Alanine scanning mutagenesis and functional analysis of the fibronectin-like collagen-binding domain from human 92-kDa type IV collagenase.

摘要: The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies the fibronectin (FN)-derived II homology unit (T2HU), T2HU-1, T2HU-2, T2HU-3. To investigate functional role this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or more CLG4B-derived T2HU. gelatin binding assays demonstrate that single copy T2HU-2 renders capable gelatin. repeats, however, differ dramatically in their capacity to bind gelatin, T2HU-1 T2HU-3 having significantly less activity than T2HU-2. Using alanine scanning mutagenesis defined amino acid residues (Arg307, Asp309, Asn319, Tyr320, Asp323) are critical for low compared was traced non-conserved Ala228-Ala Leu253-Pro. results suggest collagenase proenzyme is mediated by FN-like although presence another gelatin-binding site cannot be excluded. FN domain-mediated binding, not rate-limiting step hydrolysis enzyme.