Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.
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 P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) 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 Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-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 Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.
Knäuper
V
V
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge, UK. vk.srl@mrc-lmb.cam.ac.uk
Docherty
A J
AJ
Smith
B
B
Tschesche
H
H
Murphy
G
G
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020784:Matrix Metalloproteinase 8; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D002250:Carbon Radioisotopes; D011993:Recombinant Fusion Proteins; D003094:Collagen; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; D000409:Alanine