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Abstract

Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 m NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1–1.0 m NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5–0.8 m NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1–0.5 m NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5–0.8 m NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1–0.5 m NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.Background: The chondroitin sulfate (CS) chain of bikunin has both sulfated and unsulfated regions.Results: Bikunin CS contains heterogeneous sulfation in the chain and linkage region with CS-D containing chains facilitating HA·HC formation.Conclusion: Sulfation of the bikunin CS chain regulates HC transfer to HA.Significance: Bikunin CS chain sulfation may modulate HA tissue stabilization in disease.