使用抗基质金属蛋白酶产生的聚集蛋白聚糖新表位FVDIPEN-COOH的抗体来评估基质溶解素在兔软骨降解模型中的作用。

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

作者信息

Bayne E K, MacNaul K L, Donatelli S A, Christen A, Griffin P R, Hoerrner L A, Calaycay J R, Ayala J M, Chapman K, Hagmann W

机构信息

Merck Research Laboratories, Rahway, NJ 07065, USA.

出版信息

Arthritis Rheum. 1995 Oct;38(10):1400-9. doi: 10.1002/art.1780381007.

DOI:10.1002/art.1780381007

PMID:7575690

Abstract

OBJECTIVE

To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo.

METHODS

The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay.

RESULTS

Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor.

CONCLUSION

These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

摘要

目的

确定兔聚集蛋白聚糖球间结构域中的基质溶解素切割位点，并确定基质溶解素产生的新表位是否可作为体内基质金属蛋白酶（MMP）活性的标志物。

方法

通过逆转录-聚合酶链反应互补DNA克隆和DNA序列分析确定兔聚集蛋白聚糖的基质溶解素产生的透明质酸结合区域（HABR）的羧基末端序列，随后对HABR片段进行纯化和质谱蛋白质序列分析。将活性基质溶解素注射到兔的膝关节中，通过蛋白质印迹分析基质溶解素产生的聚集蛋白聚糖新表位，并通过间接免疫荧光进行原位定位。通过二甲基亚甲基蓝染料结合试验对关节液中的蛋白聚糖片段进行定量。