Binding of proteins to the PDZ domain regulates proteolytic activity of HtrA1 serine protease.
HtrA1, a member of the mammalian HtrA (high temperature requirement A) serine protease family, has a highly conserved protease domain followed by a PDZ domain. Accumulating evidence has indicated that PDZ domains regulate protease activity of HtrA proteins. We searched for binding partners of the PDZ domain of mouse HtrA1 by yeast two-hybrid screening, and isolated proteins that were recognized by the HtrA1 PDZ domain through their C-terminal ends with a core consensus Phi-X-Phi-[V/L/F/A]-COOH sequence (where Phi is a hydrophobic/non-polar amino acid). C-propeptides of fibrillar collagens were most frequently isolated. Type III procollagen alpha1 C-propeptide, which was used as a model protein, was digested by HtrA1. HtrA1 cleavage of the collagen C-propeptide was enhanced by reductive denaturation of the C-propeptide and partly inhibited by removal of the C-terminal four amino acids from the C-propeptide, suggesting that the substrate recognition was facilitated by the binding of the free C-terminal ends of substrates to the PDZ domain of HtrA1. The synthetic oligopeptide (GM130Pep) that fitted the consensus recognition sequence bound to HtrA1 with a high affinity (K(d)=6.0 nM). GM130Pep stimulated HtrA1 protease activity 3- to 4-fold, but did not efficiently stimulate the activity of an HtrA1 mutant lacking the PDZ domain, supporting the notion that the PDZ domain enhances protease activity upon ligand binding. The peptide derived from Type III collagen alpha1 C-propeptide specifically stimulated protease activity of HtrA1, but did not stimulate nor significantly bind to HtrA2, suggesting that the collagen C-propeptide is a specific physiological regulator of HtrA1.
Murwantoko
Division of Gene Function in Animals, Nara Institute of Science and Technology, 891605 Takayama, Ikoma, Nara 630-0101, Japan.
Yano
Masato
M
Ueta
Yoshifumi
Y
Murasaki
Ai
A
Kanda
Hidenobu
H
Oka
Chio
C
Kawaichi
Masashi
M
D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D024061:Collagen Type III / Q000378:metabolism / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D010455:Peptides / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D020798:Two-Hybrid System Techniques
C439971:COL3A1 protein, human; D024061:Collagen Type III; D010455:Peptides; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases