Caterina N C, Windsor L J, Bodden M K, Yermovsky A E, Taylor K B, Birkedal-Hansen H, Engler J A
National Institute of Dental Research, National Institutes of Health, Bldg. 30, Room 132, Bethesda, MD 20892, USA.
Biochim Biophys Acta. 1998 Oct 14;1388(1):21-34. doi: 10.1016/s0167-4838(98)00158-7.
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 Mr 72000 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 Mr 72000 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 Mr 72000 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.
