Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA.
A preparation of folate binding protein purified from human placental membranes in the presence of a variety of protease inhibitors followed by deglycosylation with N-glycanase gave a sharp band at Mr approximately 28,000 following SDS-polyacrylamide gel electrophoresis. The deglycosylated protein bound [3H]folic acid as tightly as the native protein. Peptides obtained following digestion of the purified protein with staphylococcal V8 protease and HPLC purification were sequenced. Polyclonal antibodies against the protein preparation were affinity purified and used to screen a placental cDNA expression library. A full-length cDNA for a placental folate binding protein was thus obtained and the corresponding protein sequence deduced. This result, taken together with the peptide sequence data, indicates the expression of at least two homologous folate binding proteins in placenta, one of which appears to be identical with the folate binding protein from human milk and nasopharyngeal epidermoid carcinoma (KB) cells; the cDNA sequence obtained corresponds to the other protein. The deduced protein sequence is characterized by a putative 16-residue amino-terminal signal peptide that is cleaved, resulting in a 239-residue polypeptide. The mature protein exhibits two potential sites for N-linked glycosylation at Asn-99 and Asn-179, eight potential intramolecular disulfide bonds, and a stretch of hydrophobic residues at the carboxyl terminus that could form a transmembrane domain. The protein bears a 68% sequence homology with the KB cell folate binding protein and may represent a fetal folate transport protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Ratnam
M
M
Department of Biochemistry, Medical College of Ohio, Toledo 43699-0008.
Marquardt
H
H
Duhring
J L
JL
Freisheim
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics*; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D066298:In Vitro Techniques; D007624:KB Cells; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D010920:Placenta / Q000378:metabolism*; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D010455:Peptides; D011956:Receptors, Cell Surface; D004247:DNA