Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the n- and c-termini

Nancy L. Haigwood, Guy T. Mullenbach, Gregory K. Moore, Lucy E. Desjardin, Azita Tabrizi, Sheryl L. Brown-Shimer, H. Stauß, H. A. Stöhr, Eric Paul Paques

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Mutations were directed to specific regions of the human tissue-type plasminogen activator (t-PA) gene in an effort to better define structure-function relationships of the enzyme. Three types of modifications were effected by in vitro mutagenesis: elimination of glycosylation sites; substitutions of amino acids at the cleavage site for conversion of single-chain t-PA to two-chain t-PA; and truncations of the N- and C-termini. Thirteen variants were purified from permanent CHO cell lines and analyzed for specific activity, fibrin stimulation, fibrin binding, inhibition by plasminogen activator inhibitor-2 (PAI-2) and half-life. The results of these analyses are: (i) variants with carbohydrate-depleted kringle domains possessed higher specific activities than wild-type t-PA; (ii) a cleavage site variant substituted at Arg275 with Gly had greatly reduced specific activity; (iii) two variants substituted at Lys277 exhibited altered interactions with PAI-2; (iv) the variant with a truncated C-terminus had reduced activity in the absence of fibrin; and (v) no variants had significantly altered half-lives. In order to test the effects of combining mutations, four additional variants were produced. Each combination variant retained at least one of the altered properties observed in the original variants, and in three of the variants the diverse properties were additive.

Original languageEnglish (US)
Pages (from-to)611-620
Number of pages10
JournalProtein Engineering, Design and Selection
Volume2
Issue number8
DOIs
StatePublished - Aug 1989
Externally publishedYes

Keywords

  • Cleavage site variants
  • Glycosylation variants
  • Plasminogen activator
  • Site-directed mutagenesis
  • Truncation variants

ASJC Scopus subject areas

  • Medicine(all)

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