Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis

Knauper V, Docherty AJP, Smith B, Tschesche H, Murphy G (1997)
FEBS LETTERS 405(1): 60-64.

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Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme, The mutagenesis of the amino acid residues in the P-1' position of the two autoproteolytically cleaved peptide bonds (Leu(243) and Ile(248)) to Ala showed that the mutant enzymes were more resistant to autoproteolysis, However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala(239) - Ile(240) peptide bond and the half-life of the active enzyme was increased by 50%, In contrast, mutagenesis of Pro(247) --> Ala (P-1 of the minor cleavage site Pro(247) - Ile(248)) lead to increased susceptibility of the enzyme to autoproteolysis, However,when the other P-1 position Gly(242) was altered to Ala no effect on stability was observed, The analysis of the ability of the mutant active enzymes to hydrolyse C-14-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly(242)-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro(247) sequence motif to Gly(242)-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro(247) showed that the collagenolytic activity was reduced by 68.4%, In addition, mutagenesis of the downstream sequence motif Pro(247)-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro(258) to Pro(247)-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro(258) had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%, When the Pro residues in the hinge motif (Pro(247), Pro(250), Pro(253) and P-256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme. (C) 1997 Federation of European Biochemical Societies.
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Knauper V, Docherty AJP, Smith B, Tschesche H, Murphy G. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS. 1997;405(1):60-64.
Knauper, V., Docherty, A. J. P., Smith, B., Tschesche, H., & Murphy, G. (1997). Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS, 405(1), 60-64.
Knauper, V., Docherty, A. J. P., Smith, B., Tschesche, H., and Murphy, G. (1997). Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS 405, 60-64.
Knauper, V., et al., 1997. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS, 405(1), p 60-64.
V. Knauper, et al., “Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis”, FEBS LETTERS, vol. 405, 1997, pp. 60-64.
Knauper, V., Docherty, A.J.P., Smith, B., Tschesche, H., Murphy, G.: Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis. FEBS LETTERS. 405, 60-64 (1997).
Knauper, V, Docherty, AJP, Smith, B, Tschesche, Harald, and Murphy, G. “Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis”. FEBS LETTERS 405.1 (1997): 60-64.
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