Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

Abstract

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 heterooligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage and many other forms of extracellular matrix (for review, see Ref. 1). They participate in the formation of fibrillar or filamentous structures (2-7) and mediate interactions between collagencontaining fibrils (8, 9) and other matrix constituents like aggrecan (10), small leucine-rich proteoglycans (9), or COMP (11). Matrilins form homo-and hetero-oligomers by their C-terminal coiled-coil domain. In addition, the subunits contain epidermal growth factor-like and von Willebrand factor A (VWA) 2 -like domains, where the latter are presumably the major ligand binding domains (11). Mutations in matrilin-3 in humans cause different forms of chondrodysplasia (12)(13)(14) and are also linked to the development of hand osteoarthritis (15) and intervertebral disc degeneration (16).Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Proteolysis is a major post-translational modification used to modify the function of proteins. Tissue homeostasis requires a well balanced synthesis and degradation of extracellular matrix proteins, specifically mediated by protease families like matrix metalloproteinases (17), ADAMs (18), or ADAMTSs (19). The development of degenerative diseases is often accompanied by an activation of such proteases. In addition, the cleavage sometimes releases protein fragments that have comp...

Distinct roles of two alternative splice variants of matrilin-2 in protein oligomerization and proteolysis

“…The balance between the synthesis and degradation is important to homeostasis of the extracellular network (25). The adaptor functions of matrilins may be modulated by both physiological and pathophysiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity (22)(23)(24). Third, the cleavage occasionally releases protein fragments that have completely new functions (1)(2)(3).…”

“…Second, the oligomeric proteins have a bouquet-like structure and bind to a variety of different molecules (11,22). Therefore, the avidity of their interactions is dependent on the number of subunits and domains present (22)(23)(24). The balance between the synthesis and degradation is important to homeostasis of the extracellular network (25).…”

“…These oligomers are participants of the extracellular matrix network (1-3). Second, the oligomeric proteins have a bouquet-like structure and bind to a variety of different molecules (11,22). Therefore, the avidity of their interactions is dependent on the number of subunits and domains present (22)(23)(24).…”

“…Proteolysis is a major post-translational modification used to modify the function of proteins (22)(23)(24). Proteolysis is very complex.…”

Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

“…They participate in the formation of fibrillar or filamentous structures and mediate interactions between collagen fibrils and other matrix constituents (1,2). This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity (3). Although the matrilins have been extensively studied, their exact in vivo functions are not known.…”