Characterization of FP21, a Cytosolic Glycoprotein from Dictyostelium

Kozarov, Emil; Wel, Hanke van der; Field, Melvin; Gritzali, Mikelina; Brown, Ross; West, Christopher M.

doi:10.1074/jbc.270.7.3022

Cited by 39 publications

(36 citation statements)

References 33 publications

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“…1), suggesting a frustrated substrateenzyme interaction. This is further supported by evidence that pool 1 Skp1 is incompletely galactosylated (25). Therefore, the mutations may affect recognition by ␣-GalT1 at a site separate from the sugar addition site (Pro-143).…”

Section: ␣-Galt1supporting

confidence: 54%

“…2 Concentrations were determined spectrophotometrically using an extinction coefficient for Bn glycosides of 3.4 ϫ 10 2 M Ϫ1 cm Ϫ1 at 255 nm, and for pNP glycosides of 1.15 ϫ 10 4 M Ϫ1 cm Ϫ1 at 300 nm. Concentrations of the other glycosides were determined by sugar composition analysis based on hydrolysis in 2 M trifluoroacetic acid for 4 h at 100°C, followed by chromatography in 17 mM NaOH on a PA-10 column (Dionex) and quantitation by integrated pulsed amperometry as described (25). Other pNP glycosides were from Sigma.…”

Section: Purification Of ␣-Galt1mentioning

confidence: 99%

“…At 4°C, the supernatant was made 15% saturated in (NH 4 ) 2 SO 4 and centrifuged again. The supernatant was loaded onto a 35-ml column of phenyl-Sepharose Fast Flow (high sub) (Amersham Biosciences) equilibrated in 15% (NH 4 ) 2 SO 4 in buffer C. Elution was performed with a 140-ml linear gradient of buffer C to buffer D. After a 35-ml wash with buffer D, a second, 170-ml gradient of 0 -70% (v/v) ethylene glycol in buffer E was applied followed by 35 ml of 70% ethylene glycol in buffer E. Fractions were assayed for ␣1,2-FucT using Gal␤1,3GlcNAc␤1-pNP as described (25). Active fractions were recovered from the second gradient and loaded onto a 3.3-ml DEAE 5PW column (Toso Haas) equilibrated in buffer B at 22°C.…”

Section: Purification Of ␣-Galt1mentioning

confidence: 99%

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Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium

Ketcham

Wang

Fisher

et al. 2004

Journal of Biological Chemistry

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Skp1 is an adaptor-like protein in E3SCF -ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gal␣1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn 2؉ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gal␣1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to ␣1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K m and 35-fold higher V max compared with a simple ␣-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V max advantage, but still much less active than Skp1 itself because of a K m difference. These findings indicate that ␣-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gal␣1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

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Section: ␣-Galt1supporting

confidence: 54%

Section: Purification Of ␣-Galt1mentioning

confidence: 99%

Section: Purification Of ␣-Galt1mentioning

confidence: 99%

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Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium

Ketcham

Wang

Fisher

et al. 2004

Journal of Biological Chemistry

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“…Discussion tential roles include enzyme regulation, protein folding and stability, and intracellular transport [2]. The characteristics In this paper we present evidence that the fucosyltransferase of Dcd,2FucT here reported, together with the information from the cytosol of Dictyostelium AX2 cells is able to use available on FP21 [3][4][5], suggest an even more complex role of lacto-N-biose I as acceptor forming the H type 1 trisaccharide such a cytosolic glycosylation pathway in Dictyostelium.…”

Section: Analysis Of Partially Methylated Derivativesmentioning

confidence: 90%

“…Protein content was determined by the procedure of Lowry et al [7]. COS-7 cells were cultured in Dulbecco's modified solic [5]. The enzyme was also found to use lipid-bound acEagle medium supplemented with 10% fetal bovine serum, 100 U/ml ceptors in vitro, provided they contain a terminal Gal~l-penicillin, 100 p.g/ml streptomycin, and 2 mM L-GIu.…”

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confidence: 99%

Dictyostelium cytosolic fucosyltransferase synthesizes H type 1 trisaccharide in vitro

Trinchera

Bozzaro²

1996

FEBS Letters

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A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium 2 1. Materials discoideum. This transferase requires divalent cations and is Lacto-N-biose I, LacNAc, GlcNAc, GDP-~-L-fucose, L-Fuc, inhibited by N-ethylmaleimide and detergent treatment. Appar- calculated Km values for GDP-Fuc and lacto-N-biose I are OMe, antipain shaker in axenic medium [6]. At a culture density of 5-8 × 106 cells/ ml, cells were collected by pelleting at 1300 rpm for 4 min, at 4°C. Cell pellet was resuspended with 0.25 M sucrose-TKM buffer (50 mM Tris, 1. Introduction 25 mM KCI, 5 mM MgC12, pH 8.0) by gently pipetting, and spun as above; washing procedure was repeated three times. It was then resuspended to a cell density of 0.5x109 cells/ml in 0.25 M sucroseGlycosyltransferases have been mostly reported as mem-TKM buffer containing protease inhibitors to a final concentration of brane-bound enzymes involved in the biosynthesis of the sac-20 ~tg/ml each antipain, chymostatin, leupeptin, and pepstatin, 0.2 charide chains of glycoproteins and glycolipids [1]. Glycosyl-TIU aprotinin, 37 p.g/ml TLCK, and 1 mM PMSF. The cell suspension (4-6 ml in typical experiments) was lysed by forced passage transferases found in biological fluids reach the extracellular through a polycarbonate filter with a 3 ~tm nominal pore diameter compartment from the Golgi lumen through the secretory (Nucleopore, 25 mm filter diameter), and the lysate obtained was pathway, and proteolytic cleavage eventually makes them referred to as the homogenate. It was spun at 2000×g for 6 min, at rid of the membrane anchor. On the other hand, few cytosolic 4°C, obtaining a supernatant referred to as the postnuclear fraction. glycosyltransferases have recently been demonstrated [2]. Very This fraction was centrifuged at 100000×g for 1 h at 4°C, obtaining a pellet and a supernatant. The latter was referred to as the cytosolic recently, a fucosyltransferase has been purified from the cytofraction; the pellet was resuspended to a protein concentration of 10 sol of the slime mold Dictyostelium discoideum [3]. The en-30 mg/ml with 0.25 M sucrose-TKM buffer containing the same prozyme was originally discovered by its ability to fucosylate tease inhibitors as in the filtration buffer, and referred to as the memthe FP21 glycoprotein [4], that was proved to be mostly cytobrane fraction. Protein content was determined by the procedure of Lowry et al. [7]. COS-7 cells were cultured in Dulbecco's modified solic [5]. The enzyme was also found to use lipid-bound acEagle medium supplemented with 10% fetal bovine serum, 100 U/ml ceptors in vitro, provided they contain a terminal Gal~l-penicillin, 100 p.g/ml streptomycin, and 2 mM L-GIu. 3GIcNAc disaccharide sequence [3,4]. To date, it is not known whether the enzyme utilizes simple sugar acceptors, as do Enzyme assaysmammalian fucosyltransferases, and there is no direct eviDcd,2FucT was determined in a reaction mixture containing, in a final volume of 20 ~1, 0.1 M Tris-HC1 ...

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