Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc

Abstract

Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.

Figure 1

Structures of fully synthetic three-component immunogens 1 and 2. Compounds 3-9 were employed for ELISA and inhibition ELISA to determine epitope selectivities of the antibodies.

Figure 2

Immunoblotting of three monoclonal antibodies. (a) CKII α subunit was immunoprecipitated from HEK293T cells with or without OGT overexpression. Eluates were resolved by SDS-PAGE and immunoblotted with MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14). A band corresponding to CKII α subunit was detected with signal intensity correlated with O-GlcNAc status. All blots were stripped and reprobed with antibody against CKII α subunit (only one representative blot is shown here). Also, equal amount of CKII α subunit was present in the input regardless of the status of O-GlcNAc levels. (b) HEK293T lysates with low (OGA overexpression), median (Mock transfection) and high (OGT overexpression) levels of O-GlcNAc modification were exposed to MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) respectively. The signals obtained mirror the corresponding O-GlcNAc status in each sample. Immunoblots against OGT, OGA and tubulin are also shown. While equal loading of tubulin was detected in all samples, higher OGA and OGT protein levels were detected with lysates from OGA and OGT transfections, respectively. Note: Endogenous OGT and OGA levels do appear after longer exposure. (c) O-GlcNAc proteins were immunoprecipitated from HEK293T cells treated with PUGNAc (an OGA inhibitor), resolved by SDS-PAGE and subjected to CTD110.6 (an IgM isotype O-GlcNAc specific antibody) blotting. Cross-reactivity of MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) with CTD110.6, albeit distinct in pattern, were detected. (d) Application of MAbs for O-GlcNAc-omics in cell culture: Distribution of O-GlcNAc modified proteins based on their biological process categorized in HPRD (number of individual proteins assigned to each category is displayed).

Figure 3

Application of MAbs for O-GlcNAc-omics in mammalian tissue. (a) Western blot analysis using the O-GlcNAc antibodies of rat liver extracts following Sham or TH-R treatment. (b) Distribution of O-GlcNAc modified proteins in rat liver based on their biological process following Sham or TH-R treatment (number of individual proteins assigned to each category is displayed).

Figure 3

Application of MAbs for O-GlcNAc-omics in mammalian tissue. (a) Western blot analysis using the O-GlcNAc antibodies of rat liver extracts following Sham or TH-R treatment. (b) Distribution of O-GlcNAc modified proteins in rat liver based on their biological process following Sham or TH-R treatment (number of individual proteins assigned to each category is displayed).