Detection of cytoplasmic glycosylation associated with hydroxyproline.
A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. For this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and, therefore, have been implicated in O2-sensing in Dictyostelium, as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans.
West
Christopher M
CM
Department of Biochemistry and Molecular Biology Oklahoma Center for Medical Glycobiology, Oklahoma City, Oklahoma, USA.
van der Wel
Hanke
H
Blader
Ira J
IJ
D000818:Animals; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism*; D004023:Dictyostelium / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006909:Hydroxyproline / Q000737:chemistry* / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D014122:Toxoplasma / Q000737:chemistry / Q000378:metabolism
D044786:S-Phase Kinase-Associated Proteins; D006909:Hydroxyproline