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Functional characterization of obesity-associated variants involving the ? and ? isoforms of human SH2B1.

ABSTRACT: We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1? enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1?. SH2B1? variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1?, like SH2B1?, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1? enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1? to enhance GH-induced cell motility. In contrast to SH2B1?, SH2B1? does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1? may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.

SUBMITTER: Pearce LR

PROVIDER: S-EPMC4138566 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Functional characterization of obesity-associated variants involving the α and β isoforms of human SH2B1.

Pearce Laura R LR Joe Ray R Doche Michael E ME Su Hsiao-Wen HW Keogh Julia M JM Henning Elana E Argetsinger Lawrence S LS Bochukova Elena G EG Cline Joel M JM Garg Sumedha S Saeed Sadia S Shoelson Steven S O'Rahilly Stephen S Barroso Inês I Rui Liangyou L Farooqi I Sadaf IS Carter-Su Christin C

Endocrinology 20140627 9

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1β enhancement of nerve growt ...[more]

PMID: 24971614

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Project description:In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

Dataset Information

Functional characterization of obesity-associated variants involving the ? and ? isoforms of human SH2B1.

Publications

Functional characterization of obesity-associated variants involving the α and β isoforms of human SH2B1.

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