title: Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: posttranslational modification of IalphaI-derived bikunin.
abstract: Inter-alpha-inhibitor-derived bikunin was purified and the molecular mass was determined to be approximately 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 Ser(10), (ii) glycosylation of Asn(45), and (iii) a heterogeneous truncation of the C-terminus. The Asn(45) associated glycan 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) 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., Thogersen, 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 IalphaI molecule: [GlcUA-GalNAc](a)-HC1-[GlcUA-GalNAc](b)-HC2-[GlcUA-GalNAc](c)-Gal -Gal-Xyl-Ser(10)-bikunin, (a + b + c = 12-18 disaccharides).
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA. enghi001@mc.duke.edu
authors.forename: J J
authors.initials: JJ
authors.lastname: Enghild
authors.forename: I B
authors.initials: IB
authors.lastname: Thøgersen
authors.forename: F
authors.initials: F
authors.lastname: Cheng
authors.forename: L A
authors.initials: LA
authors.lastname: Fransson
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
authors.forename: H
authors.initials: H
authors.lastname: Rahbek-Nielsen
mesh_terms: D000510:Alpha-Globulins / Q000737:chemistry*; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002809:Chondroitin Sulfates / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007536:Isomerism; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014360:Trypsin Inhibitor, Kunitz Soybean
chemical_list: D000510:Alpha-Globulins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; C011877:inter-alpha-inhibitor; D012694:Serine; D002809:Chondroitin Sulfates; D014360:Trypsin Inhibitor, Kunitz Soybean