Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1)

Nancy C.m. Caterina, L. Jack Windsor, M. Kirby Bodden, Audra E. Yermovsky, Kenneth B. Taylor, Henning Birkedal-Hansen, Jeffrey A. Engler

Research output: Contribution to journalArticle

19 Scopus citations


Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and M(r) 72 000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2-4-fold lower than that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose-response curve suggested that the interaction with the M(r) 72 000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and M(r) 72 000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)21-34
Number of pages14
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number1
StatePublished - Oct 14 1998
Externally publishedYes


  • Collagenase
  • Extracellular matrix
  • M(r) 72 000 Gelatinase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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