GLYCOSYLATION AND NH2-TERMINAL DOMAIN MUTANTS OF THE TISSUE INHIBITOROF METALLOPROTEINASES-1 (TIMP-1)

Mutants in the tissue inhibitor of metalloproteinases-(1) (TIMP-1) pro tein have been created by site-directed mutagenesis and expressed in H eLa cells, using a recombinant vaccinia virus system. Removal of eithe r or both glycosylation sites yielded proteins which retained wild-typ e inhibitory activity against both human fibroblast-type collagenase ( FIB-CL) and M-r 72 000 gelatinase (GL). However, the double glycosylat ion mutant protein was expressed at a level that was 2-4-fold lower th an that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 resi dues was also inhibitory, but the dose-response curve suggested that t he interaction with the M-r 72 000 gelatinase had been altered. A numb er of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRA K sequence which is absolutely conserved in all TIMPs in all species ( VIRAK to VIAAA), also yielded functional proteins capable of inhibitin g 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 multi ple interactions. (C) 1998 Elsevier Science B.V. All rights reserved.