Role of Individual N-Linked Glycosylation Sites in the Function and Intracellular Transport of the Human α Folate Receptor

Glycosylation is a structural feature of all three isoforms of the human folate receptor. We have used site-directed mutagenesis to study the role of individual glycosylation sites in the assembly and function of the α isoform of the human folate receptor (αhFR). Three potential N-linked glycosylation sites in the αhFR sequence were disrupted by conservative mutation of the S or T residues in the consensus sequence (N-X-S/T) to A or V, respectively. Constructs with the single mutations S71→ A (αhFR−1), T163→ V (αhFR−2), and S203→ A (αhFR−3); the double mutation S71→ A/S203→ A (αhFR−1−3); and the triple mutation S71→ A/S203→ A/T163→ V (αhFR−1−2−3) were stably transfected into Chinese hamster ovary (CHO) cells. The proteins produced in CHO cells by the mutated cDNAs have apparent molecular weights that are reduced relative to the wild type and are consistent with the loss of carbohydrate residues. The triple mutant, which lacks all three consensus glycosylation sites, yields protein that comigrates with the enzymatically deglycosylated native protein. Determinations of theKDfor folic acid by Scatchard analyses of the glycosylation mutants indicate that folic acid binding affinity is not significantly affected in the single mutants αhFR−1and αhFR−2. However, in the single mutant, αhFR−3, and the double mutant, αhFR−1−3, folic acid binding affinity is respectively 2.7- and 3.5-fold lower than that in wild type. Deglycosylation by mutation of all three consensus sites (αhFR−1−2−3) eliminates both folic acid binding and cell surface expression. In contrast, enzymatic deglycosylation of purified wild-type αhFR with endoglycosidase F does not significantly affect folate binding affinity. Thus, while carbohydrate residues are not essential for the folate binding activity of the mature folate receptor, at least one of the three core glycosylated residues is necessary for the synthesis of αhFR in its active conformation.