Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

Federica Genovese,Martin Røssel Larsen,Arkadiusz Nawrocki,Morten Asser Karsdal,Diana Julie Leeming,Lise Larsen,Jianxia Wang,Qinlong Zheng,Natasha Barascuk,Sanne Skovgård Veidal,Yili Li

doi:10.1186/1755-1536-6-9

Abstract

BackgroundThe proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR.ResultsBiglycan was cleaved in vitro by MMP-9 and -12 and the 344′YWEVQPATFR′353 peptide (BGM) was chosen as a potential neo-epitope. A technically sound competitive ELISA for the measurement of BGM was generated and the assay was validated in a bovine cartilage explant culture (BEX), in a collagen induced model of rheumatoid arthritis (CIA) and in two different rat models of liver fibrosis: the carbon tetrachloride (CCL4)-induced fibrosis model, and the bile duct ligation (BDL) model. Significant elevation in serum BGM was found in CIA rats compared to controls, in rats treated with CCL4 for 16 weeks and 20 weeks compared to the control groups as well as in all groups of rats subject to BDL compared with sham operated groups. Furthermore, there was a significant correlation of serum BGM levels with the extent of liver fibrosis determined by the Sirius red staining of liver sections in the CCL4 model.ConclusionWe demonstrated that the specific tissue remodeling product of MMPs-degraded biglycan, namely the neo-epitope BGM, is correlated with pathological ECMR. This assay represents both a novel marker of ECM turnover and a potential new tool to elucidate biglycan role during the pathological processes associated with ECMR.

Highlights

The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis
Selection of neo-epitope by mass spectrometry Purified bovine biglycan was cleaved with a variety of matrix metalloproteinase (MMP) including MMP-9 and −12, and 120 unique biglycan peptides were identified in the cleaved material
Antibodies were generated against sixteen neo-epitope sequences, and based on the reactivity against the selection peptide, the specificity for the cleaved biglycan, and the reactivity against native material, one of the antibodies recognizing one of the peptides identified by LC-MS/MS was selected for assay development

Summary

Introduction

The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR. The biglycan core protein contains leucine-rich repeats that facilitate proteinprotein interactions: this proteoglycan is able to bind to the membrane-bound proteoglycan dystroglycan and to a wide variety of proteins, being involved in, for instance, cell signal transduction during cell growth and differentiation and in regulating cytokine activity due to its capacity to bind TGF-β and TNF-α [4]. It has been demonstrated that the activity of TGF-β1 is strictly related to the presence of biglycan in vivo, as biglycandeficient mice have shown elevated levels of both total and bioactive TGF-β1 in plasma [8]

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