AIM The aim of this study was to determine whether the naturally-occurring aggrecan 32mer fragment was bioactive, and if so, to elucidate the mechanism. METHODS Native 32mer is derived from the aggrecan interglobular domain following cleavage by both MMP and ADAMTS enzymes. Mouse femoral head cartilage explants, epiphyseal chondrocytes, synovial fibroblasts or peritoneal macrophages were cultured in the presence of synthetic mouse 32mer (FFGVGGEDDITIQTVTWPDLELPLPRNVTEGE; Auspep, Australia) or a scrambled peptide comprising the same amino acids in a random order. Global gene expression in explants treated with IL-1α or 32mer was compared on Illumina mouse W6-6_V2 expression beadchips. Thereafter, changes in gene expression were validated in cell and explant cultures by qPCR and ELISA. RESULTS In the microarray experiment, 32mer-treated cartilage explants down-regulated expression of genes encoding matrix proteins and increased expression of pro-inflammatory/pro-catabolic genes with a striking correlation between genes that were regulated by IL-1α and 32mer treatment. Validation by qPCR confirmed that the 32mer, but not a scrambled 32mer peptide, regulated gene expression in articular chondrocytes, peritoneal macrophages and synovial fibroblasts from wildtype mice. The levels of IL-6 measured by ELISA were also significantly increased in cells treated with 32mer or IL-1α, but not the scrambled peptide. Neither gene expression nor levels of IL-6 cytokine were increased in cells harvested from mice lacking the Toll-like receptor (TLR) adaptor protein, MyD88, thus identifying the 32mer as an activator of TLR signalling. Using complementary hydropathy we derived an antigenic sequence for raising an antibody (αCG11) against a putative 32mer-binding protein, then used αCG11 immunoaffinity chromatography to isolate the putative 32mer-binding protein from 4M GuHCl extracts of human OA and juvenile cartilage. Mass spectrometry analyses identified PRELP as the major protein binding to and eluting from the immunoaffinity column in both samples; this was confirmed by Western blotting with αPRELP antibodies. Decorin and biglycan, the small leucine-rich proteoglycans related to PRELP, are recognised TLR2 and TL4 ligands. Experiments to resolve the biological role of PRELP in 32mer-mediated TLR activation are in progress. CONCLUSION These studies identify the aggrecan 32mer as an endogenous activator of TLR-mediated immunity with the potential to accelerate inflammation and cartilage destruction in vivo.

Item Type:	Conference Extract
Keywords:	proteomics
Research Division:	Biological Sciences
Research Group:	Biochemistry and Cell Biology
Research Field:	Signal Transduction
Objective Division:	Health
Objective Group:	Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:	Skeletal System and Disorders (incl. Arthritis)
Author:	Wilson, RR (Dr Richard Wilson)
ID Code:	93572
Year Published:	2014
Deposited By:	Central Science Laboratory
Deposited On:	2014-08-08
Last Modified:	2015-05-07
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