Abstract
Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.
Highlights
Matrix metalloproteinases (MMP)1 are a family of enzymes capable of degrading a number of components of the extracellular matrix such as proteoglycans, type I collagen, fibronectin, and laminin
A further analysis of the COOH-terminal domain demonstrated that a 67 amino acid sequence consisting of the hinge region and a part of the first hemopexin-like repeat are essential for type I collagenolytic activity of neutrophil collagenase (NC) (MMP-8) [25]
MMP-3 binds type I collagen, it does not cleave native triple helical collagen. This alignment revealed that the conserved amino acid residues Ser264, Asn266, Val268, Gln296, Gly272, Lys277, Thr284, Thr289, Asn307, and Val313 in fibroblast collagenase (FC) and NC have been substituted by Ala, Asp, Pro, Asp, Glu/Asp, Ala, Ser, Ser, Ser, and Pro in MMP-3, respectively
Summary
Plasmid Construction and Purification of Recombinant Proteins— The construction of pET/FC containing the full-length cDNA for FC has been described previously [30]. The PCR products corresponding to S264A, N266D, V268P, Q269E, and G272D mutations were digested with MaeIII and DraIII restriction enzymes and ligated with SphI/DraIII and SphI/MaeIII fragments from pET/FC to produce the final expression vectors pET/FC/S264A, pET/ FC/N266D, pET/FC/V268P, pET/FC/Q269E, and pET/FC/G272D, respectively. PET/FC/N307S expression vector was generated by digesting the N307S PCR product with DraIII and FspI restriction enzymes and ligating the PCR fragment with SalI/DraIII and SalI/FspI DNA fragments of pET/FC. The V313P PCR product was digested with restriction enzymes DraIII and NciI (a unique restriction site at the 5Ј end of the DNA fragment) and ligated with NciI/DraIII fragment from pET/FC to produce the expression vector pET/FC/V313P. The active enzymes (0.6 M) were incubated with 380 g/ml triple helical native bovine type I collagen (Fluka Biochemika, Milwaukee, WI) in an assay buffer containing 50 mM Tris, pH 7.5, 5 mM CaCl2, 150 mM NaCl at 27 °C for 18 h. The substitutions were made, and the energy of the resulting structure was minimized by the Biopolymer and Discover modules, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
