Bioactivity in an aggrecan 32mer fragment is mediated via toll-like receptors
Fosang, AJ and Golub, S and Last, K and Lees, S and Wilson, RR and Aspberg, A and Little, CB and Sutton, P, Bioactivity in an aggrecan 32mer fragment is mediated via toll-like receptors, Proceedings of the 9th Pan Pacific Connective Tissue Societies Symposium, November 24-27 2013, Hong Kong, pp. 0076. (2014) [Conference Extract]
The aim of this study was to determine whether the naturally-occurring aggrecan 32mer fragment was
bioactive, and if so, to elucidate the mechanism.
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.
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.
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.