Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-transferase in the Cytoplasm of Dictyostelium
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36392
ISSN1083-351X
AutoresPatana Teng-umnuay, Hanke van der Wel, Christopher M. West,
Tópico(s)Galectins and Cancer Biology
ResumoSkp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galα1–6Galα1-Fucα1–2Galβ1–3GlcNAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [3H]GlcNAc from UDP-[3H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH2 after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M r of ∼45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M r 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-125I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K m values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm. Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galα1–6Galα1-Fucα1–2Galβ1–3GlcNAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [3H]GlcNAc from UDP-[3H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH2 after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M r of ∼45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M r 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-125I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K m values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm. hydroxyproline 5-[3-(p-azidosalicylamido)-1-propenyl]-uridine diphosphate GlcNAc dithiothreitol D-glucosamine monoclonal antibody high pressure liquid chromatography 4-morpholineethanesulfonic acid polyacrylamide gel electrophoresis rough endoplasmic reticulum Skp1 belongs to the SCF complex that is involved in the ubiquitination of cell cycle and other regulatory proteins including transcriptional factors (1Koepp D.M. Harper J.W. Elledge S.J. Cell. 1999; 97: 431-434Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 2Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 3Li F.N. Johnston M. EMBO J. 1997; 16: 5629-5638Crossref PubMed Scopus (182) Google Scholar, 4Ingram G.C. Doyle S. Carpenter R. Schultz E.A. Simon R. Coen E.S. EMBO J. 1997; 16: 6521-6534Crossref PubMed Scopus (123) Google Scholar). In Dictyostelium discoideum, Skp1 is encoded by two similar genes, fpa1 andfpa2 (5West C.M. Kozarov E. Teng-umnuay P. Gene (Amst.). 1997; 200: 1-10Crossref PubMed Scopus (19) Google Scholar), and is found in both the cytoplasm and the nucleus based on immunofluorescence localization. 1S. Compton, K. Dobson, M. Sweetinburgh, and C. M. West, unpublished data. 1S. Compton, K. Dobson, M. Sweetinburgh, and C. M. West, unpublished data. Dictyostelium Skp1 is modified by an unusual linear pentasaccharide, Galα1–6Galα1-Fucα1–2Galβ1–3GlcNAc, attached to a HyPro2 residue at amino acid position 143 (6Teng-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). As the first glycosyltransferase in the modification of Pro-143, the hypothetical Skp1-HyPro GlcNAc-transferase is poised to control the glycosylation of Skp1 at this amino acid position. In support of such a regulatory role, a Skp1 isoform that is hydroxylated at Pro-143 but is not glycosylated can be detected when the fpa1 gene is overexpressed (6Teng-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 Skp1-HyPro GlcNAc-transferase is likely to be distinct from previously described GlcNAc-transferases based on the novelty of the GlcNAc-HyPro linkage formed. Most known protein GlcNAc-transferases are compartmentalized within the lumen of the Golgi apparatus (7Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar, 8Field M.C. Wainwright L.J. Glycobiology. 1995; 5: 463-472Crossref PubMed Scopus (98) Google Scholar, 9Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 10Bendiak B. Schachter H. J. Biol. Chem. 1987; 262: 5784-5790Abstract Full Text PDF PubMed Google Scholar). However, a Ser/Thr GlcNAc-transferase has been described that functions in the cytoplasmic compartment (11Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar, 12Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 13Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 14Hart G.W. Annu. Rev. Biochem. 1997; 66: 315-335Crossref PubMed Scopus (449) Google Scholar). Although glycosylation of Skp1 in a compartment of its function, the cytoplasm, might be simplest, there is precedence for bidirectional trafficking of proteins across the membrane of the rER for secretion or degradation (15Suzuki T. Yan Q. Lennarz W.J. J. Biol. Chem. 1998; 273: 10083-10086Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), which might also be utilized by Skp1. This more complex scenario would accommodate the hydroxylation of Pro-143, as all known prolyl hydroxylases are also located within the rER (16Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). Determining whether Skp1 is modified in the cytoplasm or a compartment of the secretory pathway is important for understanding the regulation and significance of this pathway. Studies on another glycosyltransferase in the Skp1-HyPro modification pathway, a GDP-Fuc:Galβ1–3HexNAc α1,2Fuc-transferase, strongly suggested that it is compartmentalized in the cytoplasm (17West 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). This Skp1 α1,2Fuc-transferase activity was purified to near-homogeneity from a cytosolic extract of Dictyostelium and was found to be absolutely dependent on DTT and Mg2+. Negligible activity could be detected in the vesicle fraction of the cell. The requirement for DTT implied that the enzyme normally functions in a reducing environment, i.e. the cytoplasm rather than the lumen of the secretory pathway. Furthermore, the α1,2Fuc-transferase exhibited submicromolar K m values for GDP-Fuc and Skp1. These exceptionally high affinities, relative to those of Golgi enzymes, reinforced the cytoplasmic localization model, where substrates would not be as concentrated as they are in the Golgi. A similar high affinity for its donor substrate was reported for the cytoplasmic Ser/Thr GlcNAc-transferase (11Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar). Although the compartmentalization of the Skp1 α1,2Fuc-transferase and its role in modifying Skp1 remains to be confirmed by genetic studies, the findings suggest that Skp1 is modified by a novel, sequentially acting, six-enzyme pathway residing in the cytoplasm. To investigate the properties and compartmentalization of the Skp1-HyPro GlcNAc-transferase, an assay was developed to guide its identification and purification. An activity has been detected in a cytosolic extract of Dictyostelium that appears to transfer GlcNAc from UDP-GlcNAc to Skp1-HyPro-143. By using a combination of conventional chromatography and affinity chromatography based on a resin containing a novel UDP-GlcNAc linkage, a candidateM r 51,000 protein has been purified to near-homogeneity. The kinetic properties of the purified Skp1-HyPro GlcNAc-transferase activity and its absolute dependence on a reducing environment suggest, as for the Skp1 α1,2Fuc-transferase activity described above, that the first glycosyltransferase in the Skp1 HyPro modification pathway also resides in the cytoplasm. Skp1A-Myc was purified from strain HW120 through the mAb 3F9 affinity column step as described (6Teng-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). Recombinant Skp1A-His10 was purified on a nickel chelating column as described (17West 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 further on a mAb 3F9 affinity column. Purified proteins were concentrated in Centriplus-10 ultrafiltration concentrators (Amicon) to about 10 μm, and aliquots were stored at −80 °C. Synthetic peptides corresponding to amino acids 133–155 of Skp1, KIFNIKDFTPEEEEQIRKENEW and KIFNIKNDFT(4-OH)PEEEQIRKENEW, were synthesized by the ICBR Protein Core at the University of Florida and verified by amino acid composition and matrix-assisted laser desorption-time of flight-mass spectrometry analyses. Peptides were purified by reversed-phase HPLC, dried, and dissolved in 50 mm HEPES-NaOH, pH 7.4, at 20 mg/ml. UDP-[3H-6]GlcNAc (NEN Life Science Products) had a specific activity of 34.8 Ci/mmol. GlcNAc-transferase activity was assayed by the transfer of [3H]GlcNAc from UDP-[3H]GlcNAc to Skp1A-Myc or peptide-(133–155). Typically, assays contained 1 μm Skp1A-Myc, 50 mm HEPES-NaOH, pH 7.8, 5 mm MgCl2, 5 mm DTT, 0.5 mg/ml bovine serum albumin, and 0.3–0.66 μmUDP-[6-3H]GlcNAc in a final volume of 35 μl. 1 mm ATP and 6 mm NaF were included in assays of S100 (where S100 is the cytosolic cell fraction isolated as the supernatant after centrifugation at 100,000 × g for 1 h) and DEAE column fractions. Following addition of enzyme, reactions were incubated at 30 °C for 1 h. Reactions were stopped by addition of either 500 μl of ice-cold 10 mmsodium pyrophosphate in 10% (w/v) trichloroacetic acid, or 35 μl of 2× Laemmli electrophoresis sample buffer containing 60 μg/ml soybean trypsin inhibitor (Sigma). Reactions stopped with 2× sample buffer were boiled for 3 min and resolved on a 7–20% SDS-PAGE gel. The gel was stained with 0.25% (w/v) Coomassie Blue R-250 in 45% (v/v) methanol, 10% (v/v) acetic acid for 1 h, destained in 5% (v/v) methanol, 7.5% (v/v) acetic acid overnight, and rinsed in H2O for 0.5–1 h. Gel slices corresponding to the position of soybean trypsin inhibitor (M r 20,100), which comigrated with Skp1A-Myc, or to the peptide, were excised and incubated in 10% TS-2 (Research Products International, Mt. Prospect, IL), 0.6% 2,5-diphenyloxazole, 0.015% 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene in toluene (Scintanalysis grade). After 72 h, 3H incorporation was determined by scintillation counting (Beckman LS6500). Negative control samples were quenched at zero time or lacked added Skp1, and values (15–35 dpm) were subtracted. The SDS-PAGE assay was more sensitive than the trichloroacetic acid assay and was used for the kinetic results shown. Reactions quenched with trichloroacetic acid were incubated on ice and vacuum-filtered over a GF/C (Whatman) glass fiber filter prewetted with ice-cold 10% (w/v) trichloroacetic acid. The reaction tube was rinsed with 10 mm sodium pyrophosphate in 10% trichloroacetic acid which was also transferred to the filter, and the filter was rinsed three more times with ice-cold 10% trichloroacetic acid. The filter was then rinsed 4× with 1 ml each of ice-cold acetone. Filters were counted in 10 ml of Scintiverse BioHP mixture (Fisher) as above. Negative control values, ranging from 200 to 500 dpm, were derived from reactions lacking added enzyme and were subtracted from the results given. In the assay of some particulate fractions, the reaction mixture was clarified at 100,000 × g for 70 min prior to analysis by the SDS-PAGE method. In addition, some supernatants were subjected to immunoprecipitation with mAb 9E10 conjugated to activated Sepharose CL-4B beads at 15 mg/ml (18Harlow E. Lane D. Using Antibodies: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999Google Scholar); the washed precipitates were then analyzed by the SDS-PAGE method. 5-Hg-dUMP was synthesized as described (19Zeng Y. Shabalin Y. Szumilo T. Pastuszak I. Drake R.R. Elbein A.D. Anal. Biochem. 1996; 239: 99-106Crossref PubMed Scopus (4) Google Scholar). Ethanol-precipitated 5-Hg-dUMP was washed with ethyl acetate:ethanol (1:4), dissolved in 50 ml of H2O, and loaded onto a 10-ml Chelex 100 resin (Bio-Rad) to remove residual Hg2+ as described (20Dale R.M.K. Martin E. Livingston D.C. Ward D.C. Biochemistry. 1975; 14: 2447-2467Crossref PubMed Scopus (161) Google Scholar). The flow-through fraction contained 5-Hg-dUMP to be used for the coupling reaction. The yield of mercuration, measured by the shift of absorbance spectrum from 260 to 267 nm was 71%. Thiopropyl-Sepharose 6B was prepared according to the manufacturer's protocol (Amersham Pharmacia Biotech) as follows. The dried material was swollen in 50 mm Tris-HCl, 0.1 m NaCl, pH 7.0, at room temperature overnight and washed on a Buchner funnel with a large excess of H2O. Free thiol groups were released by suspending the gel in 1% (w/v) DTT in 0.3 mNaHCO3, 1 mm Na2EDTA, pH 8.4, for 1 h. The gel was washed with a large excess of 0.5 mNaCl, 0.1 m acetic acid, 1 mmNa2EDTA, followed by H2O. The gel was then resuspended in the Chelex 100 flow-through fraction of 5-Hg-dUMP with gentle mixing on a shaking platform for 2 h at room temperature. The coupling yield was determined from the decrease of absorbance at 267 nm. Synthesis of 5-Hg-UDP-GlcNAc was performed as above for 5-Hg-dUMP except the Chelex 100 resin step was omitted, with a 70% yield. 5-(3-Amino)allyl-UDP-GlcNAc was synthesized from 5-Hg-UDP-GlcNAc and purified over a DEAE-Sepharose Fast Flow column as described (19Zeng Y. Shabalin Y. Szumilo T. Pastuszak I. Drake R.R. Elbein A.D. Anal. Biochem. 1996; 239: 99-106Crossref PubMed Scopus (4) Google Scholar). The product exhibited absorbance maxima at 240 and 287 nm and a minimum at 262 nm, typical for a compound with an exocyclic double bond with a pyrimidine ring. Fractions containing 5-(3-amino)allyl-UDP-GlcNAc were pooled giving a 63% yield, dried under vacuum centrifugation, dissolved in anhydrous dimethylformamide, and adjusted with triethylamine to pH 7.5, measured with pH paper. Two ml of NHS-activated Sepharose 4 Fast Flow gel (Amersham Pharmacia Biotech) was washed with 10 ml of dimethylformamide on a Buchner funnel, and the gel was resuspended in the 5-(3-amino)allyl-UDP-GlcNAc solution. The coupling reaction was performed for 24 h with gentle mixing on a shaking platform at room temperature; however, maximal coupling, monitored by the decrease of absorbance at 287 nm, occurred in 2 h. The dimethylformamide was then replaced by 1 maminoethanol in 50 mm Tris-HCl, pH 7.8, for 2 h. One ml of gel was packed in a HR5/5 column (Amersham Pharmacia Biotech), washed with 50 mm Tris-HCl, pH 7.8, followed by 0.1m boric acid, pH 8.0, and stored in 2 m NaCl, 0.005% thimerosol in 50 mm HEPES-NaOH, pH 7.8. Buffer pH values were adjusted at room temperature, and solutions were degassed and chilled, and filtered 1 mDTT was added just prior to use. Protease inhibitors were added to buffers A–C to the following concentrations immediately prior to use: 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Buffer A is as follows: 50 mmHEPES-NaOH, pH 7.4, 1 mm DTT, 5 mmMgCl2, 0.1 mm Na2EDTA, 15% (v/v) glycerol, protease inhibitors; buffer B is as follows: 50 mm HEPES-NaOH, pH 7.8, 5 mm DTT, 5 mm MgCl2, 0.1 mmNa2EDTA, 15% (v/v) glycerol, protease inhibitors; buffer C is as follows: 50 mm HEPES-NaOH, pH 7.8, 5 mmMgCl2, 0.1 mm Na2EDTA, 15% (v/v) glycerol, protease inhibitors; buffer D is as follows: 50 mm HEPES-NaOH, pH 7.8, 5 mm DTT, 0.1% (v/v) Tween 80; 5 mm MgCl2, 0.1 mmNa2EDTA, 15% (v/v) glycerol; buffer E is as follows: 50 mm HEPES-NaOH, pH 7.8, 5 mm DTT, 5 mm MgCl2, 0.1 mmNa2EDTA; and buffer F is as follows: 50 mmHEPES-NaOH, pH 7.8, 5 mm DTT, 0.01% (v/v) Tween 80; 5 mm MgCl2, 0.1 mmNa2EDTA, 15% (v/v) glycerol. Stationary phase strain Ax3 or HW120 cells grown in HL-5 were filter-lysed and successively centrifuged at 3000 × g for 2 min and 100,000 × g for 70 min to generate the cytosolic S100 supernatant fraction as described previously (17West 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). The pellets were resuspended in lysis buffer and re-centrifuged, yielding the P3 and P100 (where P100 is the particulate cell fraction equivalent to the pellet formed by centrifugation at 100,000 × g for 1 h) fractions, respectively. Nuclei were prepared using either Nonidet P-40 or digitonin as described (21Maclean N. Garside K. Bradley M.C. Wood C. Experientia (Basel). 1984; 40: 1207-1214Crossref Scopus (9) Google Scholar). The S100 supernatant (800 ml) was pumped onto a 450-ml DEAE-Sepharose Fast Flow column equilibrated in buffer A and eluted with the same buffer (17West 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). Skp1 was quantitatively retained on the column, whereas GlcNAc-transferase activity appeared in the wash fractions, which were frozen at −80 °C. The column was washed with 2 m NaCl in 50 mm HEPES-NaOH, pH 7.4, prior to reuse. (NH4)2SO4(ultrapure, ICN) was added to the pooled active fractions from the DEAE-Sepharose column to a final concentration of 25% saturation with stirring at 4 °C. After centrifugation at 20,000 ×g for 20 min, the supernatant was brought to 60% (NH4)2SO4 saturation. The pellet was collected by centrifugation, dissolved in 300 ml of 20 or 25% saturated (NH4)2SO4 in buffer B, and recentrifuged to clarify. The 25–60% (NH4)2SO4 cut was applied to a phenyl-Sepharose Fast Flow (low-sub) column (2.6 cm x 20 cm) pre-equilibrated with 20 or 25% saturated (NH4)2SO4 in buffer B at 4 °C, washed with the same buffer until the A 280dropped to below 1% of its maximum, and eluted with a linear decreasing 600-ml gradient to buffer B. Fractions were stored at −80 °C. The column was washed in 6 m urea in 50 mm HEPES-NaOH, pH 7.8, followed by 1 m NaOH, before reuse. The phenyl-Sepharose activity pool was loaded onto a Reactive Red-120 Fast Flow column (1.6 × 5 cm), equilibrated in buffer B, at 4 °C at 80 ml/min. After washing with buffer B until the A 280dropped to 10% of maximum, the column was eluted with a 150-ml 0–2m NaCl linear or step gradient prepared in buffer B. Fractions were assayed after desalting on 9 ml of Sephadex G-25 columns (PD-10 columns, Amersham Pharmacia Biotech) pre-equilibrated in buffer D, and stored at −80 °C. The Reactive Red-120 column was precycled according to manufacturer's instructions (Sigma) prior to reuse. The Reactive Red column activity pool was concentrated in a Centriplus-10 ultrafiltration device (Amicon) to <2 ml and was loaded onto a Superdex 200 (16/60) column (Amersham Pharmacia Biotech) equilibrated in buffer C. The column was isocratically eluted at 0.8 ml/min at 22 °C, and fractions were stored at −80 °C. The column was calibrated with the following M r standards: horse spleen apoferritin (443,000), yeast alcohol dehydrogenase (150,000), bovine serum albumin (66,000), ovalbumin (45,000), and soybean trypsin inhibitor (20,100). The Superdex 200 activity pool was loaded onto a 1-ml 5-Hg-dUMP column equilibrated in buffer C. DTT was added to the flow-through fraction to 5 mm, which was concentrated in a Centriplus-10 ultrafiltration device to <2 ml. After use, the column was cleaned with 5 mm dUMP and stored in 2m NaCl, 0.005% thimerosol in 50 mm HEPES-NaOH, pH 7.8. The concentrated flow-through fraction from the 5-Hg-dUMP column was loaded at 100 μl/min onto the aminoallyl-UDP-GlcNAc-Sepharose column described above. This column was mounted on a Smart HPLC system (Amersham Pharmacia Biotech) and equilibrated in buffer D, which contained 0.1% (v/v) Tween 80 to stabilize enzyme activity. The column was washed with buffer F until recovery of the original base line and was eluted with a 7.4-ml gradient of 0–2 mm UMP in buffer F at 22 °C and 250 μl/min. To detect enzyme activity, fractions were either desalted on PD-10 columns equilibrated in buffer D or cyclically concentrated and diluted in Microcon-10 ultrafiltration devices (Amicon) with buffer F to reduce UMP to <20 μm. For kinetic studies, active fractions were pooled and desalted on a Fast Desalting PC3.2/10 column mounted on the Smart System HPLC that was equilibrated with buffer E and run at 100 μl/min at 22 °C. The column was washed with 8 m urea, 50 mm DTT, 50 mm Tris-HCl, pH 7.5, prior to reuse. For SDS-PAGE and photoaffinity labeling, the UDP-GlcNAc affinity pool was concentrated to <50 μl in a Microcon-10 ultrafiltration device and applied to a Superdex 75 PC3.2/30 column (Amersham Pharmacia Biotech) pre-equilibrated in buffer F, which contained 0.01% Tween 80 (v/v). The column was eluted at 0.05 ml/min at 22 °C, collecting 25-μl fractions. The column was calibrated with bovine serum albumin (M r66,000), ovalbumin (M r 43,000), carbonic anhydrase (M r 29,000), and cytochrome C (M r 12,400). Protein concentration was determined by a commercial modification of a Coomassie Blue dye-binding method (22Sedmak J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Crossref PubMed Scopus (2484) Google Scholar) according the manufacturer's protocol (Pierce), using bovine serum albumin as a standard. After UDP-GlcNAc affinity chromatography, protein was alternatively estimated from itsA 280 value, assuming an extinction coefficient of 1 ml/mg for a 1-cm path length, or by analysis of phenylisothiocyanate-amino acid derivatives after acid hydrolysis of the sample at the ICBR Protein Chemistry Core Laboratory, using norleucine as an internal standard. Column fractions were diluted with equal volumes of 2× Laemmli sample buffer containing 5 mm DTT and boiled for 2 min. Iodoacetamide was added to 40 mm, and samples were applied to a 7–20% (w/v) linear gradient SDS-PAGE gel (17West 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). Gels were silver-stained (23Wray W. Boulikas T. Wray V.P. Hancock R. Anal. Biochem. 1981; 118: 197-203Crossref PubMed Scopus (2492) Google Scholar). 0.5 μmol of 5-ASA-UDP-GlcNAc (19Zeng Y. Shabalin Y. Szumilo T. Pastuszak I. Drake R.R. Elbein A.D. Anal. Biochem. 1996; 239: 99-106Crossref PubMed Scopus (4) Google Scholar), a generous gift of Dr. A. E. Elbein, was reacted with 2 mCi of carrier-free Na125I from NEN Life Science Products as described (24Holmes E.H. J. Biol. Chem. 1990; 265: 13150-13156Abstract Full Text PDF PubMed Google Scholar). 5-[125I]ASA-UDP-GlcNAc was preincubated with Superdex 75 fractions at 30 μm in the presence or absence of 700 μm UDP-GlcNAc for 15 min at 22 °C. The reaction mixture was then exposed for 2 min to 254 nm radiation from a 30-watt mineralight at a distance of 1 cm. The reaction was diluted with 2× Laemmli electrophoresis buffer and processed as described in the SDS-PAGE section above. The stained gel was then autoradiographed against Kodak Bio-Max HP film at −80 °C. Kinetic studies were performed on the UDP-GlcNAc affinity pool after passage over a Fast Desalting PC3.2/10 column. Reactions were incubated for 1 h using the SDS-PAGE assay. Gel slices containing [3H]Skp1 or [3H]4-HyPro peptide were rinsed in H2O, minced, and diluted with an equal volume of 1m NaBH4 in 0.2 m NaOH. After incubation at 45 °C for 20 h, the supernatant was neutralized with an equal volume of 0.2 m acetic acid in MeOH and dried under vacuum centrifugation. The radioactive residue was dissolved in 2 ml of H2O, adjusted to pH 9.5 with 2-amino-2-methyl-1 propanol as determined using pH paper, and loaded onto a 1-ml Dowex 1 column pretreated with 5 ml of 1 m HCl and 10 ml of H2O. The flow-through was diluted in Scintiverse-HP scintillation mixture and counted as above. Radioactive gel slices were hydrolyzed in 1 ml of 4 m trifluoroacetic acid at 100 °C for 4 h. The supernatant was dried by vacuum centrifugation, dissolved in H2O, dried again, dissolved in 100 μl of H2O containing 1 nmol of GlcNH2, and chromatographed on a PA-10 column on a DX-500 HPLC system (Dionex) in 17 mm NaOH at 1 ml/min. The eluant was monitored by pulsed amperometric detection (17West 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 fractions were counted in a liquid scintillometer. Strain HW120 expresses a c-Myc-tagged form of Skp1A under the control of the discoidin promoter (6Teng-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). Skp1A-Myc isolated from the S100 fraction of this strain contains an abundant isoform that is hydroxylated but not glycosylated at Pro-143. This unglycosylated isoform is predicted to be an acceptor substrate for the hypothetical Skp1 GlcNAc-transferase. An assay for the Skp1 GlcNAc-transferase was developed based on transfer of 3H from UDP-[3H]GlcNAc to Skp1A-Myc, in the presence of 1 mm DTT, 5 mm MgCl2, and 50 mm Tris-HCl at pH 7.5. The S100 fraction from stationary phase cells of the normal strain Ax3 was initially tested as a source of enzyme. Skp1 was purified after the reaction by SDS-PAGE, and incorporated radioactivity was measured by scintillation counting of the gel slice. Addition of Skp1A-Myc stimulated incorporation of3H into the Skp1 band over 3-fold, whereas Skp1A-His10, a recombinant form of Skp1A that is not hydroxylated at Pro-143 (6Teng-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), did not (Fig. 1 A, solid bars). No stimulation by Skp1A-Myc was seen at other M rpositions in the gel (data not shown). In addition, 10 μg/ml mAb 3F9, which is specific for Skp1 in Western blots (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), almost completely blocked stimulation of incorporation by added Skp1A-Myc (data not shown). The S100 fraction from strain HW120 exhibited a high level of spontaneous incorporation in the absence of added Skp1A-Myc (Fig. 1 B), suggesting that endogenous Skp1A-Myc was a substrate after cell lysis. Consistent with this interpretation, incorporation was only slightly further stimulated by additional Skp1A-Myc, and the high level of spontaneous incorporation was markedly reduced by mAb 3F9 (Fig. 1 B, solid bars). These results suggested the presence of a Skp1-HyPro GlcNAc-transferase in the S100 fraction of the cell. To gain further evidence that the GlcNAc-transferase activity was specific for the HyPro residue, a synthetic peptide corresponding to residues 133–155 of Skp1 with 4-HyPro at the equivalent of residue 143 was tested as a substrate. The 4-HyPro peptide induced substantial increases of 3H into the peptide band in the S100 fractions of both strains Ax3 (Fig. 1 A, open bars) and HW120 (Fig. 1 B, open bars). A synthetic peptide containing Pro in place of 4-HyPro was inactive (not shown, see Fig. 6 below). The 4-HyPro peptide markedly reduced incorporation into endogenous Skp1 of strain HW120 (Fig. 1 B). This indicated that the activity that labeled the 4-HyPro peptide was equivalent to the activity that labeled Skp1A-Myc and implied that labeling of Skp1A-Myc was associated with HyPro-143. To verify that 3H was transferred as GlcNAc
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