The Complete Amino Acid Sequence of a Human Folate Binding Protein from KB Cells Determined from the cDNA

Sadasivan, Easwara; Rothenberg, Sheldon P.

doi:10.1016/s0021-9258(18)83621-3

Cited by 111 publications

(17 citation statements)

References 25 publications

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“…(Svendsen, 1982) have been elucidated by sequencing overlapping peptides. In addition, the protein sequence of the human KB cell FBP has been deduced from the sequence of a partial cDNA (Sadasivan & Rothenberg, 1989). The sequences of the human milk and KB cell FBPs are identical.…”

Section: Resultsmentioning

confidence: 99%

Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA

Ratnam

Marquardt²,

Duhring³

et al. 1989

Biochemistry

187

136

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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)

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Section: Resultsmentioning

confidence: 99%

Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA

Ratnam

Marquardt²,

Duhring³

et al. 1989

Biochemistry

187

136

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“…While the translocation of folates by the RFC occurs directly at the plasma membrane (Sirotnak, 1985), the uptake by MFRs is thought to occur after internalization of the receptors into the cells. Several cDNAs encoding at least three different isoforms of MFRs have been isolated (Ratnam et al, 1989;Elwood, 1989;Lacey et al, 1989;Sadasivan and Rothenberg, 1989;Brigle et al, 1991;Shen et al, 1994), and it has been well established that their products are attached to membranes by a glycosyl-phosphatidyl-inositol (GPI)-anchor (Lacey et al, 1989;Luhrs and Slomiany, 1989;Alberti et al, 1990). In general, GPIlinked proteins are internalized by cells more slowly than integral membrane receptors and possibly through different internalization pathways (Low, 1989;Lisanti et al, 1990;Bamezai et al, 1992;Keller et al, 1992;Watts and Marsh, 1992).…”

mentioning

confidence: 99%

Endocytosis of GPI-linked membrane folate receptor-alpha.

Rijnboutt

Jansen

Posthuma

et al. 1996

The Journal of Cell Biology

182

114

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Abstract. GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform et in KB-cells. MFR-et was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-a was completely soluble in Triton X-100 at 0°C. In contrast, only 33 % of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37°C or in the presence of saponin at 0°C. Similar solubilization characteristics were found when MFR-et at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinityanalog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-a could be separated from soluble MFR-a on sucrose flotation gradients. Only Triton X-100 soluble MFR-a was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-a recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0°C. Using immunoelectron microscopy, we found MFR-o~ along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-et bypasses caveolae.

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“…The two known isoforms of the human cell surfaceassociated high-affinity folate receptor (FR) are homologous polypeptides of Mt ~28 000 that are N-glycosylated and attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor (Ratnam et al, 1989;Sadasivan & Rothenberg, 1989;Elwood, 1989;Lacey et al, 1989; Luhrs & Sloliamy, 1989;Verma et al, 1992). Although a dual functional role has been proposed for FR, i.e., cellular transport of folate compounds/anti-folates [reviewed in Antony (1992); Jansen etal., 1990; Westerhof etal., 1991;Luhrs etal., 1992;Matsue et al, 1992] and stimulation of cell growth in primary bone marrow cultures (Antony et al, 1991), the physiological significance of FR expression in various tissues is still unclear.…”

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confidence: 99%

Identification of a novel folate receptor, a truncated receptor, and receptor type .beta. in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity

et al. 1994

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The expression of a membrane-associated folate receptor (FR) was elevated in spleen samples from patients with chronic (CML) and acute (AML) myelogenous leukemias compared with normal spleen. Contrary to earlier reports, antibodies to a purified FR from placenta cross-reacted quantitatively with this protein in solution radioimmunoassays. Similar to FR-alpha (KB cells) and FR-beta (placenta), the protein was released from the membrane by phosphatidylinositol-specific phospholipase C, indicating a glycosylphosphatidylinositol (GPI) membrane anchor. Screening of a cDNA library from CML spleen with a heterologous murine FR cDNA and also amplification of FR cDNAs from spleen and bone marrow in CML, AML, chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL) by polymerase chain reaction (PCR) using degenerate oligonucleotides yielded cDNA clones representing FR-beta, a novel FR (type gamma), and an aberrant transcript of FR-gamma with a 2 base pair deletion resulting in a truncated 104-residue polypeptide; FR-alpha was not detected in these tissues. The cDNA for FR-gamma predicts a 243-residue polypeptide with an amino acid sequence homology of 71% and 79% with FR-alpha and FR-beta, respectively, a 23-residue amino-terminal signal peptide, and 3 potential sites for N-linked glycosylation. Transfection of COS-1 cells with the cDNA for FR-gamma resulted in low expression of a [3H]folic acid binding protein on the cell surface that was GPI-anchored. PCR analysis of total RNA from a number of normal and malignant tissues and cell lines indicated a limited tissue specificity of FR-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Complete Amino Acid Sequence of a Human Folate Binding Protein from KB Cells Determined from the cDNA

Cited by 111 publications

References 25 publications

Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA

Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA

Endocytosis of GPI-linked membrane folate receptor-alpha.

Identification of a novel folate receptor, a truncated receptor, and receptor type .beta. in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity

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