Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

Luhrs, Carol; Pitiranggon, P; Costa, Maria Da; Rothenberg, Sheldon P.; Slomiany, Bronislaw L.; Brink, Larry; Tous, Guillermo; Stein, Stanley J.

doi:10.1073/pnas.84.18.6546

Cited by 50 publications

(25 citation statements)

References 28 publications

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“…However, hydrophobicity plots of KB cell and placental cDNA species showed several hydrophobic and presumably membrane-spanning regions, without convincing evidence of sufficient length of the C-terminus for membrane insertion [2,3,7]. An alternative proposal that accounts for regional hydrophobicity holds that membrane anchoring of FBP is dependent on ester linkages to membrane fatty acids [32].…”

Section: Discussionmentioning

confidence: 99%

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

Hoozen

Ling

Halsted

1996

Biochemical Journal

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Folate-binding protein (FBP) was identified and characterized in a pig liver cDNA library by screening with a 0.6 kb fragment from the cDNA of FBP from a human KB cell cancer line. The cDNA of pig liver FBP included 1230 bp containing 759 bp in the open reading frame with 80 % similarity to the human placenta FBP. The deduced 253 amino acid sequence showed 67-73 % similarity to previous sequences and contained 16 conserved cysteine residues, 11 tryptophan potential folate-

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Section: Discussionmentioning

confidence: 99%

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

Hoozen

Ling

Halsted

1996

Biochemical Journal

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“…The membrane FBP was purified from cells cultured in FD medium using an epoxy-activated Sepharose 6B matrix coupled to folate (24), as previously described (5,25).…”

Section: Methodsmentioning

confidence: 99%

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

Luhrs¹,

Raskin²,

Durbin³

et al. 1992

J. Clin. Invest.

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KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/,ug protein, respectively. The capacity for binding 13HIfolate to the surface of transfected 3T3 cells cultured in folatedeficient medium is 7.7 pmol/ 106 cells, and this is -50% of the surface binding capacity of KB cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of 13HI folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions. (J. Clin. Invest. 1992. 90:840-847.)

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“…The possible thioester bonds were released by incubating the sample for 20 min at 37°C in the presence of 0.3 M methanolic KOH [25]. To 5 ktl of the reaction mix 5 ~tl of 0.6 M methanolic KOH were added.…”

Section: Analysand Of Oligomerization Productsmentioning

confidence: 99%

Oligomerization of VIP21‐caveolin in vitro is stabilized by long chain fatty acylation or cholesterol

et al. 1996

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VIP21-caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo-oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of eytosol to the in vitro synthesized and membrane inserted VIP21-caveolin. Here we show that long chain fatty aeyl eoenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25-hydroxy-cbolesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21-eaveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three eysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non-ionic detergent oetylglucoside, both palmitoylated and non-palmitoylated VIP21-caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non-palmitoylated protein are disrupted when analyzed by SDS-PAGE without boiling. These data suggest that the protein domains of VIP21-eaveolin are the primary determinants of oligomerization, hut that palmitoylation of cysteine residues can increase the stability of the oligomers.

show abstract

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

Cited by 50 publications

References 28 publications

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

Oligomerization of VIP21‐caveolin in vitro is stabilized by long chain fatty acylation or cholesterol

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