Abstract
Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.
Highlights
Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues
We have shown that the lysyl oxidase (LOX) precursor and the forms processed by BMP1 or ADAMTS2/14 proteases displayed similar enzymatic activity on a soluble substrate, they substantially differed in their capacity to bind collagen, with the sulfotyrosine residues playing an important role in the interaction
By using a polyclonal antibody that recognizes the C-terminal end of LOX protein, we have analyzed the electrophoretic pattern of LOX secreted from human lung fibroblasts and bovine endothelial cells incubated for 4 days in the presence or absence of the cytokine transforming growth factor-1 (TGF-1), a well-known activator of LOX expression
Summary
The mechanical strength of the extracellular collagenous matrix is highly dependent on the formation of covalent crosslinks within fibrils, a process initiated by the enzymatic action of lysyl oxidase (LOX) [4]. The molecular mechanisms leading to this behavior, which may arise by alternative proteolytic processing and because of post-translational modifications (PTM), have not been established, and its study might shed fresh light on the biological function of LOX In this context, we recently identified, using a large-scale and nonbiased approach, that LOX is a potential substrate of ADAMTS14, a complete characterization of this potential proteolysis remains to be investigated, as well as its relevance in the regulation of LOX [15]. We have shown that the LOX precursor and the forms processed by BMP1 or ADAMTS2/14 proteases displayed similar enzymatic activity on a soluble substrate, they substantially differed in their capacity to bind collagen, with the sulfotyrosine residues playing an important role in the interaction Taken together, these results point to a mechanism for the regulation of the binding and the activity of LOX on collagen, resulting from the differential cleavage by BMP1 and ADAMTS2/14 of a tyrosine sulfate domain
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