Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1).
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.
National Institute of Dental Research, National Institutes of Health, Bldg. 30, Room 132, Bethesda, MD 20892, USA.
N C
NC
Caterina
L J
LJ
Windsor
M K
MK
Bodden
A E
AE
Yermovsky
K B
KB
Taylor
H
H
Birkedal-Hansen
J A
JA
Engler
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics
D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1