Expression levels of functional folate receptors alpha and beta are related to the number of N-glycosylated sites.
In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type alpha and type beta have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn-->Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-alpha and FR-beta are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-alpha and FR-beta respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-beta the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-alpha and FR-beta.
Shen
F
F
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43699-0008, USA.
Wang
H
H
Zheng
X
X
Ratnam
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002451:Cell Compartmentation; D005347:Fibroblasts; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D010455:Peptides; D011956:Receptors, Cell Surface; D005492:Folic Acid