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.

FIG. 1. Annotation of proteins in cell surface-associated and membrane-associated extracts of the developing tendon

(A) LC-MS/MS of the surface-associated extract (gel on left) identified 453 proteins, 22% (98) were in the plasma membrane or extracellular matrix. The cellular compartments of the proteins are shown (pie chart). The plasma membrane and extracellular matrix proteins, collectively the surfaceome are classified based on molecular function (bar graph). (B) LC-MS/MS of the membrane extract (gel on left) identified 291 proteins, 30% (87) were in the plasma membrane, with diverse molecular functions (bar graph).The protein annotations are from Gene Ontology annotations in NCBI. Identified proteins function in binding, transport, structure, signal transduction, enzyme control and catalytic activity.

FIG. 2. Expression of selected proteins important in assembly of collagen and extracellular matrix

An extract of the developing FDL was probed for the integrins α1, α2, α5 and β1; collagen V; fibronectin (abbreviated here as Fn); the matrix receptors CD44 and syndecan-1; the receptor EGFR; and the protease MMP25 (with the processed enzyme and the larger proenzyme visible). All except integrin β1 and fibronectin were identified in the proteomics by no more than 2 peptides and/or with limited sequence coverage. These molecules were present in immuno-blots of the extracts and validate the proteomics data.

FIG. 3. Model of interactions involving matrix receptors, glycoproteins and collagen in surfaceome mediated regulation of tendon-specific fibril assembly

Based on the data from this work, we show the tendon fibroblast expressing diverse macromolecules that are expressed at the cell surface in early tendon development. Of these, the regulatory molecules, e.g., collagen V and integrins regulate critical steps in fibril assembly, deposition, and organization within the developing extracellular matrix. Glycoproteins could act as adaptor molecules for these interactions, e.g., fibronectin.