Structure-Function Relationship of Neutrophil Collagenase (MMP-8) Analysis of Critical Region Responsible for Substrate Specificity

• Published in: Juntendo Medical Journal Vol. 57; no. 5; pp. 504 - 511
• Main Authors: KIKUCHI, Ken, A.HASTY, KAREN, HIROSE, TOMOHIKO, KANEKO, KAZUO, YAMAUCHI, YASUO
• Format: Journal Article
• Language: English; Japanese
• Published: The Juntendo Medical Society 31.10.2011
• Subjects: binding assay; collgenase activity; MMP-3 (stromelysin 1); MMP-8 (neutrophil collagenase); substrate specificity
• ISSN: 0022-6769; 2188-2134
• DOI: 10.14789/pjmj.57.504

Objectives: To investigate the determinations for substrate specificity of collagenase.Materials and Methods: We expressed recombinant human neutrophil collagenase (MMP-8) and chimeric enzymes with human stromelysin 1 (MMP-3), fused at different positions in the non catalytic carboxy-terminal (C-terminal or hemopexin) domain or substituted the linker region by its counterpart, and examined their enzymatic activity. Furthermore, the ability of these enzymes to bind substrate was tested against native fibrillar collagen and triple helical fragments of collagen derived from collagenase degradation.Results: The results of the binding assay demonstrated that MMP-8 was bound to native collagen but not to collagenase-derived fragments. In contrast, the two chimeric enzymes in which the carboxy-terminal portions were derived from MMP-3 bound not only to native collagen but also to its fragments and showed remarkably different collagenolytic activities. The third chimera, the hinge region of which was replaced by that homolog of MMP-3, maintained the exclusive binding feature of wild type, though the collagenolytic activity of this hybrid was reduced.Conclusions: Our data indicate that the N-terminus portion of the non-catalytic domain of collagenase is critical to the recognition of the cleavage site of collagen and consequently for substrate specificity.