Two-Dimensional Gel Based Approaches for the Assessment of N-Linked and O-GlcNAc Glycosylation in Human and Simian Immunodeficiency Viruses
| Autor: | Stephen A. Whelan, David R. Graham, Gerald W. Hart, Jennifer E. Van Eyk, Dawn Chen, Megan Mitsak, Steven T. Elliott |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
PNGase F
Glycosylation viruses Blotting Western Molecular Sequence Data Carbohydrates Biology HIV Envelope Protein gp120 medicine.disease_cause Biochemistry Virus Article Mass Spectrometry Glycomics chemistry.chemical_compound Viral Proteins medicine Humans Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Electrophoresis Gel Two-Dimensional Amino Acid Sequence Molecular Biology Tropism chemistry.chemical_classification Glucosamine Staining and Labeling Viral protein processing HIV Simian immunodeficiency virus Virology chemistry Simian Immunodeficiency Virus Glycoprotein Peptides |
| Popis: | The glycosylation state of envelope glycoproteins in Human and Simian Immunodeficiency Viruses (HIV/SIV) is critical to viral infectivity and tropism, viral protein processing, and in virus evasion of the immune system. Using a rapid fluorescent two-dimensional gel based method coupled with enzymatic pre-treatment of virus with PNGase F (Peptide: N-Glycosidase F) and fluorescent 2D gels or 2D gel Western blotting, we show significant differences in the glycosylation patterns of two SIV strains widely used in animal models of HIV disease and vaccine studies. We also demonstrate the modification of a host protein important in HIV biology (HLA-DR) by O-GlcNAc. Further, this experimental pipeline allows for the identification of the modified protein and the site of N-linked glycosylation by fluorescent two-dimensional gel electrophoresis coupled with mass spectrometry (MS) and the qualitative and semi-quantitative assessment of viral glycosylation. The method is fully compatible with downstream glycomics analysis. This approach will permit correlation of virus glycosylation status with pathological severity and may serve as a rapid screen of viruses from physiological samples for further study by more advanced MS methodology. |
| Databáze: | OpenAIRE |
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