Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis

1993 ◽  
Vol 65 (21) ◽  
pp. 2953-2962 ◽  
Author(s):  
Andrew W. Guzzetta ◽  
Louisette J. Basa ◽  
William S. Hancock ◽  
Bruce A. Keyt ◽  
William F. Bennett
Keyword(s):  
Special Reference ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ion Extraction ◽  
Tissue Plasminogen ◽  
Peptide Maps

Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Keri M. Tate ◽  
Deborah L. Higgins ◽  
William E. Holmes ◽  
Marjorie E. Winkler ◽  
Herbert L. Heyneker ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Tissue ◽  
Functional Role ◽  
Proteolytic Cleavage ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Tissue Plasminogen

scholarly journals Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

2001 ◽  
Vol 67 (6) ◽  
pp. 2657-2664 ◽  
Author(s):  
Jiradej Manosroi ◽  
Chatchai Tayapiwatana ◽  
Friedrich Götz ◽  
Rolf G. Werner ◽  
Aranya Manosroi
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Stop Codon ◽  
Active Form ◽  
Site Directed Mutagenesis ◽  
Amidolytic Activity ◽  
Tissue Plasminogen ◽  
Kringle 2

ABSTRACT The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused togpIII of ΦM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII,a stop codon between K2S and the gpIIIgene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-d-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

scholarly journals Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1312-1322
Author(s):  
DT Berg ◽  
PJ Burck ◽  
DH Berg ◽  
BW Grinnell
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Functional Properties ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Tissue Plasminogen ◽  
Kringle 2

Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.

scholarly journals Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1312-1322 ◽  
Author(s):  
DT Berg ◽  
PJ Burck ◽  
DH Berg ◽  
BW Grinnell
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Functional Properties ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Tissue Plasminogen ◽  
Kringle 2

Abstract Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.

Sign in / Sign up

Export Citation Format

Share Document