Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952.
The virion basic phosphoprotein (BPP), UL32, of the human cytomegalovirus (HCMV) is a 149-kDa tegument protein that represents about 15% of the virion protein mass and is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc has been postulated to mediate subunit-subunit interaction in many different types of intracellular protein complexes, while BPP may play a role in viral assembly and/or envelopment. This report describes the identification of the major O-GlcNAc attachment sites on the HCMV (AD169) BPP. Because the amount of BPP isolated from infectious virions was insufficient to determine the site(s) of glycosylation, the full-length protein has been characterized following overexpression in recombinant baculovirus-infected insect cells. The recombinant protein (rBPP) was electrophoretically (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and immunologically (by Western immunoassaying) indistinguishable from the BPP isolated from HCMV virions. In addition, the rBPP was modified by O-GlcNAc, and a comparison of the tryptic glycopeptides from the rBPP and native virion BPP indicated that their O-GlcNAc sites are the same. Furthermore, the major sites of O-GlcNAc attachment to the rBPP were mapped on high-performance liquid chromatography-purified glycopeptides by gas phase microsequencing, manual Edman degradation, and electrospray-mass spectrometry. The results demonstrate that the major sites of O-GlcNAc attachment to the BPP are Ser-921 and Ser-952. Identification of these sites will now enable mutagenesis studies to determine the influence of O-GlcNAc on the intracellular location, protein-protein interaction, and biological function of BPP. Finally, the fidelity of the addition of O-GlcNAc to rBPP in insect cells compared with native virion BPP is documented to demonstrate the possible general applicability of the baculovirus expression system to study O-GlcNAc on other low-abundance proteins.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine and Dentistry 35294.
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D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D012867:Skin; D018411:Spodoptera; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D006020:Glycopeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C056367:pp150 protein, Cytomegalovirus; D012694:Serine; D020558:GTP Phosphohydrolases; D000117:Acetylglucosamine