Pericellular Proteins of the Developing Mouse Tendon: A Proteomic Analysis

Abstract

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.

• fibroblast surface
• collagen fibrillogenesis
• development
• extracellular matrix
• surfaceome

ACKNOWLEDGMENTS

Supported by NIH/NIAMS grants AR044745 (DEB) and NRSA Fellowship AR056937 (SMS). The Moffitt Proteomics Facility is supported by the US Army Medical Research and Material Command under Award No. DAMD17-02-2-0051 for a National Functional Genomics Center, a Cancer Center Support Grant from the National Cancer Institute under Award No. P30-CA076292, and the Moffitt Foundation. We thank Dr. Brenda Flam for helpful discussion on CD44 and generous gift of BD Biosciences anti-CD44 antibody.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.