Glycosylation and NH 2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1)

Abstract

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 NH 2-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.