Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

Mats Paulsson,Stefan Müller,Raimund Wagener,Anja Talke,Harald W.A Ehlen,Andreas R Klatt,Gerhard Sengle

doi:10.1074/jbc.m109.016568

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 hetero-oligomers, 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.

Highlights

Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles
The cleavage site lies in the hinge region between the VWA2 domain and the coiledcoil oligomerization domain (Fig. 1, B and C)
We have characterized the proteolytic processing of matrilins, a family of oligomeric proteins that have been shown to act as adapters in the assembly of the extracellular matrix [1, 8, 9, 11, 38]

Summary

Introduction

Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Determination of which extracellular proteases cleave which substrates is crucial to understand the physiological function of both [22]. Physiological cleavage has been described for most members of the matrilin family (4 – 6), but was not yet extensively studied. The adapter function of the matrilins may be modulated by physiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity [5]. We studied matrilin processing in some detail and identified another member of the ADAMTS family, ADAMTS5, as being able to cleave matrilin-3 and -4. Such cleavage is likely to alter the cohesion of the extracellular matrix

Methods

Results

Conclusion