Lark M W, Williams H, Hoernner L A, Weidner J, Ayala J M, Harper C F, Christen A, Olszewski J, Konteatis Z, Webber R
Department of Biochemical Pathology, Merck Research Laboratories, Rahway, NJ 07065, USA.
Biochem J. 1995 Apr 1;307 ( Pt 1)(Pt 1):245-52. doi: 10.1042/bj3070245.
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.
据报道，基质金属蛋白酶家族的多个成员可在天冬酰胺-341和苯丙氨酸-342之间的球间结构域切割聚集蛋白聚糖。制备了一种抗血清，其针对与基质金属蛋白酶产生的聚集蛋白聚糖G1片段（苯丙氨酸335-缬氨酸-天冬氨酸-异亮氨酸-脯氨酸-谷氨酸-天冬酰胺341）的C端序列相对应的肽偶联物。使用该抗血清开发了一种定量放射免疫测定法，检测限约为80皮摩尔。该抗血清需要C端天冬酰胺的游离羧基以实现最佳识别。如果从序列中切除C端天冬酰胺，用密切相关的氨基酸替代，或延伸至基质金属蛋白酶切割位点，则检测灵敏度会降低40至10000倍。使用从N端切割的肽段，确定该抗血清需要完整的苯丙氨酸-缬氨酸-天冬氨酸-异亮氨酸-脯氨酸-谷氨酸-天冬酰胺序列以实现最佳识别。放射免疫测定法检测基质金属蛋白酶产生的G1片段的灵敏度与苯丙氨酸-缬氨酸-天冬氨酸-异亮氨酸-脯氨酸-谷氨酸-天冬酰胺肽相似，但不识别完整的聚集蛋白聚糖。分子量为50 kDa的免疫反应性聚集蛋白聚糖G1片段由基质金属蛋白酶基质溶解素和明胶酶A产生。相比之下，在相同条件下，密切相关的金属蛋白酶明胶酶B和胶原酶，以及组织蛋白酶G、组织蛋白酶B和人白细胞弹性蛋白酶，均未产生能被该抗血清识别的G1片段。抗苯丙氨酸-缬氨酸-天冬氨酸-异亮氨酸-脯氨酸-谷氨酸-天冬酰胺血清可检测来自小鼠、豚鼠、兔子和人类的基质溶解素产生的聚集蛋白聚糖G1片段，表明该检测不具有物种特异性。这种抗血清和放射免疫测定法应有助于定量和表征来自人类及各种关节软骨破坏动物模型的关节软骨和滑液中基质金属蛋白酶产生的聚集蛋白聚糖G1片段。
