Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m313858200
ISSN1083-351X
AutoresCatherine M. Ketcham, Fei Wang, S. Zoë Fisher, Altan Ercan, Hanke van der Wel, Robert D. Locke, K. Sirajud-Doulah, Khushi L. Matta, Christopher M. West,
Tópico(s)Galectins and Cancer Biology
ResumoSkp1 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 Galα1-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 Mn2+ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galα1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to α1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and 35-fold higher Vmax compared with a simple α-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 Vmax advantage, but still much less active than Skp1 itself because of a Km difference. These findings indicate that α-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 Galα1,3Fuc linkage is deficient in expressed mutant Skp1 proteins. 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 Galα1-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 Mn2+ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galα1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to α1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and 35-fold higher Vmax compared with a simple α-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 Vmax advantage, but still much less active than Skp1 itself because of a Km difference. These findings indicate that α-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 Galα1,3Fuc linkage is deficient in expressed mutant Skp1 proteins. Skp1 is a small protein that has been found in multiple distinct heteromeric protein complexes in the cytosolic and nuclear compartments of eukaryotic cells (1Seol J.H. Shevchenko A. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (204) Google Scholar, 2Kaplan K.B. Hyman A.A. Sorger P.K. Cell. 1997; 91: 491-500Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 3Galan J.M. Wiederkehr A. Seol J.H. Haguenauer-Tsapis R. Deshaies R.J. Riezman H. Peter M. Mol. Cell. Biol. 2001; 21: 3105-3117Crossref PubMed Scopus (138) Google Scholar, 4Farras R. Ferrando A. Jasik J. Kleinow T. Okresz L. Tiburcio A. Salchert K. del Pozo C. Schell J. Koncz C. EMBO J. 2001; 20: 2742-2756Crossref PubMed Scopus (193) Google Scholar, 5West C.M. Cell. Mol. Life Sci. 2003; 60: 229-240Crossref PubMed Scopus (22) Google Scholar, 6Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1080) Google Scholar). The best characterized complex is the family of E3SCF-ubiquitin ligases that target modified proteins for polyubiquitination and subsequent degradation via the 26 S proteasome (6Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1080) Google Scholar). In this complex, Skp1 serves as an adaptor to link the catalytic E2-ligase via cullin-1 to the F-box protein specificity factor (7Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Crossref PubMed Scopus (1157) Google Scholar). In the lower eukaryote Dictyostelium, Skp1 is nearly quantitatively modified by a pentasaccharide at a hydroxyproline near its C terminus, in a region of the protein that associates with the F-box partner (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The function of glycosylation is not known, but partial glycosylation is necessary for nuclear concentration of Skp1, and cells that are genetically unable to extend the core monosaccharide are smaller and grow to higher saturation densities (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar, 10van der Wel H. Morris H.R. Panico M. Paxton T. Dell A. Thomson J.M. West C.M. J. Biol. Chem. 2001; 276: 33952-33963Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The first 3 sugars are added sequentially to Hyp-143 of Skp1 by polypeptide GlcNAcT1 1The abbreviations used are: GlcNAcT, N-acetylglucosaminyltransferase; Bn, benzyl; DTT, dithiothreitol; Fuc, l-fucose unless specified as d-; FucT, fucosyltransferase; Gal, d-galactose (pyranose); GalT, galactosyltransferase; GT, glycosyltransferase; pNP, para-nitrophenyl; β-Gal, β-galactosidase; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: GlcNAcT, N-acetylglucosaminyltransferase; Bn, benzyl; DTT, dithiothreitol; Fuc, l-fucose unless specified as d-; FucT, fucosyltransferase; Gal, d-galactose (pyranose); GalT, galactosyltransferase; GT, glycosyltransferase; pNP, para-nitrophenyl; β-Gal, β-galactosidase; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. (11Teng-umnuay P. van der Wel H. West C.M. J. Biol. Chem. 1999; 274: 36392-36402Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 12van der Wel H. Morris H.R. Panico M. Paxton T. Dell A. Kaplan L. West C.M. J. Biol. Chem. 2002; 277: 46328-46337Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and a processive bifunctional β1,3-GalT/α1,2-FucT (10van der Wel H. Morris H.R. Panico M. Paxton T. Dell A. Thomson J.M. West C.M. J. Biol. Chem. 2001; 276: 33952-33963Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 13van der Wel H. Fisher S.Z. West C.M. J. Biol. Chem. 2002; 277: 46527-46534Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). These sugar nucleotide-dependent enzymes are novel in their soluble nature and absence of rough endoplasmic reticulum targeting sequences or membrane anchor motifs. Therefore, at least the early steps of Skp1 glycosylation are mediated by a novel pathway of cytoplasmic enzymes (5West C.M. Cell. Mol. Life Sci. 2003; 60: 229-240Crossref PubMed Scopus (22) Google Scholar, 14West C.M. van der Wel H. Gaucher E.A. Glycobiology. 2002; 12: 17R-27RCrossref PubMed Scopus (31) Google Scholar). Bioinformatics studies suggest that a related modification pathway occurs in the cytoplasm of other lower eukaryotes including a diatom and an oomycete (15West C.M. van der Wel H. Sassi S. Gaucher E.A. Biochim. Biophys. Acta. 2004; (10.1016/j.bbagen.2004.04.007)Google Scholar). The Fuc terminus of the core trisaccharide of Dictyostelium Skp1 is further modified by two α-linked Galp residues whose linkages have not been established (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14West C.M. van der Wel H. Gaucher E.A. Glycobiology. 2002; 12: 17R-27RCrossref PubMed Scopus (31) Google Scholar). The mechanism of α-galactosylation is not known but it seems likely that it will involve novel sugar nucleotide-dependent enzymes in the cytoplasm or nucleus. It is not known whether the Skp1 enzymes will be more related to α-GalTs of the eukaryotic Golgi or those that extend lipopolysaccharides and other glycoconjugates in the prokaryotic cytoplasm (16Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. J. Biochem. 1998; 123: 1000-1009Crossref PubMed Scopus (136) Google Scholar). Unlike expressed wild-type Skp1, two mutant Skp1s containing amino acid substitutions in their N-terminal regions are poorly glycosylated in vivo (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar). Mass spectrometric analysis of one, Skp1A1(HW120)-myc, showed that it consists of a mixture of the unmodified protein and glycoforms terminated with GlcNAc, Fuc, or one or two α-Gal residues (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Incomplete peripheral galactosylation might result from inefficient addition of the sugar or increased susceptibility to its removal. These intermediate glycoforms of Skp1A1(HW120)-myc are potentially useful acceptor substrates for assaying the activity of the GalTs whose substrates are accumulated, as found for the Skp1 GlcNAcT activity (11Teng-umnuay P. van der Wel H. West C.M. J. Biol. Chem. 1999; 274: 36392-36402Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Although the function of the peripheral α-Gals is not known, they appear to be heterogeneous between two normal cellular pools of Skp1 (17West C.M. Kozarov E. Teng-umnuay P. Gene (Amst.). 1997; 200: 1-10Crossref PubMed Scopus (19) Google Scholar). α-Galp residues are non-reducing terminal modifications of O- and N-linked glycans of selected secretory glycoproteins in subhuman animals and microorganisms (18Taylor S.G. McKenzie I.F. Sandrin M.S. Glycobiology. 2003; 13: 327-337Crossref PubMed Scopus (62) Google Scholar, 19Pederson L.L. Turco S.J. Cell. Mol. Life Sci. 2003; 60: 259-366Crossref PubMed Scopus (163) Google Scholar), where they constitute potent xenoantigens and might contribute to specific recognition determinants. With the ultimate goal of understanding the role of peripheral α-galactosylation in the cytoplasm and assessing its phylogenetic range, we have undertaken an investigation of the enzymatic basis of this modification on Skp1. A screen of an ion exchange fractionation of a cytosolic extract of Dictyostelium cells using mutant Skp1A1(HW120)-myc as an acceptor yielded a prominent α-GalT activity that was partially purified and characterized. α-GalT1 appears to be a cytoplasmic GT like the earlier enzymes in the pathway. Based on studies of model acceptor compounds, α-GalT1 modifies the blood group H (type 1) trisaccharide of Skp1 by the addition of an α-Gal to the 3-position of Fuc. The modification is greatly potentiated by normally folded Skp1 in a manner that suggests the importance of conformation of the acceptor sugar structure. These findings provide an explanation for why mutant Skp1 is incompletely α-galactosylated in vivo. Buffers were adjusted to their pH values at 22 °C, filtered, degassed, and stored at 5 °C. DTT and protease inhibitors, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin (final concentrations), were added just before use. Buffer A was 50 mm Tris-HCl (pH 7.4), 0.25 m sucrose, and the protease inhibitors. Buffer B was 50 mm HEPES-NaOH (pH 7.4), 5 mm MgCl2, 0.1 mm NaEDTA, 15% (v/v) glycerol, 1 mm DTT, and the protease inhibitors. Buffer C was 85 mm ammonium acetate (pH 7.4), 20% (v/v) saturated (NH4)2SO4, 15% (v/v) glycerol, 5 mm MgCl2, 1 mm DTT. Buffer D was 25 mm ammonium acetate (pH 7.4), 15% (v/v) glycerol, 5 mm MgCl2, 1 mm DTT. Buffer E was 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 1 mm MnCl2, 15% (v/v) glycerol, 1 mm DTT. Buffer F was 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 1 mm MnCl2, 15% (v/v) glycerol, 0.15 m NaCl, 1 mm DTT. Cell Growth and Lysis—Dictyostelium discoideum strain HW302 (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar), which overexpresses a normal copy of Skp1B, was grown in 2 30-liter batches of HL-5 axenic growth medium at 22 °C to maximum cell density (∼107 cells/ml), collected by centrifugation at 3,000 × g for 1 min, resuspended in H2O, centrifuged again, and resuspended in buffer A at 2 × 108/ml. Cells were immediately filtered through a bed of glass wool, lysed by forced passage through a 5-μm pore diameter Nuclepore filter (20West C.M. Scott-Ward T. Teng-umnuay P. van der Wel H. Kozarov E. Huynh A. J. Biol. Chem. 1996; 271: 12024-12035Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and successively centrifuged at 3,000 × g for 2 min and 100,000 × g for 70 min to yield the cytosolic supernatant S100 fraction that was chromatographed immediately as described below. For small scale preparations, the glass wool prefilter step was skipped, and the pellet from the 100,000 × g centrifugation was resuspended in buffer B, recentrifuged, and resuspended in buffer B again. The resulting S100 and washed P100 fractions were frozen at –80 °C. DEAE-Sepharose Chromatography—Each S100 fraction (from 30 liters) was pumped onto a 450-ml column of DEAE-Sepharose Fast Flow (Amersham Biosciences) equilibrated at 4 °C in buffer B, and the column was washed with buffer B until the A280 returned to near baseline. The column was eluted with a 2.5-liter linear gradient of 0–0.25 m NaCl in buffer B, followed by 400 ml of 0.25 m NaCl in buffer B. Column fractions were frozen at –80 °C and aliquots were assayed for GalT activity (see below). Phenyl-Sepharose Chromatography—Fractions from the main GalT activity peak from both DEAE-Sepharose runs were pooled, adjusted to 20% (w/v) (NH4)2SO4, and centrifuged at 12,000 × g for 30 min. The supernatant was loaded at 4 °C onto a 2.6 × 20-cm column of phenyl-Sepharose Fast Flow (high sub) column (Amersham Biosciences) equilibrated in buffer C. The column was washed with buffer C until the A280 returned to baseline level, and eluted with a descending linear 750-ml gradient of buffer C to buffer D. The column was washed with buffer D until the A280 returned to near baseline level, and a 750-ml ascending linear gradient from 0 to 70% ethylene glycol in buffer D, followed by 200 ml of 70% ethylene glycol in buffer D was then applied. Q-Sepharose Chromatography—The active fractions from the phenyl-Sepharose column were divided into three pools, and each loaded separately, at 4 °C, onto a 5-ml Hi-Trap Q-Sepharose column (Amersham Biosciences) equilibrated in buffer E. The column was washed with buffer E until the A280 was less than 0.01. The column was eluted at 21 °C, with a 50-ml linear gradient of 0–0.2 m NaCl in buffer E, followed by a 10-ml gradient of 0.25–0.5 m NaCl in buffer E. Superdex 200 Chromatography—The pooled GalT activity from the three Q-Sepharose columns was concentrated in a Centriprep 30 ultrafiltration device (Amicon) to 2.2 ml at 4 °C, and loaded onto a 16/60 Superdex 200 column (Amersham Biosciences) equilibrated in buffer F. The column was eluted with buffer F at 1 ml/min at 21 °C. Mr values of the calibration standards were 200,000, sweet potato β-amylase; 66,000, bovine serum albumin; and 29,000, carbonic anhydrase. Protein Determination—Protein concentration was determined by the Coomassie Blue dye binding method (Pierce Coomassie Plus), or calculated from A280 values, assuming A0.1%280 = 1.0. Assay Conditions—GalT activity was assayed by the transfer of [3H]Gal from UDP-[6-3H]Gal (American Radiolabeled Chemicals) to synthetic glycosides or Skp1. Unless indicated otherwise, reactions (15–60 μl) contained 1–5 μm UDP-[3H]Gal (13,200–64,000 disintegrations/min/pmol), acceptor substrate as described below, 50 mm HEPES-NaOH (pH 7.4), 50 mm NaCl, 2 mm MnCl2, 5 mm DTT, 0.1 mg/ml bovine serum albumin, and 0.1% (v/v) Tween 20, and were incubated at 22 °C for 30–120 min. In some tests containing 3H-labeled acceptor substrates (see below), UDP-[U-14C]Gal (300 mCi/mmol; American Radiochemical Corp.) was used in place of the tritiated compound. Early purification fractions (up through Q-Sepharose) were assayed in the presence of 1 mm ATP and 1 mm NaF. To determine the pH optimum, the enzyme was diluted into the following buffers (100 mm): MES-NaOH (pH 6.0–6.6), MOPS-NaOH (pH 6.6–7.4), and HEPES-NaOH (pH 7.4–8.0). Synthetic Acceptor Substrates—Fucα1,2Galβ1,3GlcNAcβ1-pNP (21Matta K.L. Rana S.S. Piskorz C.F. Abbas S.A. Carbohydr. Res. 1984; 131: 247-255Crossref PubMed Scopus (9) Google Scholar), Galβ1,3GlcNAcβ1-pNP (22Matta K.L. Barlow J.J. Carbohydr. Res. 1975; 43: 299-304Crossref PubMed Scopus (25) Google Scholar), and Galβ1,3GlcNAcβ1-Bn (23Rana S.S. Barlow J.J. Matta K.L. Carbohydr. Res. 1981; 96: 231-239Crossref PubMed Scopus (26) Google Scholar) were synthesized previously. Galβ1,3GlcNAcα1-Bn was synthesized according to the method described for the preparation of Galβ1,3GlcNAcβ1-Bn (23Rana S.S. Barlow J.J. Matta K.L. Carbohydr. Res. 1981; 96: 231-239Crossref PubMed Scopus (26) Google Scholar). Fucα1-Me, Fucα1-allyl, and Fucα1-Bn were obtained by the reaction of l-fucose and alcohol in the presence of acidic resin Dowex 50-X (H+). For example, l-fucose on treatment with benzyl alcohol under these conditions yielded Fucα1-Bn (24Flowers H. Carbohydr. Res. 1979; 74: 177-185Crossref Scopus (17) Google Scholar). Synthesis of Fucα1,2(Galα1,6)Galβ1-Bn and Fucβ1,2(Galα1,6)Galβ1-Bn will be described later. 2R. D. Locke, Sirajud-Doulah. K, and K. L. Matta, unpublished data. Concentrations were determined spectrophotometrically using an extinction coefficient for Bn glycosides of 3.4 × 102m–1 cm–1 at 255 nm, and for pNP glycosides of 1.15 × 104m–1 cm–1 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 (25Kozarov E. van der Wel H. Field M. Gritzali M. Brown Jr., R.D. West C.M. J. Biol. Chem. 1995; 270: 3022-3030Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Other pNP glycosides were from Sigma. Fucα1,2Galβ1,3GlcNAcα1-Bn and Fucα1,2Galβ1,3GlcNAcβ1-Bn were produced by enzymatic fucosylation of Galβ1,3GlcNAcα1-Bn and Galβ1,3GlcNAcβ1-Bn, respectively, using soluble recombinant Skp1 FT85 FucT purified from Escherichia coli strain ER2566 transfected with pTY(CBD-FT85) (13van der Wel H. Fisher S.Z. West C.M. J. Biol. Chem. 2002; 277: 46527-46534Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). After induction, harvest, and lysis, the bacterial extract was centrifuged at 12,000 × g for 30 min. At 4 °C, the supernatant was made 15% saturated in (NH4)2SO4 and centrifuged again. The supernatant was loaded onto a 35-ml column of phenyl-Sepharose Fast Flow (high sub) (Amersham Biosciences) equilibrated in 15% (NH4)2SO4 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 α1,2-FucT using Galβ1,3GlcNAcβ1-pNP as described (25Kozarov E. van der Wel H. Field M. Gritzali M. Brown Jr., R.D. West C.M. J. Biol. Chem. 1995; 270: 3022-3030Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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. The column was washed with buffer B, eluted with a linear gradient of 0–0.5 m NaCl in buffer B, and active fractions were pooled. The disaccharide-Bn compounds were incubated for 24 h at 21 °C in 50 mm HEPES-NaOH (pH 7.4), 15 mm MgCl2, 2 mm MnCl2, 5 mm DTT, and 0.1 m NaCl, with a 4-fold excess of GDP-[3H]Fuc and an aliquot of purified Skp1 FucT. The reaction mixture was applied to a C18 reversed phase column and eluted as described below. The fucosylated derivative eluted in a baseline separated peak based on A254 and confirmed by scintillation counting of aliquots of the fractions. Concentration was determined from the specific activity of the incorporated [3H]Fuc. Skp1A1(HW120)-myc—Skp1A1(HW120)-myc was purified through the monoclonal antibody 3F9 step as previously described (13van der Wel H. Fisher S.Z. West C.M. J. Biol. Chem. 2002; 277: 46527-46534Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) from Dictyostelium strain HW120 (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar). Skp1A1(HW120)-myc, which contains 2 missense mutations (I34T, D71G) in its N-terminal region, consists of a mixed population of glycoforms (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar) as summarized in the text. Fucosylated Skp1 from Strain HL250 —Skp1 was purified from Dictyostelium strain HL250 as described above for Skp1A1(HW120)-myc. This strain, unable to synthesize GDP-Fuc, accumulates Skp1 containing the Galβ1,3GlcNAc-disaccharide (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The disaccharide on Skp1(HL250) was fucosylated using soluble recombinant Skp1 FT85 FucT as described above for the disaccharide-Bn compounds. Skp1(HL250) was incubated for 48 h at 21 °C with a 4.4-fold excess of GDP-fucose and a 5-fold excess of purified Skp1 FucT needed for complete fucosylation as determined in parallel aliquots supplemented with GDP-[3H]Fuc (data not shown). Fucosylated Skp1 was diluted 2-fold with water and applied to a 0.24-ml mini-Q column (Amersham Biosciences) equilibrated in buffer B at 22 °C on an Amersham Biosciences SmartSystem HPLC, and eluted with a 3-ml linear gradient of 0–0.6 m NaCl in buffer B. Skp1-containing fractions were identified by SDS-PAGE and Western blotting with monoclonal antibody 3F9 (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar). Quantitation of Reaction—Reactions containing the Skp1 substrates were terminated by the addition of 5 μl of 20 mg/ml bovine serum albumin and 700 μl of ice-cold 10 mm sodium pyrophosphate in 10% (w/v) trichloroacetic acid. After incubation on ice for 1 h, samples were vacuum-filtered through GF/C glass fiber filters pre-wetted with icecold 10% (w/v) trichloroacetic acid. The reaction tube was rinsed with 10 mm sodium pyrophosphate in 10% (w/v) trichloroacetic acid that was also applied to the filter. The filter was rinsed 3 times with 1 ml of ice-cold 10% (w/v) trichloroacetic acid and 4 times with 1 ml of ice-cold acetone, and counted in 10 ml of Scintiverse BioHP (Fisher) with a Beckman LS6500 scintillation counter. Alternatively, reaction mixtures were diluted in 2× sample buffer and separated on 7–20% SDS-PAGE gel, stained with Coomassie Blue, followed by excision of the region of the gel containing Skp1 as described (12van der Wel H. Morris H.R. Panico M. Paxton T. Dell A. Kaplan L. West C.M. J. Biol. Chem. 2002; 277: 46328-46337Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Negative control samples were either quenched at time 0, contained no acceptor substrate, or no enzyme, and subtracted as background as indicated. Reactions containing synthetic glycosides were terminated by the addition of 1 ml of ice-cold water or 1 mm EDTA. For the Bn and pNP derivatives, the mixture was filtered over a C18 Sep-Pak cartridge (Millipore) mounted on a 10-position Visiprep vacuum manifold (Supelco). After seven 5-ml water washes, each Sep-Pak was eluted with 5 ml of MeOH into a scintillation vial. 15 ml of Scintiverse LS was added and the sample was subjected to liquid scintillation counting. For methyl and allyl derivatives, the mixture was applied to a 0.5-ml column of Dowex-1 (Sigma) prewashed with 5 column volumes of 1 n HCl and 20 column volumes of water. The 1-ml flow-through fraction and three 1-ml water washes were collected in a 20-ml scintillation vial, diluted with 4 volumes of Scintiverse LC, and analyzed for disintegrations/min as above. Product Characterization—To identify the position of substitution by Gal, Fuc1α-Bn was reacted with the purified pool of α-GalT1 in the presence of 1 mm UDP-[3H]Gal for varied periods of time, to compare partially and fully modified preparations. The reaction product was recovered by elution from a C18 Sep-Pak with MeOH. The dried material was dissolved in 10 mm ammonium formate (pH 4.0) and applied to a C18-reversed phase column (4.6 × 250 mm, TSK ODS-120T, 5 μm) according to Ref. 26Toomre D.K. Varki A. Glycobiology. 1994; 4: 653-663Crossref PubMed Scopus (37) Google Scholar. The column was eluted in a gradient of 0–40% acetonitrile in the same buffer at 22 °C at 1 ml/min. The eluate was monitored by measuring A254 and liquid scintillation counting of fractions. Positional isomers of Galα1-Fucα1-Bn were synthesized in a two-step process that included: 1) condensation of the appropriately blocked benzyl-1-O-α-l-fucoside with methyl-2,3,4,6-tetra-(O-4-methoxybenzyl)-1-thio-β-d-galactopyranoside as the glycosyl donor, and 2) deprotection of the resulting disaccharide.2 Products were characterized by NMR and MS.2 To determine sensitivity of incorporated radioactivity to digestion with α-galactosidase, Fucα1,2Galβ1-pNP (0.6 mm) was reacted in the presence of the purified pool of α-GalT1 and UDP-[3H]Gal (1.8 mm) until completion as determined by incorporation of [3H]Gal. The reaction product was recovered by elution from a C18 Sep-Pak with MeOH as above and taken to dryness in a vacuum centrifuge. This material was dissolved in a preparation of α1–3/6-galactosidase from Xanthomonas manihotis (New England Biolabs; Ref. 27Wong-Madden S.T. Landry D. Glycobiology. 1995; 5: 19-28Crossref PubMed Scopus (86) Google Scholar), α-galactosidase from green coffee beans (Calbiochem), or β-galactosidase from E. coli as described (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Enzyme activities were qualitatively verified colorimetrically using Galα1-pNP or Galβ1-pNP (data not shown). After incubation at 37 °C for varying times, aliquots were applied to a C18 Sep-Pak in water. Non-hydrolyzed substrate was eluted with MeOH and quantitated by counting as above. Detection and Purification of the GalT Activity—The initial screen for Skp1 α-GalT enzyme activities was modeled after the assay for the Skp1 GlcNAcT (11Teng-umnuay P. van der Wel H. West C.M. J. Biol. Chem. 1999; 274: 36392-36402Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The acceptor substrate was a purified recombinant mutant form of Skp1, Skp1A1(HW120)-myc, which previous studies showed consisted of multiple structures including 2 glycoforms lacking one or both of the outer αGal residues (8Teng-umnuay P. Morris H.R. Dell A. Panico M. Paxton T. West C.M. J. Biol. Chem. 1998; 273: 18242-18249Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar). This heterogeneity does not occur in wild-type Skp1, which is almost homogeneously glycosylated (9Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (28) Google Scholar). The assay mixture contained UDP-[3H]Gal, 1 μm Skp1A1(HW120)-myc, divalent cations Mg2+ (5 mm) and Mn2+ (2 mm), 5 mm DTT, and 50 mm NaCl, at pH 7.4. A cytosolic (S100) fraction from growing cells was initially tested for activity as this was a source of the other known Skp1 modification enzymes (11Teng-umnuay P. van der Wel H. West C.M. J. Biol. Chem. 1999; 274: 36392-36402Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 20West C.M. Scott-Ward T. Teng-umnuay P. van der Wel H. Kozarov E. Huynh A. J. Biol. Chem. 1996; 271: 12024-12035Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Substantial incorporation of radioactivity into the Skp1 band was observed after SDS-PAGE analysis of this reaction, relative to the level found in the absence of added Skp1A1(HW120)-myc (data not shown). The S100 preparation was fractionated on DEAE-Sepharose using the method that had been previously employed for the purification of the Skp1 β1,3-GalT/α1,2-FucT (20West C.M. Scott-Ward T. Teng-umnuay P. van der Wel H. Kozarov E. Huynh A. J. Biol. Chem. 1996; 271: 12024-12035Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and Glc-NAcT (11Teng-umnuay P. van der Wel H. West C.M. J. Biol. Chem.
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