title: N-linked glycans of the human insulin receptor and their distribution over the crystal structure.
abstract: The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.
authors.affiliation: Division of Molecular and Health Technologies, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victoria 3052, Australia.
authors.forename: Lindsay G
authors.initials: LG
authors.lastname: Sparrow
authors.forename: Michael C
authors.initials: MC
authors.lastname: Lawrence
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Strike
authors.forename: Christine P
authors.initials: CP
authors.lastname: Robinson
authors.forename: Neil M
authors.initials: NM
authors.lastname: McKern
authors.forename: Colin W
authors.initials: CW
authors.lastname: Ward
mesh_terms: D015703:Antigens, CD / Q000737:chemistry*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000032:analysis*; D011972:Receptor, Insulin / Q000737:chemistry*
chemical_list: D015703:Antigens, CD; D011134:Polysaccharides; C435941:INSR protein, human; D011972:Receptor, Insulin