Posttranslational modifications of human inter-alpha-inhibitor: identification of glycans and disulfide bridges in heavy chains 1 and 2.
Inter-alpha-inhibitor (IalphaI) is a serine proteinase inhibitor found in high concentrations in human plasma. The protein is composed of a light inhibitory chain called bikunin and two heavy chains of unknown function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751]. The aim of this study was to complete the primary structure by characterizing additional covalent posttranslational modifications of the heavy chains. Analysis revealed three N-linked oligosaccharides located on Asn251 and Asn554 of heavy chain 1 and on Asn64 of heavy chain 2: all these were complex biantennary structures composed of (Asn)-GlcNAc2-Man-(Man-GlcNAc-Gal-SA)2. In addition, the IalphaI heavy chains carried several O-linked glycans located on Thr619 of heavy chain 1 and a cluster of four O-linked oligosaccharides on Thr612, Ser619, Thr621, and Thr637 of heavy chain 2. The oligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The IalphaI heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on heavy chain 1, Cys26, appears to be modified by dihexosylation. The other Cys residues exclusively form intrachain disulfide bridges. In heavy chain 1 the two disulfide bonds are formed between Cys210 and Cys213 and between Cys234 and Cys506, and in heavy chain 2, between Cys207 and Cys210 and between Cys596 and Cys597. Interestingly, three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.
Olsen
E H
EH
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rahbek-Nielsen
H
H
Thogersen
I B
IB
Roepstorff
P
P
Enghild
J J
JJ
D000510:Alpha-Globulins / Q000737:chemistry / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry; D002236:Carbohydrate Conformation; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D000510:Alpha-Globulins; D004220:Disulfides; D009005:Monosaccharides; D009844:Oligosaccharides; D010455:Peptides; D011134:Polysaccharides; C011877:inter-alpha-inhibitor; D001216:Asparagine; D003545:Cysteine