title: Role of individual N-linked glycosylation sites in the function and intracellular transport of the human alpha folate receptor.
abstract: 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 a isoform of the human folate receptor (alpha(h)FR). Three potential N-linked glycosylation sites in the alpha(h)FR 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 S(71)-->A (alpha(h)FR(-1)), T(163)-->V (alpha(h)FR(-2)), and S(203)-->A (alpha(h)FR(-3)); the double mutation S(71)--> A/S(203)-->A (alpha(h)FR(-1-3)); and the triple mutation S(71)--> A/S(203)--> A/T(163)--> V (alpha(h)FR(-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 the K(D) for folic acid by Scatchard analyses of the glycosylation mutants indicate that folic acid binding affinity is not significantly affected in the single mutants alpha(h)FR(-1) and alpha(h)FR(-2). However, in the single mutant, alpha(h)FR(-3), and the double mutant, alpha(h)FR(-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 (alpha(h)FR(-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 of the three core glycosylated residues is necessary for the synthesis of alpha(h)FR in its active conformation.
authors.affiliation: Section of Experimental Hematology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Roberts
authors.forename: M
authors.initials: M
authors.lastname: Petropavlovskaja
authors.forename: K N
authors.initials: KN
authors.lastname: Chung
authors.forename: C B
authors.initials: CB
authors.lastname: Knight
authors.forename: P C
authors.initials: PC
authors.lastname: Elwood
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D016384:Consensus Sequence / Q000235:genetics; D006224:Cricetinae; D058973:Folate Receptors, GPI-Anchored; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase