ROLE OF INDIVIDUAL N-LINKED GLYCOSYLATION SITES IN THE FUNCTION AND INTRACELLULAR-TRANSPORT OF THE HUMAN ALPHA-FOLATE RECEPTOR

Glycosylation is a structural feature of all three isoforms of the hum an folate receptor. We have used site-directed mutagenesis to study th e role of individual glycosylation sites in the assembly and function of the alpha isoform of the human folate receptor (alpha hFR), Three p otential N-linked glycosylation sites in the alpha hFR sequence were d isrupted by conservative mutation of the S or T residues in the consen sus sequence (N-X-S/T) to A or V, respectively. Constructs with the si ngle mutations S-71 --> A (alpha hFR(-1)), T-163 --> V (alpha hFR(-2)) , and S-203 --> A (alpha hFR(-3)); the double mutation S-71 --> A/S-20 3 --> A (alpha hFR(-1-3)); and the triple mutation S-71 --> A/S-203 -- > A/T-163 --> V ((alpha hFR(-1-2-3)) were stably transfected into Chin ese hamster ovary (CHO) cells. The proteins produced in CHO cells by t he mutated cDNAs have apparent molecular weights that are reduced rela tive to the wild type and are consistent with the loss of carbohydrate residues. The triple mutant, which lacks all three consensus glycosyl ation sites, yields protein that comigrates with the enzymatically deg lycosylated native protein, Determinations of the KD for folic acid by Scatchard analyses of the glycosylation mutants indicate that folic a cid binding affinity is not significantly affected in the single mutan ts alpha FR-1 and alpha hFR(-2). However, in the single mutant, alpha hFR(-3), and the double mutant, alpha hFR(-1-3), folic acid binding af finity is respectively 2.7- and 3.5-fold lower than that in wild type, Deglycosylation by mutation of all three consensus sites (alpha hFR(- 1-2-3)) eliminates both folic acid binding and cell surface expression . In contrast, enzymatic deglycosylation of purified wild-type alpha h FR 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 o f the three core glycosylated residues is necessary for the synthesis of alpha hFR in its active conformation. (C) 1998 Academic Press.