Kringle glycosylation in a modified human tissue plasminogen activator
improves functional properties
DT Berg, PJ Burck, DH Berg and BW Grinnell
Cardiovascular Research Department, Lilly Research Laboratories, Eli Lilly
and Company, Indianapolis, IN 46285-0434.
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.
Volume 81,
Issue 5,
pp. 1312-1322,
03/01/1993
Copyright © 1993 by The American Society of Hematology
