A Selective Extracellular Matrix Proteomics Approach Identifies Fibronectin Proteolysis by A Disintegrin-like and Metalloprotease Domain with Thrombospondin Type 1 Motifs (ADAMTS16) and Its Impact on Spheroid Morphogenesis

Rahel Schnellmann,Ragna Sack,Daniel Hess,Douglas S Annis,Deane F Mosher,Suneel S Apte,Ruth Chiquet-Ehrismann

doi:10.1074/mcp.ra118.000676

Abstract

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.

Highlights

The extracellular matrix (ECM)1 is a network of proteins, glycoproteins and complex carbohydrates that normally undergoes continuous remodeling via coupled proteolytic degradation and biosynthesis
Liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis of the medium of these cells detected an abundance of peptides spanning the catalytic domain to the spacer, but none derived from the C-terminal thrombospondin (TSR) repeats and the protease and lacunin (PLAC) domain (Fig. 1A, supplemental Fig. S1, supplemental Table S4)
These findings suggested that wtADAMTS16 was C-terminally processed with release of a form containing the catalytic domain through the spacer module into the medium, whereas the remaining C-terminal modules were retained within the ECM and or the cell surface

Summary

Introduction

The extracellular matrix (ECM)1 is a network of proteins, glycoproteins and complex carbohydrates that normally undergoes continuous remodeling via coupled proteolytic degradation and biosynthesis. HEK-EBNA cells stably expressing wtADAMTS16, ADAMTS16-EA or empty vector were seeded under serum-free conditions and allowed to attach for 48 h prior to fixation and phalloidin staining. ADAMTS16 is Secreted and Binds to ECM via its C-terminal Modules—Constructs expressing wt myc-tagged ADAMTS16 and their corresponding active site mutants (E432A) (Fig. 1A) were transiently or stably expressed in HEK293-EBNA cells.

Results

Conclusion