Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum.
Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum.
复制标题
使用单特异性抗肽血清对基质金属蛋白酶生成的聚集蛋白聚糖 G1 片段进行定量。
DOI：
10.1042/bj3070245
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Richard A. MUMFORDt
中科院分区：
文献类型：
--
作者：
W. Michael;Lark;H. Williams;Lori A. Hoernner;J. Weidner;J. Ayala;C. Harper;A. Christen;J. Olszewski;Z. Konteatis;R. Webber;Richard A. MUMFORDt
Several members of the matrix metalloproteinase family have been reported to cleave aggrecan in the interglobular domain between Asn-341 and Phe-342. An antiserum was prepared against a peptide conjugate corresponding to the C-terminal sequence of the matrix metalloproteinase-generated aggrecan G1 fragment (Phe335-Val-Asp-Ile-Pro-Glu-Asn341). A quantitative radioimmunoassay, with a limit of detection of about 80 pM, was developed using this antiserum. This antiserum requires the free carboxyl group of the C-terminal asparagine for optimal recognition. If the C-terminal asparagine is excised from the sequence, replaced with closely related amino acids, or extended across the matrix metalloproteinase cleavage site, there is a 40-10,000-fold loss in detection. Using peptides cleaved from the N-terminus, it was determined that the antiserum requires the entire Phe-Val-Asp-Ile-Pro-Glu-Asn sequence for optimal recognition. The radioimmunoassay detects matrix metalloproteinase-generated G1 fragments with similar sensitivity to the Phe-Val-Asp-Ile-Pro-Glu-Asn peptide, but it does not recognize intact aggrecan. Immunoreactive aggrecan G1 fragments of molecular mass 50 kDa are generated by the matrix metalloproteinases stromelysin and gelatinase A. In contrast, under identical conditions, the closely related metalloproteinases, gelatinase B and collagenase, as well as cathepsin G, cathepsin B and human leucocyte elastase, did not generate a G1 fragment recognized by the antiserum. The anti-Phe-Val-Asp-Ile-Pro-Glu-Asn serum detects stromelysin-generated aggrecan G1 fragments from mouse, guinea pig, rabbit and human, indicating that the detection is not species-specific. This antiserum and radio-immunoassay should be useful for quantifying and characterizing matrix metalloproteinase-generated aggrecan G1 fragments in articular cartilage and synovial fluids from humans and various animal models of articular-cartilage destruction.
