3 Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA
Received on April 17, 2002; revised on June 25, 2002; accepted on July 5, 2002
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Abstract |
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Key words: Chinese hamster ovary cells/EGF repeats/glucosyltransferase/glycosyltransferases/O-glucose
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Introduction |
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The O-glucose modification was originally identified on bovine blood coagulation factors VII and IX (Hase et al., 1988). Detailed analysis demonstrated a trisaccharide form of O-glucose on bovine factor IX with the structure Xyl-
1,3-Xyl-
1,3-Glc-ß1-O-Ser (Hase et al., 1990
). Similar structures were observed on human factor VII, factor IX, and protein Z (Nishimura et al., 1989
). Comparison of the sequences surrounding sites of glycosylation on these proteins led to the proposal of a putative consensus sequence for the O-glucose addition (Harris and Spellman, 1993
). The proposed consensus sequence for addition of O-glucose is C1-X-S-X-P-C2, where C1 and C2 are the first and second conserved cysteines of the EGF repeat, respectively, and X can be any amino acid (Harris and Spellman, 1993
). The actual number of proteins known to be modified with the O-glucose modification is limited, but the putative consensus sequence allows database searches (Table I) revealing that numerous secreted and cell surface proteins are predicted to bear O-glucose. We recently demonstrated that the putative consensus sequence is capable of accurately predicting whether a protein bears O-glucose by showing that the Notch receptor has this modification (Moloney et al., 2000b
) (Table I).
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Results |
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Product characterization
A series of chromatographic analyses were carried out to demonstrate that the product generated in the assays consists of glucose in O-linkage to the EGF repeat. To demonstrate that the glucose is covalently associated with the EGF repeat, the product was analyzed by reverse-phase HPLC (Figure 4a). Furthermore, to demonstrate that the glucose was attached through an O-linkage, alkali-induced ß-elimination was performed. The released sugar product from the ß-elimination migrated as a monosaccharide on gel filtration chromatography (Figure 4b). Analysis of the monosaccharide by high-performance anion-exchange chromatography (HPAEC) revealed it to be glucitol (Figure 4c), the expected product from ß-elimination of O-glucose.
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Discussion |
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Although the biological effect of modifying EGF repeats with O-glucose is not yet known, recent work on the O-fucose modifications of the Notch receptor (Moloney et al., 2000a), uPA (Rabbani et al., 1992
), and Cripto (Schiffer et al., 2001
; Yan et al., 2002
) demonstrates that carbohydrate modifications of EGF repeats can regulate receptorligand interactions. If a simple O-fucose monosaccharide can alter the interaction between receptor and ligand, the more complex O-glucose modifications on the EGF repeats may have similar effects. Several observations suggest such a role for O-glucose modifications. For instance, mutation of serine-52 (the O-glucose site) to alanine in human blood coagulation factor VII results in a decrease in factor VII clotting activity by an unknown mechanism (Bjoern et al., 1991
). The conservation across species of O-glucose consensus sites on many of the proteins either known or predicted to be modified (Table I) strongly suggests that O-glucose plays an essential role in the function of these proteins. This is most apparent in the Notch receptor. Although Notch activity is regulated by Fringe through alteration of the O-fucose residues, Notch contains more conserved O-glucose sites than O-fucose sites (Moloney et al., 2000b
). Comparison of all Notch sequences currently in the database reveals 5 evolutionarily conserved O-fucose sites (EGF modules 3, 20, 24, 26, 31) and 12 evolutionarily conserved O-glucose sites (EGF modules 4, 10, 12, 13, 14, 16, 19, 20, 21, 25, 27, 33).
The Notch receptor protein is extremely important for its role in numerous stages of development (Artavanis-Tsakonas et al., 1999). In Drosophila, Notch is one of several neurogenic genes that mediate cell fate decisions during development. Targeted disruption of the Notch1 gene in mice causes embryonic lethality (Swiatek et al., 1994
). In humans, deregulation of Notch signaling results in serious diseases, such as T cell leukemias (Ellisen et al., 1991
), a cerebral arteriopathy (Joutel et al., 1996
), spondylocostal dysostosis (Bulman et al., 2000
), and Alagille syndrome (Joutel and Tournier-Lasserve, 1998
; Li et al., 1997
). Notch receptor becomes activated by binding to its cell surface ligands, Delta or Serrate/Jagged (Mumm and Kopan, 2000
). Notch, Delta, and Serrate/Jagged all have numerous EGF repeats with multiple sites for O-glucose and O-fucose modifications (Moloney et al., 2000b
; Panin et al., 2002
). EGF repeats 11 and 12 of Notch are necessary and sufficient for interaction with ligands (Rebay et al., 1991
). Interestingly, an evolutionarily conserved O-glucose site exists on EGF repeat 12, suggesting that the O-glucose modification at this site may play a crucial role in Notchligand interactions. Experiments to examine this hypothesis are currently under way.
Glucose is not often found in oligosaccharide modifications of proteins in mammalian systems. Best studied are the three -linked glucoses attached to mannose on newly synthesized N-linked oligosaccharides. This is a transient modification that plays a role in the correct folding of glycoproteins. The glucoses are removed step by step as an indicator of proper folding (Hebert et al., 1995
). Other glucose modifications are found on glycogenin and collagen. Glycogenin, one of the two subunits of glycogen synthase complex, is an enzyme that catalyzes its own autoglucosylation, adding a
-linked glucose to itself at tyrosine 194 (Alonso et al., 1995
). The glucosylated glycogenin is the critical protein primer required for de novo glycogen synthesis. An
-linked glucose is added on to the 2'-hydroxyl of galactose during posttranslational modification of procollagen (Smith et al., 1983
). The function of the disaccharide in collagen is not clear but probably is involved in directing the correct molecular assembly of fibrils.
Only two forms of ß-linked glucose modifications are known to exist on mammalian glycoproteins, including O-glucose and the disaccharide Glcß1,3Fuc-O-Ser/Thr. Several years ago we demonstrated the presence of the Glcß1,3Fuc disaccharide O-linked to a number of proteins in CHO cells (Moloney et al., 1997) and identified a ß1,3-glucosyltransferase activity capable of adding the glucose to fucose (Moloney and Haltiwanger, 1999
). We had originally believed these disaccharides to exist on EGF repeats, because O-fucose was only known on EGF repeats at that time. A recent elegant study by Hofsteenge and co-workers (2001) using mass spectral analysis of thrombospondin-1 revealed the presence of two unusual carbohydrate modifications: C-linked mannose on several tryptophans and the O-linked disaccharide Glc-Fuc on several serine/threonine residues. This work demonstrated that Glc-Fuc disaccharides are not on EGF repeats, but instead are found on a different type of protein domain called a thrombospondin-type 1 repeat (TSR) (Adams and Tucker, 2000
). Neither the enzyme responsible for addition of O-fucose to the TSR nor the biological function of this modification is yet known.
The anomeric linkage of O-glucose was established in a single study (Hase et al., 1990) where nuclear magnetic resonance (NMR) was utilized to analyze the structure of the O-glucose modification on bovine factor IX. No other data on the anomeric linkage of O-glucose exists. In particular, no successful enzymatic cleavage of O-glucose from protein has been reported. We have been unsuccessful in several attempts to remove O-glucose from factor VII EGF repeat or glycopeptides derived from glucosylated EGF repeat using either
- or ß-glucosidases, suggesting that this linkage is uniquely resistant to enzymatic digestion. Although the O-glucose on factor VII EGF repeat is likely to be ß-linked, definitive demonstration of the linkage awaits production of sufficient product to pursue NMR analysis.
Unusual carbohydrate modifications are often related to a specific biological event (Varki, 1993). For example, mannose 6-phosphate targets lysosomal enzymes to lysosomes (Kornfeld, 1990
), sialyl Lewis x helps recruit leukocytes to sites of inflammation (Zak et al., 2000
), and polysialic acid modulates neural cell adhesion molecule interactions during neuronal development (Acheson et al., 1991
). The recent work demonstrating that O-fucose modifications on the EGF repeats of Notch, uPA, and Cripto play a role in signaling events provide other clear examples of a rare form of glycosylation performing a specific biological function. The O-glucose modifications of EGF repeats are another unusual form of glycosylation that we predict will have an equally specific role in the biology of the proteins it modifies. We have initiated attempts to purify the O-glucosyltransferase by using a combination of conventional and affinity chromatography. Purifying and cloning this enzyme will allow us to develop the tools necessary to study this unique modification.
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Materials and methods |
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Preparation of cell lysates and rat tissues
All mammalian cells were grown in 100-mm dishes in a humidified 37°C incubator with 5% CO2. Lec1 cells were grown in MEM medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Hela and NIH3T3 cells were grown in Dulbeccos modified Eagles medium (Gibco, Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum. SF-9 cells were grown at room temperature in Graces insect medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum. S2 cells were also grown at room temperature in Shields and Sang M3 insect medium (Sigma) with 10% fetal bovine serum. Cells were collected by scraping and washed three times with Tris-buffered saline (10 mM TrisHCl, pH 7.5, 0.15 M NaCl). Yeast cell pellets were generously provided by Rolf Sternglanz (Department of Biochemistry and Cell Biology, SUNY Stony Brook). Cell pellets were lysed on ice in Tris-buffered saline with 1% (w/v) Nonidet P-40 and protease inhibitor cocktails I and II (yeast cell pellets were lysed by vortexing with glass beads) (Sutton et al., 1991
). Cell debris was pelleted by centrifugation (14,000 rpm, 10 min). The supernatants were aliquoted, frozen in liquid nitrogen, and stored at 80°C. Extracts of rat tissues were prepared as described (Moloney and Haltiwanger, 1999
).
Production of recombinant factor VII EGF1 repeat and O-glucose site mutants in E. coli
DNA sequences encoding human factor VII EGF1 domain were cloned into a pET20b (+) vector (Novagen, Madison, WI) using BamHI (Invitrogen, Carlsbad, CA) and XhoI (Invitrogen) restriction sites with primers containing BamHI and XhoI restriction sites on the 5'- and 3'-ends, respectively (5'-CCGAAGGATCCGGCAAGT GATGGTGACCAG- 3', and 5'-CTGCCCTCGAGCCCGTCATCCTTGTG-3'). A plasmid encoding EGF1 from human factor VII (generously provided by Yang Wang; Wang et al., 1996) was used as template. Polymerase chain reaction was carried out for 30 cycles with the following conditions: denaturing at 95°C for 0.5 min; annealing at 65°C for 1 min; elongating at 72°C for 1 min. Serine-52 was changed to Ala (S52A) using the Strategene Quik Change Site-Directed Mutagenesis Protocol, with the primers 5'-GGTGACCAGTGT GCCGCGAGTCCATGCCAG-3' and 5'-CTGGCATGGACTCGCGGCACACTGGTCA CC-3'. Serine-53 was changed to Ala (S53A) using the primers 5'-GACCAGTGTGCCTCGGCTCCATGCCAGAATGGT-3' and 5'-ACCATTCTGGCATGGAGCCGAGGCACACTGGTC-3'. All constructs were sequenced prior to further study.
To produce protein, transformed bacteria BL21 (DE3) were grown to an absorbency of 0.6 at 600 nm in the presence of 100 mg/L ampicillin, 37°C, and induced with 1 mM isopropyl-ß-thiogalactopyranoside overnight at 25°C. Cells were harvested by centrifugation, and periplasmic shock was performed according to the Qiagen manual. The shockate was dialyzed extensively against dialysis buffer (50 mM TrisHCl pH 8.0, 10 mM imidazole) at 4°C, and purified using Qiagen Ni-NTA resin. The resin was washed with the dialysis buffer, and the protein was eluted with 50 mM TrisHCl, pH 8.0, 100 mM imidazole.
To identify properly folded EGF repeat, a portion of the purified EGF repeat was radiolabeled using baculovirus overexpressed O-FucT-1 and GDP-[3H]fucose (as described in Wang et al., 2001; Wang and Spellman, 1998
). Reverse-phase HPLC was performed to separate the properly folded EGF repeats from misfolded variants as described later, and the properly folded form was identified by the presence of [3H]fucose. The remainder of the EGF repeat was then purified in the same manner on a preparative scale. The final concentration of factor VII EGF1 was determined by a BCA assay (Pierce). The factor VII EGF1 repeats containing the S52A and S53A mutants were expressed and purified in the same manner.
O-Glucosyltransferase assay
The reaction mixture (50 µl final volume) contained 50 mM HEPES, pH 7.0, 10 mM MnCl2, 4.8 µM recombinant human factor VII EGF-1 repeat, 0.2 µM UDP-[3H]glucose, 2.8 µM UDP-glucose, and 1020 µg cellular protein. The reaction was incubated at 37°C for 20 min and stopped by addition of 450 µl 50 mM EDTA, pH 8. The EGF repeat was separated from unincorporated radioactivity on C18 cartridges using a vacuum manifold. The sample was loaded onto a C18 cartridge (100 mg). The cartridge was washed with 8 ml water, and the EGF repeat was eluted with 1.5 ml 80% methanol. Incorporation of [3H]glucose into the EGF repeat was determined by scintillation counting of the eluate. Reactions without EGF repeat were used as background control.
Reduction and alkylation of EGF repeat
Two aliquots of recombinant factor VII EGF repeat (4.8 nmole each) were dried in a Speed Vac. Both samples were dissolved in 25 µl 0.4 M NH4HCO3 containing 8 M urea (final pH between 7.5 and 8.5). Water (10 µl) was added to one sample as control. The other sample was reduced by adding 5 µl 45 mM DTT and incubating at 50°C for 15 min. After cooling to room temperature, the sample was alkylated by adding 5 µl 200 mM iodoacetamide and incubating at room temperature for another 15 min. Both the control EGF repeat and the reduced and alkylated EGF repeat were purified on reverse-phase HPLC as described.
Reverse-phase HPLC purification
Reverse-phase HPLC was performed on a 250 x 4.6 mm Dynamax C18 column using a 30-min linear gradient from 0% to 80% acetonitrile in 0.1% trifluoracetic acid at 1 ml/min. Peptide was detected at 214 nm. Fractions (1 min) were collected as necessary.
Product analysis by gel filtration chromatography and HPAEC
Radiolabeled product in the eluant from the glucosyltransferase assay was dried in a Speed-Vac. Alkali-induced ß-elimination, gel filtration on Superdex column, and HPAEC analysis of the products on a Carbopac MA-1 column (Dionex, Sunnyvale, CA) were all done as described (Moloney et al., 1997, 2000b)
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed; E-mail: Robert.Haltiwanger@stonybrook.edu
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References |
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