A Bifunctional Diglycosyltransferase Forms the Fucα1,2Galβ1,3-Disaccharide on Skp1 in the Cytoplasm ofDictyostelium
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m208824200
ISSN1083-351X
AutoresHanke van der Wel, S. Zoë Fisher, Christopher M. West,
Tópico(s)Cellular Mechanics and Interactions
ResumoSkp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucα1,2Galβ1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have β-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 β-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits β-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo. Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucα1,2Galβ1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have β-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 β-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits β-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo. Skp1 has been defined in yeast, plants, and animals as a subunit of the SCF (Skp1 cullin-1 F-box protein) 1The abbreviations used for: SCF, Skp1 cullin-1 F-box protein; CBD, chitin-binding domain; Fuc-Tase, fucosyltransferase; Gal-Tase, galactosyltransferase; GTase, glycosyltransferase; MALDI-TOF-MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; pNP, para-nitrophenyl; E3, ubiquitin-protein isopeptide ligase; mAb, monoclonal antibody; nt, nucleotide; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; UDP, uridine 5′-diphosphate. 1The abbreviations used for: SCF, Skp1 cullin-1 F-box protein; CBD, chitin-binding domain; Fuc-Tase, fucosyltransferase; Gal-Tase, galactosyltransferase; GTase, glycosyltransferase; MALDI-TOF-MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; pNP, para-nitrophenyl; E3, ubiquitin-protein isopeptide ligase; mAb, monoclonal antibody; nt, nucleotide; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; UDP, uridine 5′-diphosphate. family of E3 ubiquitin ligases, which polyubiquitinate target phosphoproteins leading to their degradation in the 26S proteasome (1Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar). Skp1 serves as an adaptor to link the F-box containing protein to the scaffold-like cullin-1, which is in turn linked to an E2 ubiquitin-conjugating enzyme via the ring H2 finger protein Roc1 (2Zheng 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 (1139) Google Scholar). Other evidence suggests that Skp1 can also be associated with Snf1-related protein kinases, the kinetochore CBF3 complex, the RAVE-complex linked to assembly of the vacuolar proton transporter, and another complex(es) possibly involved in membrane trafficking (3West C.M. Cell. Mol. Life Sci. 2002; (in press)Google Scholar). It is not known whether these complexes all draw on the same pool of Skp1 in the cell. Skp1 from the social amoeba Dictyostelium discoideum is modified by a pentasaccharide at a hydroxylated Pro residue at position 143 (4Teng-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 (70) Google Scholar). The pentasaccharide consists of a Fucα1,2Galβ1,3GlcNAc-core, 2All sugars are pyranosides and in thed-configuration except for Fuc, which is in the L-configuration. 2All sugars are pyranosides and in thed-configuration except for Fuc, which is in the L-configuration.equivalent to the blood group H type I antigen, decorated with two α-linked Gal residues probably via the Fuc residue (5West C.M. van der Wel H. Gaucher E.A. Glycobiology. 2002; 12: 17R-27RCrossref PubMed Scopus (29) Google Scholar). The majority of Skp1 in both growing and developing Dictyostelium cells appears to be fully glycosylated based on Western blotM r analysis of whole cell Skp1, and partial glycosylation of Skp1 is required for its normal accumulation in the nucleus (6Sassi S. Sweetinburgh M. Erogul J. Zhang P. Teng-umnuay P. West C.M. Glycobiology. 2001; 11: 283-295Crossref PubMed Scopus (27) Google Scholar). Assays have been developed for most of the enzymes of the Skp1 glycosylation pathway (3West C.M. Cell. Mol. Life Sci. 2002; (in press)Google Scholar, 5West C.M. van der Wel H. Gaucher E.A. Glycobiology. 2002; 12: 17R-27RCrossref PubMed Scopus (29) Google Scholar). Analysis of subcellular fractions suggests that they modify Skp1 sequentially in the cytoplasm, rather than the secretory pathway (rough endoplasmic reticulum and Golgi) as for most glycosyltransferases that modify proteins. We have taken a proteomics approach to define the glycosylation pathway of Dictyostelium Skp1 (3West C.M. Cell. Mol. Life Sci. 2002; (in press)Google Scholar, 5West C.M. van der Wel H. Gaucher E.A. Glycobiology. 2002; 12: 17R-27RCrossref PubMed Scopus (29) Google Scholar). Purification of the Skp1 fucosyltransferase (Fuc-Tase) activity led to the identification of the FT85 protein and its corresponding gene (7van 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, 8West 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). FT85 is required for Skp1 fucosylation as judged by the absence of Fuc-Tase activity in extracts of cells whose FT85 gene was modified by the replacement of its central coding region with a blastocidin resistance marker (7van 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). In addition, Skp1 made in these cells exhibits slightly increased mobility in SDS-PAGE consistent with the absence of Fuc and the outer, α-linked Gal residues. FT85 appears to be sufficient for Fuc-Tase activity based on expression studies inEscherichia coli (7van 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). With 768 amino acids, the FT85 sequence harbors two potential glycosyltransferase (GTase) domains. The C-terminal domain is sufficient, when expressed in FT85 mutant cells, to fucosylate the simple disaccharide substrate Galβ1,3GlcNAcα1-pNP. This corresponds to the core disaccharide of Skp1, therefore assigning Fuc-Tase activity to this domain. We report here that addition of the β1,3-linked Gal to Skp1 maps to the N-terminal half of FT85. The second and third sugars of the Skp1 glycosylation pathway are thus added by a single protein, which can act processively to efficiently extend the oligosaccharide chain. This molecular design has been previously observed for a class of glycosyltransferases involved in the biosynthesis of prokaryotic glycosaminoglycans (9DeAngelis P.L. Glycobiology. 2002; 12: 9R-16RCrossref PubMed Google Scholar), and is distinct from the mechanism of synthesis of the type I blood group H antigen in animals (10Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates; Composition, Structure, and Function. Marcel Dekker, New York1992: 263-332Google Scholar), which occurs in the Golgi apparatus using conventional type 2 membrane proteins with single GTase domains. For glycosidase digestions described in Fig. 2 B, Skp1 was isolated from the FT85-null strain HW260 by DEAE anion exchange chromatography of the crude S100 cell fraction, as described (8West 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). Nearly all Skp1 eluted in the pool II position as assayed by Western blot analysis using mAb 3F9 (11West C.M. Kozarov E. Teng-umnuay P. Gene. 1997; 200: 1-10Crossref PubMed Scopus (19) Google Scholar). For the Gal-Tase assays in Figs. Figure 3, Figure 4, Figure 5, the DEAE pool of Skp1 was further purified through the phenyl-Sepharose and mAb 3F9 column steps (8West 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). For the Fig. 6 Gal-Tase assays, the eluate from the mAb 3F9 step was applied directly to a Amersham Biosciences mini-Q column equilibrated in 50 mm HEPES-NaOH (pH 7.4), 10 mm MgCl2, and 2 mmMnCl2, which was mounted onto an Amersham Biosciences SmartSystem high pressure liquid chromatography unit. Skp1 was eluted with an ascending linear gradient of NaCl in the same buffer, and fractions were pooled based on A 280 values. SDS-PAGE and Western blot assays confirmed the identity and homogeneity of the pooled material. Skp1 pools were then concentrated in a Microcon centrifugal ultrafiltration cartridge (Amicon) with a nominalM r cut-off of 10,000, diluted in 20–50 mm HEPES (pH 7.4), and concentrated again to reduce NaCl levels. Protein content was estimated from A 280values. Skp1 from the GDP-Fuc synthesis mutant HL250 was purified in the same manner through the mAb 3F9 step (8West 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). Relative levels of Skp1s from strains HW260 and HL250 were determined by comparison of Western blot signals from a 2-fold dilution series of HL250 Skp1 against a known quantity of HW260 Skp1 run on the same gel.Figure 5Point mutations in the N- and C-terminal domains differentially affect β-Gal-Tase and α-Fuc-Tase activities. A, diagram of predicted domains of the expressed CBD-FT85 fusion protein. Positions of amino acid substitutions are indicated. B, SDS-PAGE and Coomassie Blue staining of soluble (S13) extracts of strains expressing normal and mutant CBD-FT85s. Lanes are labeled according to the position of the Asp residue mutated. None, parental strain ER2566; wt, normal CBD-FT85. C, Western blot analysis of the samples shown in panel B after probing with anti-CBD antibody. D, parallel aliquots of the samples shown in B and C were assayed for β-Gal-Tase activity using purified Skp1-GlcNAc (as in the legend to Fig. 3) as the acceptor or α-Fuc-Tase activity using either pNP-GlcNAc-Gal or purified Skp1-GlcNAc-Gal as the acceptor, as indicated. Incorporation into Skp1 was determined using the trichloroacetic acid precipitation assay. Results are normalized to the activity of normal CBD-FT85 determined in the same experiment.View Large Image Figure ViewerDownload (PPT)Figure 4Skp1 β-Gal-Tase activity is associated with the N-terminal domain. A, S13 extracts containing equal amounts of protein (∼0.5 mg) fromDictyostelium strain Ax3 (A3), strain HW260 (FT85 mutant, 0), or HW260 cells transfected with either pV(FT85N) (Na and Nb; 2 transfections) or pV(FT85C) (Ca and Cb; 2 transfections), encoding the N- or C-terminal domain of FT85, were assayed for β-Gal-Tase activity in the presence of 1 μm UDP-[3H]Gal and 0.5 μm purified Skp1-GlcNAc (as in the legend to Fig. 3), or no substrate, for 3 h. Incorporation of radioactivity into protein was assayed using the trichloroacetic acid precipitation assay, and no-substrate control values were subtracted. B andC, the S13 fraction from the Na strain was adsorbed to a DEAE column and eluted with a gradient of NaCl as described in "Experimental Procedures." Selected fractions as indicated were analyzed for Gal-Tase activity (B) in the presence or absence of added purified Skp1-GlcNAc as in A, except that the SDS-PAGE assay was used. Total bar height is the value with added Skp1-GlcNAc. Partially overlapping fractions were assayed for the N-terminal domain polypeptide by SDS-PAGE and Western blotting using mAb 9E10 against its C-terminal myc-tag and for Skp1 using mAb 3F9 (C). The N-domain polypeptide eluted at two apparentM r positions as explained in the "Results."View Large Image Figure ViewerDownload (PPT)Figure 3Purified FT85 exhibits both β-Gal-Tase and α-Fuc-Tase activities. A, purified DictyosteliumFT85 was incubated with 0.2 μm Skp1-GlcNAc, 0.2 μm Skp1-GlcNAc-Gal, or both 0.2 μmSkp1-GlcNAc and 1 μm Skp1-GlcNAc-Gal, each of which had been purified through the mAb 3F9 step, in the presence of 1 μm UDP-[3H]Gal for 1 h as described under "Experimental Procedures." Incorporation of radioactivity into Skp1 was determined by the SDS-PAGE assay. B, purified Dictyostelium FT85 was incubated as in panel A except with GDP-[3H]Fuc in place of UDP-[3H]Gal and the addition of unlabeled UDP-Gal as indicated. Incorporation was assayed by the same method.View Large Image Figure ViewerDownload (PPT)Figure 6Kinetic analysis of FT85 β-Gal-Tase and α-Fuc-Tase activities. FT85 was incubated with varying concentrations of purified Skp1-GlcNAc or Skp1-GlcNAc-Gal in the presence of 2 μm UDP-[14C]Gal (open circles), 2 μm GDP-[3H]Fuc (open squares), or both (filled symbols), as indicated, for 30 min. The reaction scheme is depicted at the right, and the legend (inset) shows the reaction product assayed with the radioactive label in bold. Less than 10% of donor and acceptor substrates were consumed in the reactions. Similar results were obtained in an independent trial based on separate Skp1 purifications, except that the Gal-Tase and Fuc-Tase activities in the tandem reaction were more similar to one another (data not shown).View Large Image Figure ViewerDownload (PPT) For the MS studies, the Skp1 pool from the mAb 3F9 column was denatured with 8 m urea, reduced and alkylated with iodoacetamide (8West 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), applied to an Amersham Biosciences ProPep 5/2 C8reversed phase column, and eluted with an ascending gradient of acetonitrile in 0.1% trifluoroacetic acid. The pool of reversed phase purified Skp1 was concentrated by vacuum centrifugation, mixed with an equal volume of 20 mg/ml recrystallized α-cyano 4-hydroxycinnamic acid (Aldrich) in 0.1% trifluoroacetic acid in 50% acetonitrile, and air-dried for analysis by MALDI-TOF-MS, on a ABI PerSeptive Biosystems Voyager DE-Pro instrument operated in the positive ion and reflector modes (4Teng-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 (70) Google Scholar). Singly and doubly charged ions of bovine carbonic anhydrase II (Sigma; average mass = 28,980 daltons) analyzed in the same fashion were used as an external standard. Average masses of carbonic anhydrase and Skp1 were estimated from the centroid of the upper region of the primary signal after smoothing. For analysis of peptides, the Skp1 pool was concentrated by vacuum centrifugation, digested with endo-Lys-C (Wako) as described (4Teng-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 (70) Google Scholar), and fractionated on a Amersham Biosciences C18/C22.1/10 reversed phase column as for the full-length protein. Samples were analyzed as above using known Skp1 peptides (4Teng-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 (70) Google Scholar) for calibration. Monoisotopic masses were determined, and in-source decay (12Reiber D.C. Brown R.S. Weinberger S. Kenny J. Bailey J. Anal. Chem. 1998; 70: 1214-1222Crossref PubMed Scopus (46) Google Scholar) was induced in the linear mode by increasing the grid voltage. Dictyostelium FT85 was purified from normal strain Ax3 cells through the final Superdex 200 column step as described previously (7van 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, 8West 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 material was subsequently adsorbed to a mini-Q ion exchange column (AmershamBiosciences) equilibrated in 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 0.1 mm EDTA, 5 mm dithiothreitol, and 0.02% Tween 20 (Calbiochem), and Fuc-Tase activity was eluted as a single, sharp peak early in an ascending gradient to 0.25 m NaCl in the same buffer. This pool was applied to a mini-S column (Amersham Biosciences) equilibrated in 50 mm MES-NaOH (pH 6.6), 5 mmMgCl2, 0.1 mm EDTA, 5 mmdithiothreitol, and 0.02% Tween 20, and collected as slightly retarded flow-through fraction. Recombinant FT85 was isolated as a soluble fraction of E. coli strain ER2566 cells transfected with pTY(CBD-FT85) after induction and lysis as described (7van 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). Soluble cell extracts were prepared by centrifugation at 13,000 g × 30 min. Samples were electrophoresed on a 7–20% SDS-PAGE gel and stained with Coomassie Blue or transferred to nitrocellulose and probed with anti-CBD (chitin-binding domain) antibody as described (13van 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). Dictyostelium cells (FT85-null strain HW260) transfected with either pV(FT85N) or pV(FT85C) (7van 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) were grown to near saturation density, lysed in 50 mm Tris-HCl (pH 7.4), 250 mm sucrose, and protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin), and a 13,000 g × 60 min supernatant (S13) was prepared. The S100s were applied to a Fast-Flow DEAE-Sepharose (Amersham Biosciences) column equilibrated in lysis buffer and eluted using a linear ascending gradient of 0–0.4 M NaCl in the same buffer, as for FT85 (8West 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). Fractions were pooled based on the presence of a Western blot band that was immunoreactive with mAb 9E10 directed against the c-myc epitope that was engineered at the C terminus of the domains. Protein was estimated using a Coomassie Blue dye-binding assay with bovine serum albumin as a standard (Pierce). The DEAE pool of the expressed C-terminal domain was purified further by addition of (NH4)2SO4 to 15% (w/v), followed by centrifugation at 10,000 g × 30 min and application of the supernatant to a Hi-sub Phenyl-Fast Flow-Sepharose column equilibrated in 50 mm HEPES-NaOH (pH 7.4), 5 mm dithiothreitol, 10 mmMgCl2, 0.1 mm EDTA, and protease inhibitors (see above). FT85C was eluted near the end of a linear descending gradient of 15–0% (NH4)2SO4 in the same buffer followed by 5 mm NH4Ac (pH 7.5). Fractions reactive with mAb 9E10 were pooled in the same way, and applied to a Amersham Biosciences Resource-Q column equilibrated in 50 mm HEPES-NaOH (pH 7.4), 5 mm dithiothreitol, 10 mm MgCl2, 0.1 mm EDTA, and protease inhibitors (see above). The flow-through fraction was frozen at −80 °C. The amino acid sequence of recombinant FT85 was altered by site-directed mutagenesis of pTY(CBD-FT85) as described previously (7van 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. 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 described under "Results." The nt sequence encoding amino acids 52–53 (DD) was changed from GATGAT to GACAAT; the nt sequence encoding Asp-226 was changed from GAT to AAT; the nt sequence encoding Asp-497 was changed from GAT to AAC; the nt sequence encoding amino acid Asp-508 was changed from GAT to AAC; and the nt sequence encoding Asp-653 was changed from GAT to AAT. The modified plasmids were transfected into E. coli strain ER2566, expression was induced, and cells were lysed also as before (7van 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). α1,2-Fuc-Tase activity was assayed as described previously (7van 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) using GDP-[2-3H]Fuc (17.3 Ci/mmol, diluted 5–20-fold with unlabeled GDP-Fuc; New England Nuclear) as the donor and either 0.36 mmGalβ1,3GlcNAcα1-pNP (pNP-GlcNAc-Gal) or Skp1-GlcNAc-Gal purified to near homogeneity from Dictyostelium strain HL250, which is unable to synthesize GDP-Fuc (14Gonzalez-Yanes B. Mandell R.B. Girard M. Henry S. Aparicio O. Gritzali M. Brown R.D. Erdos G.W. West C.M. Develop. Biol. 1989; 133: 576-587Crossref PubMed Scopus (31) Google Scholar). Reactions were conducted in a 50 μl volume containing 500 μg/ml bovine serum albumin, 50 mm HEPES-NaOH (pH 7.5), 100 mm NaCl, 10 mm MgCl2, 2 mm MnCl2, 0.1 mm EDTA, 5 mm dithiothreitol, and 0.05% Tween 20 at 30 °C for the time indicated. Incorporation was measured after precipitation of protein by 10% trichloroacetic acid, as described (13van 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), or by cutting out the Skp1 band after separation on an SDS-PAGE gel (13van 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). Assays performed in crude extracts were supplemented with 1 mm ATP, 3 mm NaF, and protease inhibitors (see above). β1,3-Gal-Tase activity was assayed similarly except that the donor was 1 μm UDP-[4,5-3H]Gal (30 Ci/mmol, diluted 5–20-fold with unlabeled UDP-Gal; New England Nuclear) or 2 μm UDP-[U-14C]Gal (300 mCi/mmol; American Radiochemical Corporation) as indicated, and the acceptor was Skp1-GlcNAc isolated from strain HW260. Linearity of reactions was established based on time-course studies. The radioactive product from the mixture of FT85, Skp1-GlcNAc purified from FT85 mutant (HW260) cells through the DEAE column step (see above), and UDP-[3H]Gal, was desalted by two passages over a PD10 column (Sephadex G-25; Amersham Biosciences) equilibrated in 25 mm NH4Ac (pH 7.1). The void volume was distributed into multiple tubes each receiving 2,300 dpm, which were each taken to dryness in a vacuum centrifuge, redissolved in water, and taken to dryness again. The sample was redissolved in 3 μl of 8m urea in 50 mm NH4HCO3and heated at 95 °C for 5 min. After 8-fold dilution in 50 mm NH4HCO3, a stock solution of 0.5 mg/ml TPCK trypsin (Worthington) in 1 mm HCl was added to a final concentration of 20 μg/ml and incubated at 37 °C for 8 h. The sample was heated at 100 °C for 5 min and taken to dryness by vacuum centrifugation, rehydrated, and dried again. Samples were redissolved in 100 μl of a solution containing the glycosidase to be tested dissolved in its appropriate buffer as described (4Teng-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 (70) Google Scholar), supplemented with 100 μg/ml bovine serum albumin, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 5 μg/ml Ovomucoid trypsin inhibitor (Sigma). Glycosidases were bovine kidney β-galactosidase (cleaves Galβ1,3/4 > 6; Oxford Glycosciences), recombinant β-galactosidase (cleaves Galβ1,3/6; Glyko), Xanthomonas manihotis β-galactosidase (cleaves Galβ1,3; New England Biolabs), and green coffee bean α-galactosidase (cleaves Galα1,4 > 2/3≫6; Roche Molecular Biochemicals). Digestions were incubated at 37 °C for 5 h, passed over 0.8 ml Dowex-2 columns in water, and analyzed in a liquid scintillation counter. Activity of α-galactosidase was verified by treatment of Skp1 glycopeptides that had been labeled in their α-linked Gal residues in vitro. 3H. van der Wel and C.M. West, unpublished data. Skp1 from FT85 mutant cells (strain HW260) migrates slightly more rapidly than normal Skp1 on SDS-PAGE gels consistent with the loss of Fuc and 2 Gal residues thought to be attached to Fuc (7van 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). To verify the structure of the glycan formed, Skp1 was purified to homogeneity as described under "Experimental Procedures." The A 280 profile of the final purification step on a C8-reversed phase column is shown in Fig. 1 A. MALDI-TOF-MS analysis of this material yielded an ion with anm/z value of 19,130 (Fig. 1 B), similar to the predicted M r of 19,132 of Skp1 in which the N-terminal Met has been removed, the new N terminus has beenN-acetylated, and Pro-143 has been hydroxylated and modified by a GlcNAc residue. These N-terminal modifications are consistent with previous results based on Edman degradation (11West C.M. Kozarov E. Teng-umnuay P. Gene. 1997; 200: 1-10Crossref PubMed Scopus (19) Google Scholar). To confirm the modification on Pro-143, Skp1 was digested with endo-Lys-C, and the peptides were separated on a C18 reversed phase column.A 215 peaks were assigned based onm/z values determined by MALDI-TOF-MS (Fig.1 C). Peak 7 had a singly charged monoisotopicm/z value of 1853.8 (Fig. 1 D), which is identical to the MH+ value of the previously identified Skp1 peptide 139–151 (4Teng-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 (70) Google Scholar) modified at Pro143 by hydroxylation and GlcNAc. This peptide eluted as expected between the positions of peptide 139–151 modified at Pro-143 by GlcNAc-Gal (2017) and the unmodified peptide (1635) as indicated in Fig. 1 B and had no A 280 absorbance, consistent with the absence of amino acids that absorb at this wavelength. To confirm the identity of the glycopeptide, the grid voltage was increased to promote in-source decay (Fig. 1 D). This yielded new ions atm/z 1652, corresponding to the loss of a GlcNAc (1651 predicted), and 1725, corresponding to the loss of the C-terminal Lys (1724 predicted). A 215 peaks corresponding to alternative glycoforms were not detected (Fig. 1 C). Therefore, in the absence of FT85, Skp1 was missing its β-linked Gal in addition to Fuc and the outer sugars. The unexpected absence of the β1,3-linked Gal in addition to the outer sugars of the Skp1 glycan might be due to an absence of Skp1 β1,3-Gal-Tase activity in FT85 mutant cells. To test for Skp1 Gal-Tase activity, crude S100 extracts of FT85 mutant HW260 cells were incubated with UDP-[3H]Gal for 2 h, and incorporation of radioactivity into Skp1 was measured by running the extract on an SDS-PAGE gel, excising the gel band containing endogenous Skp1, and counting in a liquid sci
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