TYPE V COLLAGENASE IN RHEUMATOID ARTHRITIS

细胞外基质的降解需要协调

The degradation of the extracellular matrix requires the coordinated action of enzymes which constitute a family of metalloproteinases. The enzymes which make up this family are related in that they are released from the cell in a latent form, they contain zinc and require divalent cations for biological activity. Together, these enzymes have the ability to degrade virtually all components of the extracellular matrix. Nowhere is this degradative ability more apparent than in rheumatoid arthritis. In this disease, excessive production of metalloproteinases mediates the degradation of articular cartilage and subchondral bone, resulting in severe deformity. Type V collagenase is a member of this multigene family of enzymes which exhibits high specificity for denatured collagen, degrades native type V collagen and cleaves type XI collagen which is a structural component of cartilage. The main objective of the proposed research is to understand the regulation of this enzyme and to define its precise role and significance in rheumatoid arthritis. Towards the achievement of this goal we propose to 1. study the mechanism of activation of type V collagenase by identifying the sequences in the structure of the enzyme responsible for this process using site-directed mutagenesis and NMR spectroscopy. This will be done by generating a series of mutations in the conserved amino acid sequence (PRCGVPD) and assessing the effect of the substitutions on enzyme latency and conformation. Amino acid side chain moieties interacting with catalytic zinc ion will be identified and confirmed by 113Cd-NMR spectroscopy of Cd-substituted wild type and mutant enzymes, 2. examine the function and significance of the presumptive active site residues in catalysis and ligand binding. A series of mutations will be created in the active site of the latent enzyme and the recombinant mutant enzymes will be assayed for their ability to degrade and/or bind to type V and XI collagen substrates. The mutant proteins will be evaluated for their ability to bind Zn2+ and Ca2+ and divalent cation binding will be correlated with enzymatic activity and substrate binding. The number of Ca ions associated with the enzyme will be determined by NMR spectroscopy, 3. identify the acidic amino acids that function as the active base catalyst in this enzyme by site-directed mutagenesis and X-ray crystallography and, 4. investigate the role of the fibronectin binding domain and collagen-like domain in substrate specificity of type V collagenase by generating deletion mutants and constructing chimeric proteins of neutrophil collagenase/type V collagenase. These studies will further our understanding on the action of this enzyme and may lead to new therapeutic approaches which would focus on protection of articular tissues from degradation in rheumatoid arthritis.