ◽

Sodium Borate ◽

Isoelectric Precipitation ◽

Fibrin Plate ◽

Blood Clots ◽

Human Plasminogen ◽

Plasma Glycoprotein ◽

Sodium Borate Buffer ◽

Polypeptide Chains

Background: Plasminogen is a blood plasma glycoprotein of molecular weight about 92,000 Daltons. Physiologically, it incorporates into blood clots and after its activation by plasminogen activators to plasmin can perform a fibrinolytic function. Microplasmin is truncate polypeptide chain derivate of plasmin may be increase the fibrinolytic activity. Objective: To study the amino acid sequence of two polypeptides chains derivate to the plasminogen with fibrinolytic activity. Methods: he two polypeptides chains were prepared by isoelectric precipitation of human plasma in sodium borate buffer. The sample in a second step was subjected to affinity and ionic interchange chromatography and denaturalized electrophoresis was carried out on the sample previous heat 70ºC. Results: Two polypeptide chains of 29.000 and 35.000 Daltons by autolysis controlled were obtained with 25 UI of fibrinolytic activity in fibrin plate. Conclusion: Microplasmin was obtained with cleavage in different amino acid bounds and rearrangement of amino acids by autolysis with controlled alkaline precipitation.

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