Zhen Eugene Y, Brittain Isabelle J, Laska Dennis A, Mitchell Peter G, Sumer Eren U, Karsdal Morten A, Duffin Kevin L
Eli Lilly & Company, Greenfield, IN 46140, USA.
Arthritis Rheum. 2008 Aug;58(8):2420-31. doi: 10.1002/art.23654.
To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage.
Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry.
Complete sequences of the peptides proteolyzed from human articular cartilage, including N- and C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases.
The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.
鉴定、表征和比较人关节软骨金属蛋白酶消化产生的蛋白水解肽产物。
通过添加外源性金属蛋白酶，包括基质金属蛋白酶2、3、8、9、12和13以及聚集蛋白聚糖酶ADAMTS - 4和ADAMTS - 5来消化人关节软骨。使用质谱通过蛋白质组学方法鉴定蛋白水解肽产物。
确定了从人关节软骨中蛋白水解得到的肽的完整序列，包括N端和C端以及翻译后羟基化修饰。分析了金属蛋白酶消化后源自I型、II型和III型胶原蛋白、双糖链蛋白聚糖、脯氨酸富含蛋白、纤调蛋白、纤连蛋白、核心蛋白聚糖、软骨寡聚基质蛋白、软骨中间层蛋白、巨核细胞刺激因子、 mimecan、聚集蛋白聚糖和光蛋白聚糖的多种肽。肽的释放随时间、酶特异性和丰度而变化。在选定蛋白酶释放后，观察到特定的II型胶原蛋白肽生物标志物，包括那些含有全长四分之三片段切割位点的以及那些含有II型胶原蛋白螺旋肽结构域和II型胶原蛋白C端肽结构域的。
在测定中使用完整软骨而非纯化的蛋白质底物，使得在更生理相关的条件下能够鉴定单个酶的新型潜在底物和切割位点。这些软骨基质肽的表征可能有助于软骨降解药效学生物标志物的开发，也可能有助于理解软骨细胞信号传导中重要的生物活性肽。
