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Protein Engineering vol. 9 no. 3 pp. 283-290, 1996
© 1996 Oxford University Press

RESEARCH-ARTICLE

Locating the unpaired cysteine of tissue-type plasminogen activator

Louis c. Sehl1, Hung V. Nguyen, Lea T. Berleau, Patricia Arcila, William F. Bennett1 and Bruce A. Keyt2

Department of Cardiovascular Research, Genentech, Inc., 460 Point San Bruno Blvd, South San Francisco, CA 94080, USA

2To whom correspondence should be addressed

Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module. C75A, C83A, C84A and CC83–84AA variants of t-PA were expressed transiently in human embryonic kidney cells. The biochemical properties of these variants provided experimental evidence to identify the unpaired cysteine in t-PA. Assays of amidolytic activity, plasminogen activation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for comparing these variants with wild-type t-PA. In all assays, C83A t-PA was biochemically equivalent to wild-type t-PA. C75A t-PA, C84A t-PA and CC83-84AA t-PA variants exhibited reduced activities in a variety of functional assays. These variants displayed two- to threefold lower activity in fibrinogen or fibrin stimulated plasminogen activation, and fivefold reduced plasma clot lysis activity compared with that of wild-type t-PA. The affinity of C75A t-PA and C84A t-PA for fibrin was decreased more than two orders of magnitude compared with C83A t-PA or wild-type t-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-fold in mice. The C75A, C84A and CC83–84AA variants displayed significantly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes. These data are consistent with a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as an unpaired sulfhydryl. The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.

Keywords: free sulfhydryl group/site-specific mutants/tissuetype plasminogen activator/unpaired cysteine

Received August 9, 1995; revised November 7, 1995; accepted November 13, 1995.


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