Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia.
Mol Cell Proteomics. 2010 Jun;9(6):1296-313. doi: 10.1074/mcp.M000014-MCP201. Epub 2010 Feb 26.
Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.
关节软骨对于关节功能不可或缺，但自我修复能力有限。因此，体外工程化生成新的软骨成为关节炎自体软骨修复的主要目标。先前对新生成软骨的分析主要针对细胞组织和特定的分子成分。然而，蛋白质组学尚未研究新生成软骨中细胞外基质（ECM）的复杂性。为了解决这个问题，我们开发了一种产生软骨细胞外基质的小鼠新生成软骨培养系统。使用基于溶解度的蛋白质分组分析 3 周龄新生成软骨和 3 天龄新生鼠软骨的组织蛋白质组，靶向新生成软骨发育相关的成分，包括 ECM 成熟。最初，通过 SDS-PAGE 分析连续提取液，发现在从幼年软骨中高比例易溶性（NaCl 提取）蛋白到新生成软骨中高比例难溶性（盐酸胍提取）蛋白的转变。随后使用无标记定量质谱（LTQ-Orbitrap）和统计分析来筛选三个显著的蛋白质组：根据提取条件富集的蛋白质、幼年软骨和新生成软骨之间差异丰富的蛋白质以及两种组织类型之间具有不同溶解度特性的蛋白质。使用生物信息学对 NaCl 和盐酸胍提取物中差异丰富的蛋白质（n = 403）进行分类，有效分离了易溶性成分和较大蛋白质复合物的亚基。在新生成软骨中显著富集的蛋白质（n = 78）包括先前未报道或在软骨中功能未知的蛋白质（整合素结合蛋白 DEL1；卷曲螺旋域蛋白 80；弹力蛋白 1 和色素上皮衍生因子）。在幼年软骨和新生成软骨之间具有不同提取能力的蛋白质包括 ECM 成分（腱糖蛋白-2、蛋白聚糖、胶原 VI、基质金属蛋白酶 3、纤维连接蛋白和血小板反应蛋白 1），并且蛋白质的提取能力与 ECM 超微结构组织之间的关系得到了电子显微镜的支持。此外，一种盐酸胍提取物特异性的新生成软骨蛋白，蛋白酶抑制剂 1，通过免疫组织化学被确认为体内关节软骨发育的新成分。该提取谱和基质相关免疫染色提示蛋白酶抑制剂 1 参与了体外和体内软骨发育。
