Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: Posttranslational modificationof I alpha I-derived bikunin

Inter-alpha-inhibitor-derived bikunin was purified and the molecular mass w as determined to be similar to 8.7 kDa higher than the prediction based on the protein sequence, suggesting extensive posttranslational modifications. These modifications were identified and characterized by a combination of protein and carbohydrate analytical techniques. Three modifications were id entified: (i) glycosylation of Ser(10), (ii) glycosylation of Asn(45), and (iii) a heterogeneous truncation of the C-terminus. The Asn45 associated gl ycan was shown to be a homogenous "complex type" biantennary structure. The chondroitin-4-sulfate (CS) chain attached to Ser(10) was analyzed by both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) an d acrylamide gel electrophoresis after partial chondroitin ABC lyase digest ion. The analyses showed that the CS chains were composed of 15 +/- 3 [GlcU A-GalNAc] disaccharide units. On average, every forth disaccharide was sulf ated, and these sulfated disaccharides appeared to be more common near the reducing end. Anion exchange chromatography at pH 3.4 of intact bikunin res ulted in the isolation of four isotypes shown to differ only in the amount of sulfation. Heavy chain 1 (HCl) and heavy chain 2 (HC2) are attached to t he CS by a novel cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Th phi gersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], and the order in which the two heavy chains are positioned o n the CS was examined. The results indicate that HCl is in close proximity to HC2 and both are near the less sulfated nonreducing end of the CS. Taken to,together, the data show the following organization of the I alpha I mol ecule: [GlcUA-GaWAc](a)-HCl-[GlcUA-GalNAc](b)-HC2-[GlcUA-GalNAc](b)-Gal-Gal -Xyl-Ser(10)-bikunin (a + b + c = 12-18 disaccharides).