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Mutational analysis of the N-linked glycosylation sites of the human insulin receptor.

ABSTRACT: Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

SUBMITTER: Elleman TC

PROVIDER: S-EPMC1221015 | biostudies-other | 2000 May

REPOSITORIES: biostudies-other

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Project description:The addition of asparagine (N)-linked polysaccharide chains (i.e., glycans) to the gp120 and gp41 glycoproteins of human immunodeficiency virus type 1 (HIV-1) envelope is not only required for correct protein folding, but also may provide protection against neutralizing antibodies as a "glycan shield." As a result, strong host-specific selection is frequently associated with codon positions where nonsynonymous substitutions can create or disrupt potential N-linked glycosylation sites (PNGSs). Moreover, empirical data suggest that the individual contribution of PNGSs to the neutralization sensitivity or infectivity of HIV-1 may be critically dependent on the presence or absence of other PNGSs in the envelope sequence. Here we evaluate how glycan-glycan interactions have shaped the evolution of HIV-1 envelope sequences by analyzing the distribution of PNGSs in a large-sequence alignment. Using a "covarion"-type phylogenetic model, we find that the rates at which individual PNGSs are gained or lost vary significantly over time, suggesting that the selective advantage of having a PNGS may depend on the presence or absence of other PNGSs in the sequence. Consequently, we identify specific interactions between PNGSs in the alignment using a new paired-character phylogenetic model of evolution, and a Bayesian graphical model. Despite the fundamental differences between these two methods, several interactions are jointly identified by both. Mapping these interactions onto a structural model of HIV-1 gp120 reveals that negative (exclusive) interactions occur significantly more often between colocalized glycans, while positive (inclusive) interactions are restricted to more distant glycans. Our results imply that the adaptive repertoire of alternative configurations in the HIV-1 glycan shield is limited by functional interactions between the N-linked glycans. This represents a potential vulnerability of rapidly evolving HIV-1 populations that may provide useful glycan-based targets for neutralizing antibodies.

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Mutational analysis of the N-linked glycosylation sites of the human insulin receptor.

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