Glycosylation of the rabbit intestinal brush border Na+/glucose cotransporter.
The glycosylation of the mature form of the rabbit intestinal Na+/glucose cotransporter was investigated by using both glycosidases and chemical treatment. The protein was identified on Western blots using polyclonal antibodies directed against peptide sequences from the cloned transporter as a Mr 68,000 polypeptide. The effect of these treatments on the size of the transporter is consistent with the major post-translational processing being a single N-linked glycosylation of either the tri- or tetra-antennary complex type. Either method of deglycosylation reduced the SDS-PAGE size by 11,000 to Mr 57,000. These results also suggest that O-linked glycosylation, if present, contributes little to the apparent size of the transporter. The relative size of the deglycosylated mature protein appears to be greater than that of the in vitro primary transcript (Mr 45,000), suggesting either a difference in a stable conformational state insensitive to reduction and denaturation by SDS or an additional post-translational modification. In addition, deglycosylation of the native transporter does not affect transport activity in brush border membrane vesicles. The transporter, an integral membrane protein having several membrane-spanning regions, has an anomalous mobility in SDS-PAGE as shown by Ferguson analysis. We estimate that the actual size of the mature Na+/glucose cotransporter is 86,000, and that N-linked glycosylation contributes about 15,000 to the mass.
Hirayama
B A
BA
Department of Physiology, UCLA School of Medicine 90024-1751.
Wright
E M
EM
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007413:Intestinal Mucosa / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits
D009004:Monosaccharide Transport Proteins