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Originally published In Press as doi:10.1074/jbc.M605912200 on October 10, 2006

J. Biol. Chem., Vol. 281, Issue 48, 36742-36751, December 1, 2006
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Identification and Characterization of abeta1,3-Glucosyltransferase That Synthesizes the Glc-beta1,3-Fuc Disaccharide on Thrombospondin Type 1 Repeats*

Krisztina Kozma{ddagger}, Jeremy J. Keusch{ddagger}, Björn Hegemann{ddagger}1, Kelvin B. Luther§, Dominique Klein{ddagger}, Daniel Hess{ddagger}, Robert S. Haltiwanger§, and Jan Hofsteenge{ddagger}2

From the {ddagger}Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland and §Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215

Received for publication, June 20, 2006 , and in revised form, September 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombospondin type 1 repeats (TSRs) are biologically important domains of extracellular proteins. They are modified with a unique Glcbeta1,3Fuc{alpha}1-O-linked disaccharide on either serine or threonine residues. Here we identify the putative glycosyltransferase, B3GTL, as the beta1,3-glucosyltransferase involved in the biosynthesis of this disaccharide. This enzyme is conserved from Caenorhabditis elegans to man and shares 28% sequence identity with Fringe, the beta1,3-N-acetylglucosaminyltransferase that modifies O-linked fucosyl residues in proteins containing epidermal growth factor-like domains, such as Notch. beta1,3-Glucosyltransferase glucosylates properly folded TSR-fucose but not fucosylated epidermal growth factor-like domain or the non-fucosylated modules. Specifically, the glucose is added in a beta1,3-linkage to the fucose in TSR. The activity profiles of beta1,3-glucosyltransferase and protein O-fucosyltransferase 2, the enzyme that carries out the first step in TSR O-fucosylation, superimpose in endoplasmic reticulum subfractions obtained by density gradient centrifugation. Both enzymes are soluble proteins that efficiently modify properly folded TSR modules. The identification of the beta1,3-glucosyltransferase gene allows us to manipulate the formation of the rare Glcbeta1,3Fuc{alpha}1 structure to investigate its biological function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein glycosylation is a common co- and post-translational modification that has important biological functions at the cellular and organism level. In eukaryotes, O-glycosylation is the most diverse class of protein glycosylation as any amino acid with a hydroxyl group (Ser, Thr, Tyr, hydroxylysine, and hydroxyproline) can be modified with one of eight different monosaccharides (GalNAc, GlcNAc, Gal, Glc, Man, Fuc, xylose, arabinose) in either an {alpha} or beta configuration (1). The unusual protein O-linked fucosylation has been described on two different extracellular protein domains, epidermal growth factor-like (EGF)3 repeat (2–8) and thrombospondin type 1 repeat (TSR) domains (9, 10).

These domains are cysteine knot motifs of between 30–40 and ~60 amino acids in length for an EGF repeat and TSR, respectively. The TSR superfamily contains different extracellular matrix and transmembrane proteins involved in cell-cell and cell-matrix interactions (11). There are TSRs present in 41 different human proteins and 27 different Caenorhabditis elegans proteins including thrombospondin-1 (TSP-1), multiple ADAMTS proteases, F-spondin, and UNC-5 (12). Significantly, the TSR domain within these diverse proteins has been shown to be the site of protein-protein or protein-carbohydrate interaction and is nearly always essential for function. The crystal structure of TSR2 and TSR3 from human TSP-1 expressed in Drosophila S2 cells shows that each domain consists of three antiparallel strands (12). The first is a longer irregular strand containing the three conserved tryptophans, which are known to be C-mannosylated (9). This A strand is connected to two regular beta-strands, B and C. Three disulfide bridges and, alternately, stacked layers of tryptophan and arginine residues define this unique structure. Recently the solution structures of TSR1 and TSR4 domains of rat F-spondin were determined (13).

We have identified three members of the TSR superfamily (human TSP-1, human properdin, and rat F-spondin) as the first proteins that carry the disaccharide Glc-Fuc on Ser/Thr (9, 10). The disaccharide structure observed in TSRs was confirmed as Glcbeta1,3Fuc (14), a structure first discovered as an amino acid glycoside in human urine (15). TSR O-fucosylation occurs in the putative consensus sequence WX5CX2–3S/TCX2G (10). The crystal structure revealed that the Ser or Thr carries a fucosyl residue and is located in the short loop between the A and B strands (12). This modification is synthesized by protein O-fucosyltransferase 2 (POFUT2) in C. elegans (16), mammalian cells (14), and in Drosophila S2 cells, where it has been shown to be localized within the endoplasmic reticulum (ER) (17).

In an alternative glycosylation pathway, O-fucosylation has been identified in a number of proteins containing EGF repeats including the Notch receptors (8) and its ligands (7). This modification is carried out in the ER by protein O-fucosyltransferase 1 (POFUT1) (18, 19), a paralogue of POFUT2. The role of POFUT1 is essential in Notch signaling, and mutant mice defective in POFUT1 expression are embryonic lethal with a phenotype similar to Notch1 deficiency (20). We recently reported that an enzymatically inactive mutant of the gene for POFUT2 in C. elegans results in an abnormal shape of the anterior gonadal arm (16).

Furthermore, the O-fucose on Notch serves as a substrate for a beta1,3-N-acetylglucosaminyltransferase, Fringe, a known modulator of Notch function (21, 22). The product of the Fringe reaction is then further extended by the addition of a galactose and sialic acid to form the tetrasaccharide (NeuAc{alpha}2, 6Galbeta1, 4GlcNAcbeta1, 3Fuc-{alpha}1-O-Ser, NeuAc is N-acetyl neuraminic acid) (8, 23). This mature Notch glycoform leads to strong activation of Notch signaling via its interaction with the Delta ligand, whereas there is inhibition of Notch signaling via the Jagged ligand (24). These data demonstrate that the particular O-glycosylation in an EGF repeat or TSR module on the protein can have significant functional and biological consequences.

Although there are similarities between these two O-fucosylation pathways in that both fucosyltransferases are present in the ER, use GDP-fucose as the donor sugar, and require properly folded acceptor substrate, there does not appear to be any biosynthetic cross-talk (14). In contrast to O-fucosylation of the EGF repeat, the second and final step in the TSR O-fucosylation pathway involves the addition of a capping beta1,3-glucose residue.

As a first step to understanding the functional significance of the Glcbeta1,3Fuc disaccharide structure in TSR, we wanted to identify the missing beta1,3-glucosyltransferase. A soluble enzyme activity capable of forming a Glcbeta1,3Fuc linkage has been reported in cell lines from different species and multiple rat tissues (25). We postulated that the beta1,3-glucosyltransferase may share sequence similarity with known beta1,3-glycosyltransferase members of family 31 in the carbohydrate active enzymes data base (CAZy) (26). One putative human beta1,3-glycosyltransferase, B3GTL, that shares 28% sequence identity to the human radical Fringe was recently reported (27). In this paper we identify and characterize this protein as the missing beta1,3-glucosyltransferase and we will refer to it as beta3Glc-T.4 This enzyme is localized in the ER compartment of HEK 293T cells and shows high specificity toward TSR-fucose in comparison to fucosylated EGF repeat in vitro.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and GDP-beta-L-fucose were purchased from Sigma-Aldrich. UDP-[6-3H]glucose (60 Ci/mmol) and GDP-[2-3H(N)]fucose (17.5 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer Life Sciences, respectively. Oligonucleotides for PCR were synthesized by Microsynth (Balgach, Switzerland). Oasis® HLB cartridges (30 mg) were purchased from Waters Associates (Rupperswil, Switzerland). Antibodies used in Western blots included mouse anti-myc (Sigma-Aldrich), rabbit anti-calnexin (Abcam, Cambridge, UK), and horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). All other chemicals were of the highest quality available.

Subcloning of beta3Glc-T and Site-directed Mutagenesis—We identified a full-length cDNA clone of 3467 bp (accession number BC068595 [GenBank] , IMAGE 4837250, obtained from Geneservice, Cambridge, UK) that has a single base substitution of an A to a G at position 852 in its 1497-bp coding sequence compared with the B3GTL sequence (accession number AY190526 [GenBank] ) (27). This results in a conservative change from an isoleucine to a methionine at position 284. The open reading frame encoding the 498-amino acid-long human beta3Glc-T (protein ID AAH68595 [GenBank] .1) was amplified by PCR from the plasmid pBluescript-B3GTL (IMAGE 4837250) with the forward primer (5'-TGCTCTAGAACCACCATGCGGCCGCCCGCCTGCTG-3') and the reverse primer (5'-GCGGATCCTAACTCCTCTCGAAAACCTTTCTG-3') to create XbaI and BamHI restriction sites (underlined) at the 3'- and 5'-end of the gene, respectively. The PCR product was subcloned into pcDNA3.1TM (–)-myc-His A (Invitrogen) to give the plasmid pcDNA3.1-beta3Glc-T-Myc-His. The 349DDD351 motif in beta3Glc-T was mutated to 349ADD351 and to 349ADA351 by inverse PCR using Pfu DNA polymerase (Promega, Wallisellen, Switzerland) with overlapping primers containing the desired point mutations (28). The coding regions of all expression plasmids were sequenced to verify the presence of the desired mutations and the absence of unwanted ones.

Cell Culture—HEK 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin, and streptomycin. HEK 293T cells that were stably transfected with a plasmid encoding C. elegans POFUT1 or POFUT25 were grown in the same medium supplemented with 200 µg/ml hygromycin B.

Preparation of Cell Lysates—The plasmids encoding either the wild type or mutant forms of beta3Glc-T were transiently transfected into HEK 293T cells using Lipofectamine (Invitrogen) following the manufacturer's instructions. Sixty hours post-transfection the cells were washed and scraped from the plates in ice-cold 10 mM Tris-HCl, 150 mM NaCl, pH 7.5, complemented with EDTA-free Complete® protease inhibitor mixture (PIC, Roche Applied Science). The cells were centrifuged at 170 x g for 5 min and resuspended in 10 mM Tris-HCl, 30 mM NaCl, pH 7.4, buffer containing PIC to a density of ~3 x 107 cells/ml and disrupted by sonication at 4 °C with three 30-s bursts (35% probe energy, using a 3-mm Microtip from Branson). The lysate was centrifuged at 3320 x g for 10 min at 4 °C. The supernatant was ultracentrifuged at 100,000 x g for 1 h at 4 °C. The high speed supernatant was collected, separated into aliquots, and stored at –80 °C until use. Protein concentration was determined using the Bradford Protein Assay (Bio-Rad). Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad). The membranes were developed using the enhanced chemiluminescence (ECL Western blotting, GE Healthcare) kit.

Expression and Purification of TSR4—The protein rat F-spondin TSR4 (residues 615–666 (29)) fused to a tobacco etch virus protease cleavage site followed by a Myc epitope and a His affinity tag (TKLCLLSPWSEWSDCSVTCGKGMRTRQRMLKSLAELGDCNEDLEQAEKCMLPECPENLFQGSRGPEQLISEEDLNSAVDHHHHHH, T indicates Thr at position 18) was prepared by standard PCR methods using a rat F-spondin cDNA as the template (a gift from Dr. A. Klar, Hebrew University, Jerusalem, Israel). The PCR product was subcloned into the bacterial expression plasmid pET22b. Bacteria, strain BL21 (DE3), that had been transformed with pET22b-F-spondin-TSR4-tobacco etch virus-Myc-His were grown to an optical density of 1.0 at 600 nm and induced with 1 mM isopropyl-beta-thiogalactopyranoside for 3 h. The cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, 0.5% Tween 20, PIC, and 10 mM imidazole), and lysed in a French pressure cell. The lysate was cleared by centrifugation and loaded onto nickel nitrilotriacetic acid beads (Qiagen). The resin was washed in lysis buffer with 20 mM imidazole, and the TSR4 was eluted in lysis buffer containing 500 mM imidazole. The protein was gel-filtered on a Sephacryl FF S-200 column in 50 mM NH4HCO3, and the fractions containing monomeric TSR4 were lyophilized. Final purification was achieved by ion-exchange chromatography on a Mono Q 5/50 fast protein liquid chromatography column (GE Healthcare) in 20 mM Tris-HCl, pH 8.0. The protein eluted at 120 mM NaCl in a linear gradient of 0–200 mM NaCl over 30 min in the same buffer. The concentration of the purified protein was determined from its absorbance at 280 nm using a calculated molar absorption coefficient of 12962 M–1cm–1. The overall yield of the procedure was 0.3 mg/liter of culture. Aliquots were stored at –80 °C.

Characterization of the TSR4 Protein—The molecular mass of purified TSR4 and of TSR4 after treatment with 102 mM iodoacetamide in 500 mM Tris-HCl, pH 8.6, containing 6 M guanidinium-HCl and 10 mM EDTA with and without prior reduction with 54 mM dithiothreitol were determined by LC-MS (9).

Far-UV CD spectra of TSR4 and TSR4-fucose (0.3–0.8 mg/ml) in 20 mM Tris-HCl, pH 7.5, were measured on an Aviv 62 DS or a Shimadzu circular dichroism spectropolarimeter in 1- or 2-mm cuvettes, respectively. The reported data (molar ellipticity mean residue weight, [{theta}]MRW) are the average of six spectra and have been base line-corrected. Thermal denaturation and renaturation curves were measured in 1 °C steps at the maxima of ellipticity.

Fluorescence spectra of TSR4 (0.06 mg/ml) in the same buffer were measured on an Aminco Bowman Series 2 luminescence spectrometer in a 1-cm quartz cuvette. Excitation was at 295 nm with a band pass of 2 nm, whereas the emission was recorded with a width of 4 nm. The spectra were quantum-corrected, and contributions from the buffer solution were subtracted.

Production of Fucosylated TSR and EGF Repeat—High speed supernatant from sonicated HEK 293T cells that stably express C. elegans POFUT2 (25 µg of total protein) was incubated with 20 µM TSR4 and 100 µM GDP-fucose in 10 mM imidazole-HCl, pH 6.9, 5 mM MnCl2 for 2 h at room temperature. The fucosylated product was purified by fast protein liquid chromatography using a HisTrap HP (GE Healthcare) affinity column and desalted. The mass of the TSR4-fucose was 10,106 Da, as determined using an internally calibrated MALDI-TOF-MS (Brucker Daltonics, Ultraflex II) and sinapinic acid as the matrix. Recombinant EGF repeat from human factor VII was produced in Escherichia coli and purified as described (30). Complete fucosylation of the EGF repeat (EGF-fucose) was performed in a similar way except that the enzyme source was from HEK 293T cells stably expressing C. elegans POFUT1 and 20 µM EGF repeat was used instead of TSR4 as the acceptor substrate. The fucosylated product was purified by reversed-phase HPLC on a C18 column (Zorbax, 1.0 x 150 mm, 3.5 µm, Agilent Technologies) with a 30-min linear gradient of 0–30% buffer B (buffer A, 2% CH3CN and 0.1% trifluoroacetic acid; buffer B, 80% CH3CN and 0.085% trifluoroacetic acid). The purified glycoproteins were examined by MALDI-TOF-MS using 2,5-dihydroxybenzoic acid as the matrix. The masses of the unmodified and fucosylated EGF repeat were 5688.6 and 5834.4 Da, respectively.

Glucosyltransferase Assays—Reaction mixtures for assaying the glucosyltransferase activity of recombinant human beta3Glc-T contained 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MnCl2, PIC, 2.64 µM UDP-glucose, 0.33 µM UDP-[6-3H]glucose, 10 µM TSR4-fucose, and high speed supernatant (25 µgof total protein) in a volume of 25 µl. After incubation for 1 h at 37 °C the reaction was terminated by adding 200 µl ice-cold 0.2% trifluoroacetic acid. In the case of blank reactions, the acceptor substrate was added after the reactions were stopped. The reaction mixtures were loaded onto Oasis® HLB cartridges (30 mg) that were previously equilibrated in 0.2% trifluoroacetic acid and washed with 1.2 ml of 0.2% trifluoroacetic acid followed by 1.2 ml of 0.2% trifluoroacetic acid, 20% CH3CN. The glucosylated product was eluted with 600 µl of 0.2% trifluoroacetic acid, 40% CH3CN. With the unmodified and fucosylated EGF repeats (EGF-fucose) as acceptor substrates, the cartridges were washed with 0.2% trifluoroacetic acid and 0.2% trifluoroacetic acid, 5% CH3CN, and the product was eluted as above. Incorporation of [3H]glucose into the acceptor substrate was determined by scintillation counting. Product analysis of glucosylated TSR4-fucose by reversed-phase HPLC, alkali-induced beta-elimination, gel filtration, and high pH anion exchange chromatography on a Dionex MA1 column was performed as described (17, 31).

Donor Substrate Specificity of beta3Glc-T—The glucosyltransferase assay was carried out as above, except that 20 µM concentrations of the different UDP-sugar donors were used. The products were analyzed by reversed-phase HPLC using the C18 Zorbax column with a 35-min linear gradient of 0–35% buffer B (buffer A, 2% CH3CN, 0.1% formic acid; buffer B, 80% CH3CN, 0.1% formic acid) on an Agilent 1100 HPLC system interfaced to an upgraded API 300. The product peak in the 280-nm absorbance trace was integrated.

In Vitro Glucosylation of TSR4-fucose and Mass Spectrometric Analysis—The glucosylation reaction was carried out as above, except that 200 µM UDP-glucose was used, and the incubation was for 2 h. The nearly fully glucosylated product was purified by reversed-phase HPLC on the C18 Zorbax column with a 30-min linear gradient of 0–48% buffer B using the trifluoroacetic acid system described above. Fractions containing glucosylated TSR4-fucose were neutralized, dried, reduced, and carboxymethylated as described (32), except that the EDTA was omitted from the buffer and digested with endoproteinase Lys-C (20% w/w, Wako) in 50 mM sodium phosphate, pH 8, 700 mM guanidinium-HCl for 12 h at 37 °C. The peptide of interest was isolated by reversed-phase HPLC-MS on a C18 column (GraceVydac 1.0 x 230 mm) with a 2-h linear gradient of 0–50% buffer B using the formic acid system, neutralized, dried, and digested overnight at 37 °C with endopeptidase Asp-N (Roche Applied Science) in 50 mM ammonium bicarbonate, pH 8.

Nanospray Mass Spectrometry of O-Fucosylated and Glucosylated Peptide—Static nanoelectrospray mass spectrometry was performed on a quadrupole linear 4000 Q TRAP instrument (Applied Biosystems, Foster City, CA) in the positive ion mode. In the mass spectrometry with collision-induced dissociation experiments the precursor ion was selected in the quadrupole analyzer and fragmented in the collision cell. Masses were analyzed in the linear ion trap. For MS3 analysis, the daughter ion was isolated in the linear trap and fragmented, and the granddaughter ions were analyzed using Analyst software. The mass accuracy of all measurements was better than 0.3 m/z units.

Subcellular Fractionation—Subcellular fractions of wild type HEK 293T cells were obtained by ultracentrifugation on a nonlinear Nycodenz gradient, and marker proteins for ER and Golgi subfractions were determined as described.6 Fractions were assayed for beta3Glc-T activity as described above. POFUT2 activity was determined as described elsewhere.5


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Properly Folded TSR4 and TSR4-fucose—Previous studies have shown that O-fucosylation of EGF repeats (33) and TSRs (14) requires their correct three-dimensional structure. Therefore, we prepared properly folded recombinant TSR4 from rat F-spondin in E. coli as a suitable acceptor substrate for the assays described below. The highly purified protein showed a single band both on reducing and non-reducing SDS-polyacrylamide gels that had been stained with colloidal Coomassie Brilliant Blue. Furthermore, a single peak was detected by reversed-phase HPLC (not shown).

F-spondin TSR4 consists predominantly of beta-strands connected by turns and contains three disulfide bridges (13). Mass spectrometric analysis of TSR4 in our purified preparations revealed a molecular mass of 9960 Da, which would be consistent with the presence of three disulfides and the absence of free thiol groups (expected mass 9960 Da). This interpretation was confirmed by the observation that the mass of TSR4 did not change upon treatment with iodoacetamide in the presence of 6 M guanidinium-HCl. In contrast, reduction with dithiothreitol before alkylation resulted in a mass of 10,308 Da, consistent with the incorporation of six carboxamidomethyl moieties (not shown).

We examined the secondary structure of TSR4 by spectroscopic techniques that have previously been used to ascertain the correct folding of recombinant TSRs from human TSP-1 (34). At 25 °C the far-UV CD spectrum of TSR4 displayed positive ellipticity with maxima at 212 and 231 nm (Fig. 1A). As expected for a fully denatured protein, the spectrum obtained at 81 °C was dominated by strongly negative ellipticity (Fig. 1A). Determination of the thermal denaturation curves at either ellipticity maximum yielded very similar transition temperatures, 47 and 48 °C for 212 and 231 nm, respectively (Fig. 1B). This indicated that the maxima arise from the same structural motif. Furthermore, the denaturation process was fully reversible based on the observation that the renaturation curves have the same shape as the ones for denaturation. The positive ellipticity of the far-UV CD spectrum of native TSR4 is in agreement with a structure that largely consists of beta-strands and turns and contains clustered aromatic amino acids. In fact, the spectra are very similar to that found for properdin, a protein isolated from human plasma that consists of six TSRs (35) and for recombinant TSRs from TSP-1 (34).

The tryptophan side chains in TSRs form alternately stacked layers with arginines and are partially shielded from solvent. We examined the local environment of the tryptophans in folded and unfolded TSR4 by fluorescence spectroscopy. A clear red shift of the emission maximum from 330 to 344 nm was observed (Fig. 1C), indicating that the tryptophans move from a rather non-polar environment to a more solvent-exposed environment upon unfolding of TSR4. In summary, the electrophoretic, chromatographic, chemical, and spectral data provide strong evidence that TSR4 in our preparations contains three disulfide bridges and is correctly folded.

TSR4 could be 100% converted into TSR4-fucose (10,106 Da) using GDP-fucose and recombinant POFUT2 from C. elegans. Importantly, we determined the modified amino acid residue by peptide mapping and tandem MS analysis to be Thr-18 (TSR4 numbering), the same residue as in the full-length protein (10) (data not shown).5 The far-UV CD spectrum of TSR4-fucose was very similar to that of TSR4 (Fig. 1A), indicating that the O-fucosylation did not cause a significant change in secondary structure.

beta3Glc-T Transfers Glucose onto the Fucosyl Residue in TSR4-fucose—B3GTL was identified by Heinonen et al. (27) as a putative beta1,3-glycosyltransferase-like protein. We tested whether the overexpressed protein7 in HEK 293T cells has activity for TSR4-fucose. Incubation of TSR4-fucose with UDP-[3H]glucose and high speed supernatant from HEK 293T cells overexpressing beta3Glc-T resulted in glucose incorporation into the acceptor (Fig. 2). The amount of activity was 4.7-fold higher than the endogenous activity found in cells transfected with the empty control vector. Importantly, this increase fully depended on the presence of a fucosyl residue in the acceptor, since it was not observed with unmodified TSR4 (Fig. 2).


Figure 1
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  		FIGURE 1.
Circular dichroism and fluorescence spectra of folded and unfolded TSR4. A, the far-UV CD spectra of native recombinant TSR4 (solid line) and TSR4-fucose (dotted line) were measured at 25 °C and that of the completely unfolded TSR4 at 81 °C (dashed line). The molar ellipticity data are the average of six spectra. B, the thermal unfolding and refolding of TSR4 was monitored at the maxima of ellipticity (212 and 231 nm). The data were fitted to the Hill equation. C, the fluorescence spectra of folded TSR4 (solid line) and reduced and carboxamidomethylated TSR4 (broken line) were determined at 25 °C at an excitation wavelength of 295 nm. [{theta}]MRW, molar ellipticity mean residue weight.

 
This observation was confirmed by experiments in which we extended the incubation time and increased the concentration of (unlabeled) UDP-glucose. This resulted in the nearly complete conversion of TSR4-fucose into product exhibiting a mass (10,271 Da, as determined using a triple quadrupole mass spectrometer) in close agreement with that expected for glucosylated TSR4-fucose (Fig. 3A, solid line). No such product was observed when the acceptor substrate was added after stopping the reaction (Fig. 3A, dotted line), and comparatively little of it was formed by the endogenous enzyme alone (Fig. 3A, dashed line). The purified product was used to determine the site of attachment of the glucosyl residue by LC-MS peptide mapping. The peptide containing the Thr-18 had a mass of 2593 Da (Fig. 3B), consistent with the presence of a fucosyl and a glucosyl residue. Because this peptide contained multiple hydroxyl amino acids that could carry the glucose it was necessary to obtain a smaller peptide suitable for analysis by tandem MS. Cleavage with endopeptidase Asp-N yielded a peptide with a mass of 1234.5 Da, whose tandem MS spectrum confirmed it to have the sequence DCSVTCGK (T is Thr-18) and to be modified with fucose and glucose (+308 Da; Fig. 3C). Due to the extreme lability of the fucosyl-peptide linkage under the MS-MS conditions of the triple-quadrupole mass spectrometer, the vast majority of the parent ion as well as most of the fragments had lost their 308-Da modification. However, weak ions corresponding to y4 and y5 fragments were observed, suggesting the presence of a disaccharide on Thr-18. We confirmed this by MS3 analysis of the y4 fragment, which showed a prominent loss of 308 Da and a minor one of 162 Da. This behavior is typical of Glc-Fuc-peptides and has previously been observed in tandem MS experiments with peptides obtained from natural proteins (9). Because Thr-18 is the only hydroxyl amino acid in the y4 fragment, we conclude that a Glc-Fuc disaccharide is attached to this residue.


Figure 2
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  		FIGURE 2.
beta3Glc-T transfers glucose to TSR4-fucose. Glucosyltransferase activity of beta3Glc-T with either unmodified or fucosylated factor VII EGF repeat or TSR4 as the acceptor substrates (10 µM) was determined with the radiochemical assay described under "Experimental Procedures." The activity in high speed supernatants of HEK 239T cells overexpressing beta3Glc-T (gray bars) and from cells transfected with empty vector (black bars) was assayed. Background activity, determined by adding the acceptor substrate after stopping the reaction (<5%), has been subtracted. Values are the mean (±S.E.) of triplicate determinations from one of three independent experiments. The inset shows the linearity of the assay as a function of total protein concentration.

 


Figure 3
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  		FIGURE 3.
beta3Glc-T transfers glucose onto the fucosyl residue in TSR4-fucose. A, purification of TSR4-fucose-glucose on a C18 reversed-phase HPLC column (solid line). TSR4-fucose (10 µM), modified with fucose on Thr-18, was glucosylated in vitro as described under "Experimental Procedures." None or only a small amount of product formation was observed in the enzyme blank (dotted line) or control reaction (dashed line). B, the reaction product eluting at 20.4 min in panel A was reduced, alkylated, and cleaved with endoproteinase Lys-C. The digest was fractionated by LC-MS on a reversed-phase C18 column. The eluate was monitored by absorbance at 214 and 280 nm. The glycosylated peptide, eluting at 79.5 min, was identified by its mass. C, static nanoelectrospray MSMS of the glycosylated subpeptide obtained by cleavage of the 2593-Da peptide with endopeptidase Asp-N. D, MS3 analysis of the y4 fragment (m/z 773) from panel C. The losses of Glc (162 Da) and the disaccharide Glc-Fuc (308 Da) have been indicated by dashed arrows.

 
To determine the stereochemistry of the linkage between the glucosyl and fucosyl residue, the disaccharide was released from 3H-glucosylated TSR4-fucose by reductive beta-elimination, isolated by gel filtration, and compared with disaccharides of known configuration by high pH anion exchange chromatography (14). This analysis showed a single radioactive peak that co-migrated with Glcbeta1,3Fucitol (Fig. 4). The absence of [3H]glucitol in the gel filtration profile (not shown) excluded the possibility that a glucosyl monosaccharide is present in addition to the disaccharide. Taken together, the results in Figs. 3, C and D, and 4 demonstrate that beta3Glc-T attaches a glucose residue in a beta1,3-linkage to the fucosyl residue on Thr-18 in TSR4-fucose.


Figure 4
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  		FIGURE 4.
The disaccharide formed by the beta3Glc-T-catalyzed reaction is Glc-beta1,3-Fuc. TSR4-Fuc was incubated with UDP-[6-3H]Glc and high speed supernatants of HEK 293T cells overexpressing beta3Glc-T. The product of the reaction was purified by reversed-phase HPLC, and the disaccharide was released by alkali-induced beta-elimination. The radiolabeled and reduced disaccharide was purified by gel filtration and compared with sugar standards by high pH anion exchange chromatography.

 
Mutation of the DXD Motif Abolishes the Enzymatic Activity of beta3Glc-T—To ascertain that beta3Glc-T itself contains the glucosyltransferase activity and that the increase in activity we observed in overexpression experiments (Fig. 2) was not resulting from an endogenous transferase, we mutated the DXD motif (349DDD351) in its putative catalytic domain. This sequence occurs in most glycosyltransferases that use nucleotide-activated sugars and is crucial for activity (36–38). Extracts from cells expressing the single (349ADD351) or double mutant (349ADA351) of beta3Glc-T did not exhibit glucosyltransferase activity above background level (Fig. 5). This indicates that the increased activity is due to the protein encoded by the transgene.

beta3Glc-T Glucosylates TSR-fucose and Strongly Prefers UDP-Glc as the Sugar Donor—As pointed out in the Introduction, EGF repeats can also be modified with an O-linked fucosyl residue, which can undergo subsequent elongation reactions. It was, therefore, of interest to examine whether beta3Glc-T would transfer glucose to a fucosylated EGF repeat. We prepared and purified the fully fucosylated EGF repeat (EGF-fucose) from human factor VII (5834.4 Da) and tested it as an acceptor substrate for beta3Glc-T with UDP-glucose as the donor in the radiochemical assay. No activity was found (Fig. 2).

In addition to O-fucosylation, EGF repeats can be core O-glucosylated by an as yet unidentified glucosyltransferase (30). Using EGF repeat from human factor VII as the acceptor substrate, we did not find any evidence for such an activity in beta3Glc-T (Fig. 2).

To test the sugar donor specificity of beta3Glc-T, we determined its activity in high speed supernatants of transfected HEK 293T cells with different nucleotide sugars using the HPLC-based assay. As shown in Fig. 6, beta3Glc-T has a strong preference for UDP-glucose. There is less than 10% of the activity with UDP-Gal, whereas it is hardly above background with UDP-GlcNAc and UDP-GalNAc.


Figure 5
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  		FIGURE 5.
Effect of mutations in the catalytic domain of beta3Glc-T. A, wild type and two mutated (349ADD351 and 349ADA351) forms of beta3Glc-T were expressed in HEK 293T cells. Approximately equal amounts of enzyme, as judged from Western analysis (B), were assayed for glucosyltransferase activity with TSR4-fucose (10 µM) as the acceptor. The amount of product was determined with the radiochemical assay as described under "Experimental Procedures." To compensate for the different beta3Glc-T protein expression levels, 3-fold more total protein was used in assays from the mutants compared with wild type cell extracts. As a control, high speed supernatant from cells transfected with empty vector was analyzed. The reported values are the mean (±S.E.) of triplicate determinations from one of two independent experiments. Values have been corrected for background (<5%) as described in the legend to Fig. 2.

 


Figure 6
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  		FIGURE 6.
Sugar donor specificity of beta3Glc-T. The glucosyltransferase activity of beta3Glc-T in high speed supernatants of transfected HEK 293T cells was determined with different sugar donors (20 µM) and TSR4-fucose (10 µM) as the acceptor. The products of the transferase reaction were quantified by reversed-phase HPLC as described under "Experimental Procedures." High speed supernatant from cells transfected with empty vector was used as a control. The reported values are the mean (±S.D.) of duplicate determinations from one of two independent experiments.

 
beta3Glc-T Activity Is Localized in the Endoplasmic Reticulum— Contrary to expectation, it has recently been found that POFUT2 is located in the ER (17). Therefore, it was of interest to investigate whether beta3Glc-T is present in the same subcellular compartment. To avoid possible artifacts of overexpressing the enzyme, we determined the endogenous activity along with that of POFUT2 in cellular subfractions that were obtained from HEK 293T cells by density gradient centrifugation. The gradient that we used has been specifically developed to separate ER, intermediate compartment, and Golgi complex (39). The ER sedimented as a wide peak, as judged by the position of its marker, calnexin (39), that was well separated from Golgi and plasma membranes (Fig. 7). We found the specific activity of both beta3Glc-T and POFUT2 in the ER fractions as a double peak. Significantly, the profiles were essentially superimposed. The reason for the double peak is unknown but could be due to separation of the rough and smooth ER.


Figure 7
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  		FIGURE 7.
beta3Glc-T and POFUT2 activities colocalize in the endoplasmic reticulum. Isotonic homogenates of wild type HEK 293T cells were fractionated on a non-linear Nycodenz density gradient. The activity of beta3Glc-T and POFUT2 in the fractions was determined with the radiochemical assay described under "Experimental Procedures." The position of markers for the Golgi (mannosidase II) and plasma membrane (alkaline phosphatase) subfractions was determined by enzymatic activity, whereas that of ER marker calnexin was determined by Western blot analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report we have identified and characterized beta3Glc-T as the beta1,3-glucosyltransferase that adds a single glucose from UDP-glucose to a specific {alpha}-linked fucose in properly folded TSRs. The closest relatives to beta3Glc-T are beta1,3-N-acetylglucosaminyltransferase (Fringe), core 1 beta1,3-galactosyltransferase, and its chaperone COSMC-1, proteins involved in the addition of the second sugar in different protein O-glycosylation pathways. The beta3Glc-T protein sequence contains 12 amino acids in the putative catalytic domain that are invariant in other members of the CAZy family 31 beta1,3-glycosyltransferases (27). This includes the highly conserved DXD motif, which is involved in coordinating the divalent cation and UDP-sugar in many different glycosyltransferases (40, 41) and is also necessary for beta3Glc-T catalytic activity.

The putative recognition sequences for POFUT1, CX4–5S/TC, and POFUT2, WX5CX2–3S/TCX2G, are clearly different (7, 10), with the modification of the EGF repeats occurring in a beta-strand, whereas TSR domains are modified in the loop connecting the A and B strands. The O-fucose in an EGF repeat can be extended by one of three Fringe glycosyltransferases (lunatic, manic, and radical) in the Golgi (23, 42). However, the sites are incompletely modified and exhibit distinct effects on Notch function (43). In contrast, almost all the TSR modules expressed in mammalian systems are fully substituted with a Glc-Fuc disaccharide. beta1,3-Glucosyltransferase activity was originally identified using the small artificial acceptor para-nitrophenyl-{alpha}-1-fucose (25). Here, we show that TSR-O-{alpha}-1-fucose is a natural acceptor for beta3Glc-T but not EGF-O-{alpha}-1-fucose. This suggests that the beta3Glc-T recognizes a particular fold rather than a specific amino acid sequence.

We found the beta3Glc-T and POFUT2 enzyme activities to be soluble rather than membrane-bound, consistent with other reports (17, 25). Both enzymes appear to have a signal sequence, the first 24 amino acids in the case of beta3Glc-T rather than a single transmembrane domain as predicted (27). The in vitro POFUT2 and beta3Glc-T reactions can be easily driven to completion on fully folded TSR modules. We also observed full occupancy of the glycosylation sites in mammalian protein in vivo (9). This and the observation that the activity profiles of POFUT2 and beta3Glc-T closely superimpose in ER subfractions (Fig. 7) suggests that the two glycosylation reactions may be tightly coupled in vivo. Unusual for a glycosyltransferase, beta3Glc-T contains a KDEL-like REEL sequence at its C terminus. We are currently examining whether the ER localization of POFUT2, which lacks such an ER retrieval signal, is dependent on the presence of beta3Glc-T.

Because both the POFUT1 and POFUT2 enzymes are localized in the ER and recognize properly folded protein domains, it has been proposed that these glycosyltransferases may be involved in quality control of protein folding (17, 19). The localization of POFUT1 is essential for its function in vivo. Furthermore, the ability of POFUT1 to bind to its substrate, Notch, in the ER and to help its folding does not require fucosyltransferase activity (19). Because O-fucosylation of a TSR domain requires proper folding (14), beta3Glc-T also acts on properly folded TSR. Therefore, if beta3Glc-T were involved in quality control of folding, this would not be at the level of the individual TSR domain. Rather it could help in the assembly of the supra-domain structure of multiple TSR domains. In this context it is important to note that multiple TSRs often occur in proteins that contain other types of domains. Some of the most extreme examples occur in the ADAMTS-9 and ADAMTS-20, human metalloproteases, where there are 15 TSRs (44). These are orthologs of GON-1, a protease required for gonadal morphogenesis in C. elegans (45).

The pofut2 gene in C. elegans (pad2) has been deleted, and we observed an altered shape of the anterior gonadal arm resulting from abnormal distal tip cell migration (16). This is in contrast to a study using RNA-mediated interference where gross morphological changes were reported (46). We are currently investigating whether the phenotype we observed in the pofut2 null worm is due to loss of functional POFUT2 protein, the fucosyl moiety, or the disaccharide Glcbeta1,3Fuc on a particular class of proteins.

Now that the pertinent glycosyltransferases have been cloned, it is also possible to test whether these structures in TSR domains modulate protein-protein interactions. For example, the anti-angiogenic activity of TSP-1 and TSP-2 is mediated through the interaction of their TSR domains with the cell surface receptor, CD36 (47, 48). These studies used synthetic peptides and identified the CSVTCG sequence (derived from TSP-1 TSR2, and TSR3) as the binding region (49). In addition, the TSP-1 and -2/CD36 interaction is antagonized by a histidine-rich glycoprotein that also binds to the CSVTCG sequence (50, 51). Naturally this sequence occurs in TSRs with the cysteine residues engaged in disulfide bonds and the Thr residue modified with the disaccharide Glc-Fuc (9). It is, therefore, worthwhile testing if the glycosylation of the TSR affects the binding to CD36.

TSRs of TSP have been implicated in the activation of TGFbeta through their interaction with the inhibitory latency binding protein. Consequently, it is interesting to note that the beta3Glc-T gene was identified in a differential display-PCR screen in which T84 human intestinal epithelial cells were induced to differentiate in response to TGFbeta (27). It remains to be determined if beta3Glc-T activity modulates TGFbeta activation by TSP.

It is worth remarking on the changes that have been reported in the level of expression of the two transferases involved in the biosynthesis of the Glcbeta1,3Fuc{alpha}1 disaccharide. The beta3Glc-T gene is located on human chromosome 13q12.3, and its promoter region contains several potential binding sites for Smads, proteins that transduce TGFbeta signals (27). beta3Glc-T is widely expressed in human tissues as two transcripts of 4.2 and 3.4 kilobases whose relative levels differ in a tissue-specific manner (27). Changes in both beta3Glc-T and POFUT2 expression have also been found in cancer. Jacques et al. (52) observed that the level of beta3Glc-T transcript increases in human thyroid cancer, whereas POFUT2 is overexpressed in human glioblastomas (53). Interestingly, the genes for a number of tumor suppressors (BRCA2, RB1, and FLT3) are in close proximity to the beta3Glc-T gene (54), and a critical region (marker D13S893) in chromosome 13q12 that excludes the BRAC2 gene but includes the beta3Glc-T gene has been implicated in tumorigenicity (55).

Protein O-fucosylation is often accompanied by other unusual forms of glycosylation. Within the EGF repeats of factors VII, IX, Notch, and TSP, an O-beta-linked glucose capped with either one or two Xyl{alpha}1,3 (Xyl, xylose) residues is found just nine amino acids away from the O-fucosylated glycans (56, 57). Similarly, we reported the disaccharide Glc-Fuc in TSR domains of TSP-1 while studying C-mannosylation, another unusual type of glycosylation that is located just five amino acids N-terminal to the O-fucosylation site (9). This paper and our previous observations show that glucosylation of fucosylated TSR can occur in the absence of C-mannosylation (and vice versa) (9).

Clearly distinct glycosyltransferases act in the EGF repeat and TSR glycosylation pathways. The ability to detect and modify different forms of glycosylation in these biologically important protein domains will help to determine their functional significance.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant GM61126. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria. Back

2 To whom correspondence should be addressed. Tel.: 41-61-6974722; Fax: 41-61-6973976; E-mail: jan.hofsteenge{at}fmi.ch.

3 The abbreviations used are: EGF, epidermal growth factor-like; Asp-N, endoproteinase of Pseudomonas fragi mutant; beta3Glc-T, beta1,3-glucosyltransferase; B3GTL, beta3-glycosyltransferase-like; Fuc, fucose; GalNAc, N-acetylgalactosamine; Lys-C, endoproteinase from Achromobacter lyticus; MS, mass spectroscopy; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; m/z, mass over charge; POFUT, protein O-fucosyltransferase; TSP, thrombospondin; TSR, TSP type 1 repeat; TSR4, fourth TSR repeat in rat F-spondin; ER, endoplasmic reticulum; HEK cells, human embryonic kidney cells; PIC, protease inhibitor mixture; LC, liquid chromatography; HPLC, high performance LC. Back

4 During the revision of this manuscript a paper describing the characterization of B3GTL as a novel beta1,3-glucosyltransferase appeared (58). Back

5 S. Canevascini, D. Klein, J. Althaus, K. Kozma, R. Chiquet and J. Hofsteenge, manuscript in preparation. Back

6 J. J. Keusch, T. Smilda, J. Krieg, and J. Hofsteenge, manuscript in preparation. Back

7 Please note that there is a single point mutation in the cDNA used here as compared to the clone identified by Henionen et al. (27). See "Experimental Procedures" for details. Back


   ACKNOWLEDGMENTS
 
We thank Alexandra Bezler for the initial experiments on the glucosylation of TSR-fucose, Jasmin Althaus for setting up the POFUT2 radiochemical assay and help in the TSR4 purification, and Ragna Sack for help with the MALDI-TOF-MS analysis. The cDNA encoding rat F-spondin was a generous gift from Dr. A. Klar (Hebrew University, Jerusalem, Israel). We are thankful to Drs. T. Ahrens and A. Bachmann (Biocenter, University of Basel, Switzerland) for help with and advice on measuring the CD and fluorescence spectra. The Friedrich Miescher Institute is part of the Novartis Research Foundation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Spiro, R. G. (2002) Glycobiology 12, 43–56
  2. Bjoern, S., Foster, D. C., Thim, L., Wiberg, F. C., Christensen, M., Komiyama, Y., Pedersen, A. H., and Kisiel, W. (1991) J. Biol. Chem. 266, 11051–11057[Abstract/Free Full Text]
  3. Buko, A. M., Kentzer, E. J., Petros, A., Menon, G., Zuiderweg, E. R., and Sarin, V. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3992–3996[Abstract/Free Full Text]
  4. Harris, R. J., Leonard, C. K., Guzzeta, A. W., and Spellman, M. W. (1991) Biochemistry 30, 2311–2314[CrossRef][Medline] [Order article via Infotrieve]
  5. Harris, R. J., Ling, V. T., and Spellman, M. W. (1992) J. Biol. Chem. 267, 5102–5107[Abstract/Free Full Text]
  6. Nishimura, H., Takao, T., Hase, S., Shimonishi, Y., and Iwanaga, S. (1992) J. Biol. Chem. 267, 17520–17525[Abstract/Free Full Text]
  7. Panin, V. M., Shao, L., Lei, L., Moloney, D. J., Irvine, K. D., and Haltiwanger, R. S. (2002) J. Biol. Chem. 277, 29945–29952[Abstract/Free Full Text]
  8. Moloney, D. J., Shair, L. H., Lu, F. M., Xia, J., Locke, R., Matta, K., and Haltiwanger, R. S. (2000) J. Biol. Chem. 275, 9604–9611[Abstract/Free Full Text]
  9. Hofsteenge, J., Huwiler, K. G., Macek, B., Hess, D., Lawler, J., Mosher, D. F., and Peter-Katalinic, J. (2001) J. Biol. Chem. 276, 6485–6498[Abstract/Free Full Text]
  10. Gonzalez de Peredo, A., Klein, D., Macek, B., Hess, D., Peter-Katalinic, J., and Hofsteenge, J. (2002) Mol. Cell. Proteomics 1, 11–18[Abstract/Free Full Text]
  11. Tucker, R. P. (2004) Int. J. Biochem. Cell Biol. 36, 969–974[CrossRef][Medline] [Order article via Infotrieve]
  12. Tan, K., Duquette, M., Liu, J. H., Dong, Y., Zhang, R., Joachimiak, A., Lawler, J., and Wang, J. H. (2002) J. Cell Biol. 159, 373–382[Abstract/Free Full Text]
  13. Paakkonen, K., Tossavainen, H., Permi, P., Rakkolainen, H., Rauvala, H., Raulo, E., Kilpelainen, I., and Guntert, P. (2006) Proteins 64, 665–672[CrossRef][Medline] [Order article via Infotrieve]
  14. Luo, Y., Nita-Lazar, A., and Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385–9392[Abstract/Free Full Text]
  15. Hallgren, P., Lundblad, A., and Svensson, S. (1975) J. Biol. Chem. 250, 5312–5314[Abstract/Free Full Text]
  16. Canevascini, S., Kozma, K., Grob, M., Althaus, J., Klein, D., Chiquet-Ehrismann, R., and Hofsteenge, J. (2006) European Worm Meeting, April 29–May 3, 2006, p. 71, Hersonissos, Crete, Greece
  17. Luo, Y., Koles, K., Vorndam, W., Haltiwanger, R. S., and Panin, V. M. (2006) J. Biol. Chem. 281, 9393–9399[Abstract/Free Full Text]
  18. Luo, Y., and Haltiwanger, R. S. (2005) J. Biol. Chem. 280, 11289–11294[Abstract/Free Full Text]
  19. Okajima, T., Xu, A., Lei, L., and Irvine, K. D. (2005) Science 307, 1599–1603[Abstract/Free Full Text]
  20. Shi, S., and Stanley, P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5234–5239[Abstract/Free Full Text]
  21. Haines, N., and Irvine, K. D. (2003) Nat. Rev. Mol. Cell Biol. 4, 786–797[Medline] [Order article via Infotrieve]
  22. Haltiwanger, R. S., and Stanley, P. (2002) Biochim. Biophys. Acta 1573, 328–335[Medline] [Order article via Infotrieve]
  23. Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wislon, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S., and Vogt, T. F. (2000) Nature 406, 369–375[CrossRef][Medline] [Order article via Infotrieve]
  24. Chen, J., Moloney, D. J., and Stanley, P. (2001) Proc. Natl. Acad. Sci. 98, 13716–13721[Abstract/Free Full Text]
  25. Moloney, D. J., and Haltiwanger, R. S. (1999) Glycobiology 9, 679–687[Abstract/Free Full Text]
  26. Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat, B. (1997) Biochem. J. 326, 929–939[Medline] [Order article via Infotrieve]
  27. Heinonen, T. Y., Pasternack, L., Lindfors, K., Breton, C., Gastinel, L. N., Maki, M., and Kainulainen, H. (2003) Biochem. Biophys. Res. Commun. 309, 166–174[CrossRef][Medline] [Order article via Infotrieve]
  28. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51–59[CrossRef][Medline] [Order article via Infotrieve]
  29. Klar, A., Baldassare, M., and Jessell, T. M. (1992) Cell 69, 95–110[CrossRef][Medline] [Order article via Infotrieve]
  30. Shao, L., Luo, Y., Moloney, D. J., and Haltiwanger, R. (2002) Glycobiology 12, 763–770[Abstract/Free Full Text]
  31. Moloney, D. J., Lin, A. I., and Haltiwanger, R. S. (1997) J. Biol. Chem. 272, 19046–19050[Abstract/Free Full Text]
  32. Krieg, J., Hartmann, S., Vicentini, A., Gläsner, W., Hess, D., and Hofsteenge, J. (1998) Mol. Biol. Cell 9, 301–309[Abstract/Free Full Text]
  33. Wang, Y., and Spellman, M. W. (1998) J. Biol. Chem. 273, 8112–8118[Abstract/Free Full Text]
  34. Huwiler, K. G., Vestling, M. M., Annis, D. S., and Mosher, D. F. (2002) Biochemistry 41, 14329–14339[CrossRef][Medline] [Order article via Infotrieve]
  35. Smith, C., Pangburn, M., Vogel, W., and Muller-Eberhard, J. (1984) J. Biol. Chem. 259, 4582–4588[Abstract/Free Full Text]
  36. Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 275, 25308–25314[Abstract/Free Full Text]
  37. Munro, S., and Freeman, M. (2000) Curr. Biol. 10, 813–820[CrossRef][Medline] [Order article via Infotrieve]
  38. Wiggins, C. A. R., and Munro, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7945–7950[Abstract/Free Full Text]
  39. Hammond, C., and Helenius, A. (1994) J. Cell Biol. 126, 41–52[Abstract/Free Full Text]
  40. Gastinel, L. N., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 3546–3557[CrossRef][Medline] [Order article via Infotrieve]
  41. Unligil, U. M., Zhou, S., Yuwaraj, S., Sarkar, M., Schachter, H., and Rini, J. M. (2000) EMBO J. 19, 5269–5280[CrossRef][Medline] [Order article via Infotrieve]
  42. Rampal, R., Li, A. S., Moloney, D. J., Georgiou, S. A., Luther, K. B., Nita-Lazar, A., and Haltiwanger, R. S. (2005) J. Biol. Chem. 280, 42454–42463[Abstract/Free Full Text]
  43. Rampal, R., Arboleda-Velasquez, J. F., Nita-Lazar, A., Kosik, K. S., and Haltiwanger, R. S. (2005) J. Biol. Chem. 280, 32133–32140[Abstract/Free Full Text]
  44. Somerville, R. P., Longpre, J. M., Jungers, K. A., Engle, J. M., Ross, M., Evanko, S., Wight, T. N., Leduc, R., and Apte, S. S. (2003) J. Biol. Chem. 278, 9503–9513[Abstract/Free Full Text]
  45. Blelloch, R., and Kimble, J. (1999) Nature 399, 586–590[CrossRef][Medline] [Order article via Infotrieve]
  46. Menzel, O., Vellai, T., Takacs-Vellai, K., Reymond, A., Mueller, F., Antonarakis, S. E., and Guipponi, M. (2004) Genomics 84, 320–330[CrossRef][Medline] [Order article via Infotrieve]
  47. Dawson, D. W., Pearce, S. F., Zhong, R., Silverstein, R. L., Frazier, W. A., and Bouck, N. P. (1997) J. Cell Biol. 138, 707–717[Abstract/Free Full Text]
  48. Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L., and Bouck, N. (2000) Nat. Med. 6, 41–48[CrossRef][Medline] [Order article via Infotrieve]
  49. Asch, A. S., Silbiger, S., Heimer, E., and Nachman, R. L. (1992) Biochem. Biophys. Res. Comm. 182, 1208–1217[CrossRef][Medline] [Order article via Infotrieve]
  50. Simantov, R., Febbraio, M., Crombie, R., Asch, A. S., Nachman, R. L., and Silverstein, R. L. (2001) J. Clin. Investig. 107, 45–52[Medline] [Order article via Infotrieve]
  51. Simantov, R., Febbraio, M., and Silverstein, R. L. (2005) Matrix Biol. 24, 27–34[Medline] [Order article via Infotrieve]
  52. Jacques, C., Baris, O., Prunier-Mirebeau, D., Savagner, F., Rodien, P., Rohmer, V., Franc, B., Guyetant, S., Malthiery, Y., and Reynier, P. (2005) J. Clin. Endocrinol. Metab. 90, 2314–2320[Abstract/Free Full Text]
  53. Dong, S., Nutt, C. L., Betensky, R. A., Stemmer-Rachamimov, A. O., Denko, N. C., Ligon, K. L., Rowitch, D. H., and Louis, D. N. (2005) J. Neuropathol. Exp. Neurol. 64, 948–955[Medline] [Order article via Infotrieve]
  54. Dunham, A., Matthews, L. H., Burton, J., Ashurst, J. L., Howe, K. L., Ashcroft, K. J., Beare, D. M., Burford, D. C., Hunt, S. E., Griffiths-Jones, S., Jones, M. C., Keenan, S. J., Oliver, K., Scott, C. E., Ainsc