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Mutagenesis investigation of the molecular determinants of collagen triple helicase activity in neutrophil collagenase (MMP-8) Pelman, Gayle R.

Abstract

Collagen is a triple helical macromolecule that is one of the most complex proteins of the extracellular matrix and one of the most difficult to cleave and degrade. The physiological turnover of collagen is important for growth and repair, with abnormal breakdown of collagen contributing to numerous pathological conditions including arthritis and tumour metastasis. The mechanism of. collagenase activity has been studied extensively through the use of chimeric proteins and more recently through site-directed mutagenesis of specific residues thought to play key roles. Despite these efforts, the mechanism of collagenase cleavage of collagen remains unresolved. The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are thought to cleave all extracellular matrix proteins as well as bioactive molecules. Though the collagen triple helix is resistant to most proteases, a number of MMPs display collagenase activity. Neutrophil collagenase (MMP-8) degrades collagen types I, II and III, the major collagen components of bone, tendon, and cartilage, and is implicated in the pathogenesis of rheumatic disease. We have chosen to systematically alter a range of residues in the S₃' substrate specificity subsite of MMP-8 by site-directed mutagenesis. The aims of this project are to decipher which S₃' residues are involved in the catalytic function of the enzyme and to resolve any specific molecular determinants for collagen catalysis. Assays of synthetic peptide substrate and native type I collagen cleavage were used to measure the activity of mutant proteins in comparison to wildtype MMP-8 control. Results indicate a specific role for Tyr¹⁸⁹ in type I collagen cleavage. While the Tyr¹⁸⁹Ala mutant was catalytically competent and retained 88% of wildtype synthetic peptide activity, its specificity for type I collagen dropped to one-third of that of wildtype. Mutations of Asn¹⁸⁸ affected both the catalytic and collagenolytic activity of MMP-8. Our findings support the importance of the S₃' residues in the general catalytic competency of MMP-8 as well as its specificity for native type I collagen.

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