Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor.
A cDNA clone encoding rat intrinsic factor (IF), pIFQ, has been inserted into the eukaryotic expression vector pSVL and used to transfect COS-1 cells. The IF produced by the transfected cells was secreted nearly exclusively into the medium at concentrations of 0.1-0.2 micrograms/ml. Tunicamycin treatment (1-10 micrograms/ml) completely blocked N-linked glycosylation but had no effect on IF secretion. The secreted glycosylated IF retained all the properties of native IF, i.e., high affinity for cobalamin (Cbl) and for the IF-Cbl receptor and relative resistance to low pH and to proteolysis. The nonglycosylated IF also retained these properties except that it was more protease sensitive. The protease degradation was prevented by the presence of the ligand Cbl. The presence of carbohydrate may play a role in protecting IF from digestion by pancreatic proteases in the intestinal lumen.
Gordon
M M
MM
Division of Gastroenterology, Washington University Medical School, St. Louis, Missouri 63110.
Hu
C
C
Chokshi
H
H
Hewitt
J E
JE
Alpers
D H
DH
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006031:Glycosylation; D007437:Intrinsic Factor / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C028199:intrinsic factor receptor; D014415:Tunicamycin; D007437:Intrinsic Factor