Department of Pathology and Cell Biology, University of South Florida College of Medicine, Tampa, FL, USA.
Connect Tissue Res. 2012;53(1):2-13. doi: 10.3109/03008207.2011.602766. Epub 2011 Aug 18.
Tendon fibroblasts synthesize and assemble collagen fibrils, the basic structural unit of tendons. Regulation of fibrillogenesis is essential for tendon development and function. Fibril assembly begins within extracellular micro-domains associated with the fibroblast surface. We hypothesize that molecules crucial to the regulation of fibril assembly are membrane associated and/or within the pericellular micro-environment. This report defines proteins in the surfaceome, that is, plasma membrane and pericellular matrix, from mouse flexor digitorum longus tendons. Proteomic analysis identified a set of surfaceome molecules including collagens, fibronectin, integrins, proteoglycans, and receptors in extracts from mouse tendons at postnatal day 1, a developmental stage when collagen protofibril nucleation and initial steps in fibril assembly predominate. The proteomic results were validated for molecules identified with a small number of unique peptides and/or low sequence coverage. For these analyses, proteins were selected based on their potential roles in fibril nucleation, that is, collagen V; organization of fibrillogenesis, that is, integrins and fibronectin; and known localization to the plasma membrane with potential to impact matrix assembly, that is, CD44, syndecan-1, epidermal growth factor receptor, and matrix metalloproteinase 25. These molecules were all detected in extracts of the developing tendon, demonstrating that the surfaceome included molecules hypothesized to regulate fibrillogenesis as well as many with no known function in this capacity. This report, therefore, generates an unbiased set of cell surface-associated molecules, providing a resource to identify novel or unexpected regulatory molecules involved in collagen fibril and matrix assembly.
肌腱成纤维细胞合成和组装胶原原纤维，这是肌腱的基本结构单位。纤维原形成的调节对于肌腱的发育和功能至关重要。纤维的组装始于与成纤维细胞表面相关的细胞外微区。我们假设对纤维组装调节至关重要的分子与膜相关或位于细胞周微环境中。本报告定义了从小鼠屈趾长肌腱中提取的表面体（即质膜和细胞周基质）中的蛋白质。蛋白质组学分析鉴定了一组表面体分子，包括在出生后第 1 天的小鼠肌腱提取物中的胶原蛋白、纤连蛋白、整合素、蛋白聚糖和受体，这是胶原原纤维成核和纤维组装初始步骤占主导地位的发育阶段。对于用少数独特肽和/或低序列覆盖率鉴定的分子，对蛋白质组学结果进行了验证。对于这些分析，根据它们在纤维核形成中的潜在作用选择蛋白质，即胶原蛋白 V；纤维形成的组织，即整合素和纤连蛋白；以及已知定位于质膜并有可能影响基质组装的蛋白质，即 CD44、 syndecan-1、表皮生长因子受体和基质金属蛋白酶 25。这些分子都在发育中的肌腱提取物中被检测到，表明表面体包括假设调节纤维原形成的分子以及许多在该功能中没有已知功能的分子。因此，本报告生成了一组无偏的细胞表面相关分子，为鉴定参与胶原原纤维和基质组装的新的或意外的调节分子提供了资源。
