Hyaluronan (HA) provides structural organisation to many extracellular matrices (ECM). HA is stabilised in the ECM through a trans-esterification process where the heavy chain (HC) proteins of inter-α-trypsin inhibitor (IαI) become covalently bound to HA and serve to crosslink HA through HC-HC interactions. IαI is a complex between two HCs, designated HC1 and HC2, covalently bound to the chondroitin sulphate (CS) chain attached to bikunin. This study demonstrated that the bikunin CS chain sulphation controls the formation of HA-HC.
Bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography, where fractions were analysed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions in urine that were 2B6 positive were found to contain 10% un-sulphated, 30% 4-sulphated, 20% 6-sulphated and 40% di-sulphated CS disaccharides. In contrast, the 2B6 negative fraction contained 66% un- and 34% 4-sulphated CS disaccharides. Complexes of bikunin in plasma that were 2B6 positive were able to promote the trans-esterification of HCs to HA in vitro in the presence of TSG-6 while 2B6 negative fractions had a significantly reduced capacity to transfer HCs to HA. Thus the bikunin CS chain structure is a key determinant of the stabilisation of HA-rich matrices. These data also suggest that the transfer of HCs to HA is mediated by CS chains with high levels of sulphation and we hypothesise that the sulphated regions mediate binding of co-factors required for the trans-esterification process.
Analysis of proteoglycan-enriched synovial fluid from pooled samples from individuals with rheumatoid arthritis indicated low levels of 2B6 reactivity, suggesting a limited ability to form HA-HC. These data have implications for the ability of HA to be stabilised during inflammatory processes which is essential to stabilise the matrix and prevent loss of matrix components.