Organization of the Inter-α-Inhibitor Heavy Chains on the Chondroitin Sulfate Originating from Ser10 of Bikunin: Posttranslational Modification of IαI-Derived Bikunin
- 19 August 1999
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 38 (36), 11804-11813
- https://doi.org/10.1021/bi9908540
Abstract
Inter-α-inhibitor-derived bikunin was purified and the molecular mass was determined to be ∼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 identified: (i) glycosylation of Ser10, (ii) glycosylation of Asn45, and (iii) a heterogeneous truncation of the C-terminus. The Asn45 associated glycan was shown to be a homogenous “complex type” biantennary structure. The chondroitin-4-sulfate (CS) chain attached to Ser10 was analyzed by both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and acrylamide gel electrophoresis after partial chondroitin ABC lyase digestion. The analyses showed that the CS chains were composed of 15 ± 3 [GlcUA-GalNAc] disaccharide units. On average, every forth disaccharide was sulfated, and these sulfated disaccharides appeared to be more common near the reducing end. Anion exchange chromatography at pH 3.4 of intact bikunin resulted in the isolation of four isotypes shown to differ only in the amount of sulfation. Heavy chain 1 (HC1) and heavy chain 2 (HC2) are attached to the CS by a novel cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thøgersen, 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 on the CS was examined. The results indicate that HC1 is in close proximity to HC2 and both are near the less sulfated nonreducing end of the CS. Taken together, the data show the following organization of the IαI molecule: [GlcUA-GalNAc]a-HC1-[GlcUA-GalNAc]b-HC2-[GlcUA-GalNAc]c-Gal-Gal-Xyl-Ser10-bikunin, (a + b + c = 12−18 disaccharides).Keywords
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