Identification of Prodomain Determinants Involved in ADAMTS-1 Biosynthesis

Jean-Michel Longpré,Richard Leduc

doi:10.1074/jbc.m313151200

Abstract

The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys106, Tyr108, Gly110, Cys125, and Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis.

Highlights

Proteolysis of extracellular substrates by the ADAMTS1 family, which consists of at least 19 members, is an important mechanism regulating events such as cartilage biosynthesis, angiogenesis, and cell motility and growth [1]
Proteolysis of the extracellular matrix and cell surface proteins mediated by metalloproteases, including MMPs, ADAMs, and ADAMTSs, is of vital importance for tissue-remodeling processes during normal and pathological conditions such as tissue morphogenesis, wound healing, inflammation, and tumor cell invasion and metastasis [1, 32]
For proADAMTS precursors, members of the proprotein convertase family would be responsible for the proteolytic cleavage of the prodomain at the junction of the catalytic domain

Summary

Introduction

Proteolysis of extracellular substrates by the ADAMTS1 (a disintegrin and metalloprotease with thrombospondin type I motif) family, which consists of at least 19 members, is an important mechanism regulating events such as cartilage biosynthesis, angiogenesis, and cell motility and growth [1]. The first member, ADAMTS-1 (peptidase M12.222, Merops data base), identified as a cachexia-associated gene expressed in colon tumor cells [2], along with ADAMTS-4 and ADAMTS-5 ( called aggrecanases), degrades to different extents the cartilage proteoglycan aggrecan and lectican or aggrecan-like proteins such as brevican and versican. This suggested a significant participation of these enzymes in conditions such as arthritis [3,4,5,6]. These observations could be of broad significance to understand the regulation of the ADAMTS proteases

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Conclusion