Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties.
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.
Cardiovascular Research Department, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0434.
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Berg
P J
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Burck
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Berg
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Grinnell
D001483:Base Sequence; D005337:Fibrin / Q000378:metabolism / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D006031:Glycosylation; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D008239:Lysine