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Originally published In Press as doi:10.1074/jbc.M511974200 on February 7, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9385-9392, April 7, 2006
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Two Distinct Pathways for O-Fucosylation of Epidermal Growth Factor-like or Thrombospondin Type 1 Repeats*

Yi Luo, Aleksandra Nita-Lazar, and Robert S. Haltiwanger1

From the Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215

Received for publication, November 7, 2005 , and in revised form, February 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) are both small cysteine-knot motifs known to be O-fucosylated. The enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (POFUT1), has been identified and shown to be essential in Notch signaling. Fringe, an O-fucose beta1,3-N-acetylglucosaminyltransferase, elongates O-fucose on specific EGF repeats from Notch to form a disaccharide that can be further elongated to a tetrasaccharide. TSRs are found in many extracellular matrix proteins and are involved in protein-protein interactions. The O-fucose moiety on TSRs can be further elongated with glucose to form a disaccharide. The discovery of O-fucose on TSRs raised the question of whether POFUT1, or a different enzyme, adds O-fucose to TSRs. Here we demonstrate the existence of a TSR-specific O-fucosyltransferase distinct from POFUT1. Similar to POFUT1, the novel TSR-specific O-fucosyltransferase is a soluble enzyme that requires a properly folded TSR as an acceptor substrate. In addition, we found that a previously identified fucose-specific beta1,3-glucosyltransferase adds glucose to O-fucose on TSRs, but it does not modify O-fucose on an EGF repeat. Similarly, Lunatic fringe, Manic fringe, and Radical fringe are all capable of modifying O-fucose on an EGF repeat, but not on a TSR. Taken together, these results suggest that two distinct O-fucosylation pathways exist in cells, one specific for EGF repeat and the other for TSRs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
O-Fucose is an unusual form of glycosylation in which a fucose is covalently attached to the hydroxyl group (O-linkage) of a serine or threonine residue. It was originally found in amino acid fucosides isolated from human urine (1). Later, O-fucose was shown to exist on epidermal growth factor-like (EGF)2 repeats of several proteins, including urinary-type plasminogen activator, tissue-type plasminogen activator, Factor VII, Factor IX, Notch, and Cripto (2-5). EGF repeats are small cysteine-knot motifs of ~40 amino acids in length defined by six conserved cysteines forming three disulfide bonds in a specific pattern: Cys1-Cys3, Cys2-Cys4, and Cys5-Cys6 (6). Several studies have shown that O-fucose on some proteins is further elongated to a tetrasaccharide, NeuAc-{alpha}2,3/6-Gal-beta1,4-GlcNAc-beta1,3-Fuc-O-Ser/Thr, whereas on others O-fucose is elongated to the disaccharide, Glc-beta1,3-Fuc-O-Ser/Thr (7-9). Based on the presence of two different forms of elongated O-fucose, we had originally proposed that the O-fucose glycosylation pathway was branched, and that the enzymes modifying O-fucose, the fucose-specific beta1,3-glucosyltransferase and the fucose-specific beta1,3-N-acetylglucosaminyltransferase, would compete with the one another (9).

More recently, a significant role for O-fucosylation of EGF repeats within the Notch receptor protein has been revealed (10, 11). Fringe, a known modulator of Notch signaling, is a fucose-specific beta1,3-N-acetylcosaminyltransferase, capable of elongating O-fucose on Notch (12, 13). O-Fucose itself has an essential and Fringe-independent role in Notch signaling. Protein O-fucosyltransferase 1 (abbreviated POFUT1 for humans, OFUT1 for Drosophila) is responsible for adding O-fucose to EGF repeats (14, 15). RNA interference-mediated reduction of Ofut1 expression or mutants in Ofut1 result in Notch phenotypes in Drosophila (16, 17). Furthermore, ablation of the mouse Pofut1 gene causes an embryonic lethal phenotype similar to Notch1 deficiency (18). Mutations in specific O-fucose glycosylation sites on mouse Notch1 alter Notch activity (19). Reduction of O-fucosylation on Notch through down-regulation of Ofut1 significantly reduces the binding between Notch and its ligands, suggesting that O-fucosylation plays an important role in Notch-ligand interactions (20). Recent results also suggest that Drosophila OFUT1 can function as a chaperone and play an important role in proper folding and cell surface expression of Notch (21). Taken together, these studies demonstrate that O-fucosyltransferase 1 and O-fucose modifications of EGF repeats play essential roles in Notch function.

In addition to EGF repeats, Hofsteenge and co-workers (22, 23) have shown that O-fucose occurs in a totally different protein context: thrombospondin type 1 repeats (TSRs). TSRs are small cysteine-knot modules of ~60 amino acids in length containing six conserved Cys residues, as well as conserved Trp, Ser, and Arg residues (24). Like EGF repeats, the cysteines of TSRs participate in disulfide bonds stabilizing the whole structure, although their disulfide bonding patterns are distinct (24). Hofsteenge and co-workers (22, 23) found O-fucose on 11 TSRs from three proteins: human thrombospondin 1 (TSP-1), human properdin, and rat F-spondin. By comparing sequence contexts surrounding the modified residues, a consensus sequence was proposed: WX5C1X2-3S/TC2X2G. They also showed that O-fucose on TSRs could be further elongated by glucose to form a Glc-Fuc disaccharide, although the linkage between fucose and glucose was not determined. This suggested that the previously described Glc-beta1,3-Fuc disaccharide may occur on TSRs instead of EGF repeats (9).

The presence of O-fucose on TSRs raised the question of whether POFUT1 or a novel enzyme is responsible for adding O-fucose to TSRs. Because TSRs are also cysteine-knot motifs, it is plausible that POFUT1 could fucosylate both EGF repeats and TSRs. Here we examine whether POFUT1 modifies both EGF repeats and TSRs or whether a unique enzyme exists for fucosylation of TSRs. In addition, we examine whether the glucose-fucose disaccharide seen by Hofsteenge and co-workers (22, 23) on TSRs is the Glc-beta1,3-Fuc disaccharide we previously reported in CHO cells (9). Finally, we investigate whether there is cross-talk between elongation of O-fucose on EGF repeats and TSRs by testing if any of the fringe enzymes modify O-fucose on TSR repeats or if the fucose-specific beta1,3-glucosyltransferase modifies O-fucose on EGF repeats.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—GDP-beta-L-Fucose was purchased from Oxford GlycoSystems. L-[6-3H]Fucose (60 Ci/mmol) and GDP-[1-3H(N)]fucose (17.3 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Human factor VII EGF was either kindly provided by Dr. Yang Wang or synthesized as described (25). The Lec1-CHO cell line was developed by Dr. Pamela Stanley (26). Solid Phase Extraction C18 cartridges were purchased from Agilent Technologies. Alditol sugar standards were prepared by reduction of the corresponding sugar with sodium borohydride as described (27). Reverse-phase C18 HPLC columns (4.6 x 250 mm) were purchased from Vydac. Generation of soluble and membrane fractions of COS1 cells, extraction of Lec1-CHO cells, and preparation of recombinant POFUT1, Lunatic fringe, Manic fringe, and Radical fringe were described elsewhere (28, 29). All other reagents were of the highest quality available.

Plasmid Construction—Constructs for expressing the third TSR from human thrombospondin-1 (TSP1-TSR3) in mammalian cells (pSecTag-hTSP1-TSR3) and Escherichia coli (pET20b+-hTSP1-TSR3) were prepared as follows. DNA sequences encoding TSP1-TSR3 (amino acids 472-530) were amplified using primers containing HindIII and XhoI sites (5'-TCGCTAAAGCTTCCATCAATGGAGGCT-3' and 5'-TCGACGATCTCGAGGAATTGGACAGTCCTG-3', for pSecTag-hTSP1-TSR3) or primers containing BamHI and XhoI sites (5'-ACCGAAGGATCCCATCAATGGAGGCTGGGG-3' and 5'-TGAAATCTCGAGAATTGGACAGTCCTGCTTGTTGC-3', for pET20b+-hTSP1-TSR3). A plasmid encoding the three TSRs of human TSP1 (kindly provided by Dr. Deane Mosher, University of Wisconsin) was used as template. The amplified fragments were then subcloned into pSecTag2C vector in-frame with a C-terminal Myc and His6 coding sequence using HindIII and XhoI sites or pET20b+ vector in-frame with a C-terminal His6 coding sequence using BamHI and XhoI sites. To mutate the O-fucose site in both constructs, threonine 489 was changed to Ala (T489A) using the Stratagene QuikChange Site-directed Mutagenesis Protocol, with primers 5'-CATCTGTTCTGTCGCCTGTGGAGGAGGG-3' and 5'-CCCTCCTCCACAGGCGACAGAACAGATG-3'. All constructs were sequenced prior to further study.

Analysis of O-Fucose Saccharide Structure on the Third TSR of Human Thrombospondin 1—The plasmid encoding wild type or the T489A mutant of TSP1-TSR3 (pSecTag-hTSP1-TSR3) was transiently transfected into Lec1-CHO cells using Geneporter (Gene Therapy Systems) essentially as described previously (12, 30). Following transfection (24 h), the medium was replaced with fresh medium containing 20 µCi/ml [6-3H]fucose. After 48 h, the medium was collected, and the fragments were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography as described previously (30). Western blots and fluorography were then performed as described (3). O-Fucose saccharides on the fragments were released by alkali-induced beta-elimination and analyzed by gel filtration chromatography and high pH anion exchange chromatography as described (3, 9).


Figure 1
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  		FIGURE 1.
TSP1-TSR3 expressed in Lec1-CHO cells is modified with Glc-beta1,3-Fuc. Lec1-CHO cells were transiently transfected with constructs encoding wild type or T489A mutant of the third TSR repeat from human thrombospondin 1 (TSP1-TSR3), followed by metabolic radiolabeling of the cells with [3H]fucose. Secreted proteins were purified from media using Ni-NTA chromatography as described under "Experimental Procedures." A, wild type and T489A mutant TSP1-TSR3 were detected by immunoblot using anti-MYC antibody, and the [3H]fucose labeling level of proteins was detected by fluorography. B, the [3H]fucose-labeled O-linked saccharides were released by alkali-induced beta-elimination from affinity purified wild type TSP1-TSR3 and analyzed by gel filtration to determine the size. Elution positions of partially hydrolyzed dextran standard (in glucose units) are indicated by diamonds at the top of the graph (12). The eluted fractions corresponding to disaccharide (peak, highlighted by a circle) and monosaccharide (shoulder, highlighted by a circle) were then pooled and further analyzed by high-pH anion exchange chromatography (C). Samples from the disaccharide are indicated by diamonds, and those from the shoulder are indicated by squares. Elution positions of several sugar standards are indicated by inverted triangles.

 
Production of Recombinant Human TSP1-TSR3 Repeat and O-Fucose Site Mutants in E. coli—TSP1-TSR3 was expressed in the BL21(DE3) E. coli strain using the pET20b+-hTSP1-TSR3 vector, and the TSP1-TSR3 protein was partially purified using Ni-NTA-agarose essentially as described previously (25). To identify the properly folded TSP1-TSR3 repeat, a portion of the partially purified TSP1-TSR3 repeat was radiolabeled using detergent-containing extracts of Lec1-CHO cells (source of TSR-specific O-fucosyltransferase) using O-fucosyltransferase assay conditions as described (15, 31). Reverse-phase HPLC was performed to separate the properly folded TSP1-TSR3 repeats from misfolded variants following conditions described in Ref. 25. We have used the same conditions to purify recombinant EGF repeats in the past, and the properly folded form was identified by the presence of [3H]fucose. The remainder of the TSP1-TSR3 repeat was then purified in the same manner on a preparative scale. The final concentration of TSP1-TSR3 was determined by a BCA assay (Pierce). The TSP1-TSR3 repeats containing the T489A mutation were expressed and purified in the same manner. Reduction and alkylation of TSRs were carried out as reported previously (25).


Figure 2
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  		FIGURE 2.
TSR O-fucosyltransferase activity in Lec1-CHO extracts is dependent on the concentration of properly folded TSP1-TSR3. A, increasing amounts of bacterially expressed TSP1-TSR3 (wild type or T489A mutant) were incubated for 30 min with constant amounts (~8 µg) of Lec1-CHO lysate and GDP-[3H]fucose in in vitro O-fucosyltransferase assays as described under "Experimental Procedures." B, after reduction and alkylation (see "Experimental Procedures"), TSP1-TSR3 was purified through reverse phase HPLC. Control TSP1-TSR3 from a reaction without reducing/alkylating reagents was also purified. O-Fucosyltransferase assays were performed as in panel A with control and reduced/alkylated TSP1-TSR3 as acceptor substrates and Lec1-CHO cell lysates as enzyme source.

 
O-Fucosyltransferase Assays—The POFUT1 assays were performed as described previously (32). The assay for TSR O-fucosyltransferase activity is essentially the same with the POFUT1 assay except that recombinant TSR instead of the EGF repeat was used as acceptor substrate. A typical assay was carried out by incubating 8 µg of recombinant TSR with 0.1 µCi of GDP-[3H]fucose, 50 mM imidazole-HCl (pH 7.0), 50 mM MnCl2, and enzyme sources in a total volume of 50 µlat37 °Cfor 1 h. Product characterization of fucosylated TSRs from the assay was performed as described (15).


Figure 3
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  		FIGURE 3.
Product characterization demonstrates fucose is covalently attached to TSP1-TSR3 through an O-linkage. A, TSP1-TSR3 was labeled with [3H]fucose as described in the legend to Fig. 2 and purified using reverse phase HPLC. The arrow indicates the elution position of TSP1-TSR3 based on absorbance at 214 nm. B, alkali-induced beta-elimination was performed to release the glycans from radiolabeled TSP1-TSR3 and the product was analyzed by gel filtration chromatography. The diamonds indicate the elution positions of the partially hydrolyzed dextran standards (in glucose units) (12). C, the monosaccharide from panel B was further analyzed by high-pH anion exchange chromatography and identified as fucitol. The migration positions of sugar standards are indicated by arrows.

 
Reduction of Endogenous POFUT1 by RNA Interference in HeLa Cells—A 21-nucleotide siRNA duplex with 3'-TT (sense strand) and 3'-TC (antisense strand) overhangs corresponding to the human POFUT1 mRNA-translated region (GGAUUUCAUGGAGAAGCUG) was synthesized (Ambion). A "scrambled" siRNA duplex (Ambion) was used as the RNA interference specificity control. The siRNA duplexes were transfected into HeLa cells on 35-mm dishes by using Oligofectamine (Invitrogen) following the manufacturer's protocol. Following transfection (48 h), the media were removed and the cells were washed three times with Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 0.15 M NaCl). The cells on the dishes were then lysed in Tris-buffered saline with 1% (w/v) Nonidet P-40 and protease inhibitor mixtures (Roche). Cell debris were removed by centrifugation. POFUT1 activity and TSR-specific O-fucosyltransferase activity in the supernatants were then determined using the assays as described above.


Figure 4
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  		FIGURE 4.
A novel O-fucosyltransferase distinct from POFUT1 adds O-fucose to TSR repeats. A, O-fucosyltransferase assays were performed with recombinant POFUT1, GDP-[3H]fucose, and ~20 µM of either recombinant Factor VII EGF repeat 1 (EGF) or TSP1-TSR3 (TSR). An assay without either acceptor substrate was performed as the control. B, O-fucosyltransferase assays using ~20 µM factor VII EGF repeat 1 (EGF) or TSP1-TSR3 (TSR) as acceptor substrates were performed with extracts from control and POFUT1 siRNA-treated HeLa cells.

 
In Vitro Fucosylation of TSR and Mass Spectrometry Analysis—HPLC purified recombinant TSP1-TSR3 was reconstituted in water to a concentration ~0.5 mg/ml. In vitro O-fucosylation reaction was carried out by incubating ~0.35 mg of TSP1-TSR3 with 0.6 mM GDP-fucose, 50 mM imidazole-HCl (pH 7.0), 50 mM MnCl2, and 0.25 mg of high speed supernatants of sonicated Lec1-CHO cells in a total volume of 1 ml at 37 °C for 12 h. The reaction was then continued for 4 more h after adding 0.12 mg of fresh Lec1-CHO lysate. Fucosylated TSRs were then re-purified using reverse-phase HPLC as described above. The extent of O-fucosylation was determined by mass spectrometry using direct infusion into an Agilent XCT ion trap mass spectrometer as described previously (29).

Other Methods—beta4GalT and beta1,3-glucosyltransferase assays were performed as described previously (12, 33). Fringe assays were performed as described previously (29), except that the assays with Manic fringe contained five times more protein than those with Lunatic or Radical fringe to incorporate sufficient radioactivity for the comparisons. Soluble and membrane fractions of COS1 cells were prepared as described (28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lec1-CHO Cells Possess an Enzymatic Activity Capable of O-Fucosylating the Third TSR of Human Thrombospondin 1—We previously described two different elongated forms of O-fucose on proteins isolated from Lec1-CHO cells: the tetrasaccharide, NeuAc-{alpha}2,3-Gal-beta1,4-GlcNAc-beta1,3-Fuc-O-Ser/Thr, and the disaccharide, Glc-beta1,3-Fuc{alpha}1-O-Ser/Thr (9). Lec1-CHO cells are a convenient model system for the study of O-fucose glycans because they lack N-acetylglucosaminyltransferase I and, therefore, cannot synthesize complex or hybid-type N-glycans (34, 35). Because most [3H]fucose becomes metabolically incorporated into complex or hybrid-type N-glycans in wild type CHO cells, Lec1-CHO cells have the advantage that the majority of [3H]fucose is metabolically incorporated into O-fucose glycans (9, 36). At the time of our earlier studies, O-fucose was only known to exist on EGF repeats (2). As a result, we proposed that both the di- and tetrasaccharides would modify EGF repeats. We later discovered that the tetrasaccharide exists on EGF repeats of the Notch1 protein (3). Subsequently, Hofsteenge and co-workers (22, 23) reported an O-linked disaccharide, Glc-Fuc-O-Ser/Thr, on TSR repeats from platelet thrombospondin 1, properdin, recombinant thrombospondin 1-TSRs expressed in insect cells, and recombinant F-spondin expressed in COS1 cells. These results suggested that the Glc-beta1,3-Fuc disaccharide found in Lec1-CHO cells may actually exist on TSR repeats, whereas the tetrasaccharide form of O-fucose exist on EGF repeats.

To confirm this speculation, a secreted form of the third TSR of human thrombospondin 1 (TSP1-TSR3) was expressed in Lec1-CHO cells. As a control, the O-fucose site (Thr489) was abolished by site-directed mutagenesis of threonine to alanine. Wild type and mutant constructs were transiently transfected into Lec1-CHO cells, followed by metabolic radiolabeling with [3H]fucose. The secreted wild type and T489A mutants were purified from media by Ni-NTA-agarose and analyzed by Western blot and fluorography. Whereas wild type TSP1-TSR3 protein was radiolabeled with [3H]fucose, the T489A mutant was not (Fig. 1A). To confirm that the fucose was O-linked to the hydroxyl group of Thr489, the O-linked sugars were released from TSP1-TSR3 using alkali-induced beta-elimination. Gel filtration analysis revealed the majority of released glycan as disaccharide, with a very small amount of monosaccharide (Fig. 1B). High-performance anion-exchange chromatography analysis showed that the disaccharide was Glc-beta1,3-fucitol and the small amount of monosaccharide was fucitol (Fig. 1C). Thus, TSRs expressed in Lec1-CHO cells are modified with the Glc-beta1,3-Fuc disaccharide. The fact that this disaccharide has never been detected on an EGF repeat isolated from Lec1-CHO cells (3, 12, 19, 30) nor from any other context (4, 5, 7, 37-39) argues that the disaccharide is specific for TSRs. The lack of any tetrasaccharide on the TSR (Fig. 1B and Ref. 22) suggests that the tetrasaccharide is specific for EGF repeats. These results also indicate that Lec1-CHO cells possess the enzymatic activity capable of adding O-fucose to Thr489 of TSP1-TSR3. Thus, extracts of these cells can be used as enzyme sources to develop an in vitro assay using a bacterially expressed (unfucosylated) TSP1-TSR3 as acceptor substrate.

Development of an in Vitro Assay for O-Fucosylation on TSP1-TSR3—To develop an in vitro assay for the enzymatic activity responsible for O-fucosylating TSRs, we utilized GDP-[3H]fucose as the donor substrate and bacterially expressed recombinant TSP1-TSR3 as the acceptor substrate. The product of the assay was then separated from the unincorporated radiolabel using a C18 cartridge. Using extracts of Lec1-CHO cells as the enzyme source, O-fucosyltransferase activity showed approximately linear dependence with respect to the amount of added TSP1-TSR3. Moreover, a recombinant TSP1-TSR3 with a mutated O-fucosylation site (T489A) did not serve as substrate (Fig. 2A). Thus, bacterially expressed TSP1-TSR3 functions as an acceptor substrate for the Lec1-CHO O-fucosyltransferase in vitro.

Product analysis was performed to demonstrate that TSP1-TSR3 is modified with O-fucose. Reverse-phase HPLC analysis was used to demonstrate that the fucose was covalently associated with TSP1-TSR3 (Fig. 3A). To demonstrate that the monosaccharide fucose was attached through an O-linkage, alkali-induced beta-elimination was performed. The released sugar product from the beta-elimination migrated as a monosaccharide on gel filtration chromatography (Fig. 3B). Analysis of the monosaccharide by high-pH anion exchange chromatography revealed it to be fucitol, the expected product from beta-elimination of O-fucose (Fig. 3C). These results indicate that the TSP1-TSR3 product of in vitro O-fucosylation assays is modified with the monosaccharide form of O-fucose.

POFUT1 Is Not Responsible for O-Fucosylation of TSP1-TSR3—POFUT1 is known to add O-fucose to EGF repeats and to be present in Lec1-CHO cells (15). To investigate whether POFUT1 is also responsible for O-fucosylation of TSP1-TSR3, we performed in vitro assays using recombinant human POFUT1 as enzyme source and recombinant, bacterially expressed TSP1-TSR3 and Factor VII EGF1 repeat as substrates. As expected, recombinant POFUT1 was able to O-fucosylate Factor VII EGF1 (Fig. 4A). In contrast, no O-fucosylation of TSP1-TSR3 was detected. This result strongly suggests that TSR O-fucosyltransferase is a distinct enzyme from POFUT1. To add further support to this claim, we knocked down POFUT1 activity in HeLa cells using siRNA (Fig. 4B). Whereas POFUT1 activity in extracts of these cells (measured using factor VII EGF repeat as substrate) is reduced by ~40% compared with the control, TSR O-fucosyltransferase activity (using TSP1-TSR3 as substrate) decreases only slightly. We were unable to completely eliminate POFUT1 activity, probably because of the limited efficacy of RNA interference oligonucleotides used in the experiments or limited transfection efficiency. Nonetheless, the fact that TSR O-fucosyltransferase activity is unaffected by POFUT1 knockdown strongly suggests that a novel O-fucosyltransferase distinct from POFUT1 is responsible for O-fucosylation of TSRs.


Figure 5
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  		FIGURE 5.
Endogenous TSR O-fucosyltransferase is a soluble protein. High speed supernatant and pellet were generated from sonicated COS1 cells as described under "Experimental Procedures." Both fractions were assayed for TSR O-fucosyltransferase (A) and beta4GalT (B) activity.

 
The TSR O-Fucosyltransferase Requires Correctly Folded TSP1-TSR3 as Substrate—Previous studies on POFUT1 demonstrated that the EGF repeat needs to be properly folded for the enzyme to recognize the consensus site (14). To examine whether the TSR O-fucosyltransferase also requires a folded substrate, TSP1-TSR3 was denatured in urea, and reduced and alkylated using dithiothreitol and iodoacetamide. Control TSP1-TSR3 was treated with urea but not with dithiothreitol or iodoacetamide. Both were re-purified by reverse-phase HPLC, where the reduced and alkylated form migrated slightly later because of the carboxyamidomethylation (data not shown). Both the native and reduced/alkylated TSP1-TSR3 were analyzed as substrates in the O-fucosyltransferase assay, where only the native TSP1-TSR3 repeat was functional. The reduced and alkylated TSP1-TSR3 did not serve as a substrate (Fig. 2B). These results indicate that like POFUT1, the TSR O-fucosyltransferase requires a correctly folded TSP1-TSR3 repeat for recognition.


Figure 6
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  		FIGURE 6.
Lunatic, Radical, and Manic fringe do not modify O-fucose on TSRs, and the fucose-specific beta1,3-glucosyltransferase does not modify O-fucose on EGF repeats. A, electrospray mass spectral analysis of unmodified (top panel) and O-fucosylated (bottom panel) TSP1-TSR3. Charge state and m/z for the most abundant species are shown. Md, deconvoluted mass. B, TSR-O-fucose (~25 µM) and EGF26-O-fucose (~25 µM) were analyzed in GlcNAc transferase assays with affinity purified recombinant mouse Lunatic fringe, Radical fringe, or Manic Fringe as enzyme source, and in beta1,3-glucosyltransferase assays using detergent containing extracts of Lec1-CHO as the enzyme source.

 
Endogenous TSR O-Fucosyltransferase Is a Soluble Protein—Recent studies have revealed that O-fucosyltransferase 1 is a soluble enzyme localized to the endoplasmic reticulum (21, 28). To investigate whether TSR O-fucosyltransferase is a soluble or membrane-bound protein, COS1 cells were disrupted by sonication and subjected to ultracentrifugation to separate soluble (high-speed supernatant) and membrane (high-speed pellet) fractions. The activity of TSR O-fucosyltransferase was assayed in both fractions. The majority of the activity was found in the soluble fraction (Fig. 5A). As a control, we also compared the enzyme activity of a known membrane-bound protein, beta4GalT, and found the majority of its activity in the membrane fraction (Fig. 5B). These results strongly suggest that like POFUT1, the TSR O-fucosyltransferase is a soluble protein.

There Is No Cross-talk between EGF Repeat and TSR O-Fucosylation Pathways—The fact that O-fucose occurs in two contexts, EGF repeats and TSRs, raises the question of whether enzymes capable of modifying O-fucose can do so in either context. The enzymes responsible for modifying O-fucose on EGF repeats and TSRs, the beta1,3-GlcNAc transferases of the Fringe family and the beta1,3-glucosyltransferase, respectively, both use low molecular weight acceptors such as p-nitrophenol-{alpha}-L-fucose as substrate (12, 29, 33). From prior studies, it is not clear whether the underlying protein plays a significant role in substrate recognition. To examine whether the EGF repeat or the TSR determines specificity for these enzymes, O-fucosylated EGF repeat and TSR were generated for use in in vitro assays using either one of the Fringe enzymes (Lunatic, Manic, or Radical) or the fucose-specific beta1,3-glucosyltransferase activity of CHO cell extracts. We have previously generated O-fucosylated EGF repeat 26 from mouse Notch1 and shown it to be an excellent in vitro substrate for the Fringes (29). To prepare O-fucosylated TSR, bacterially expressed TSP1-TSR3 was incubated with non-radioactive GDP-fucose and a source of TSR O-fucosyltransferase (high speed supernatants of sonicated Lec1-CHO cells, see "Experimental Procedures" for details). The TSR was re-purified by reverse-phase HPLC and analyzed by electrospray mass spectrometry to demonstrate modification (Fig. 6A). Based on this analysis, the O-fucosylated TSP1-TSR3 was nearly completely modified with O-fucose. TSP1-TSR3-O-fucose and EGF26-O-fucose were then used as acceptor substrates in in vitro assays for either one of the Fringes or the fucose-specific beta1,3-glucosyltranferase. As expected, EGF26-O-fucose was an excellent substrate for all three Fringes, whereas TSR-O-fucose was not (Fig. 6B). This indicated that none of the Fringes add GlcNAc to the O-fucose on TSRs. To determine whether EGF-O-fucose could be modified by a beta1,3-glucose, detergent containing extracts of Lec1-CHO cells were incubated with UDP-[3H]glucose as donor substrate and either EGF-O-fucose or TSR-O-fucose. Consistent with our previous findings, extracts of Lec1-CHO extracts possess a fucose-specific beta1,3-glucosyltransferase activity (33), although it was capable of modifying O-fucose on TSR but not on EGF (Fig. 6B). In addition, Lunatic fringe did not display any glucosyltransferase activity in the presence of high concentrations of TSR-O-fucose or EGF-O-fucose (data not shown). Taken together, these results suggest that the Fringe enzymes modify O-fucose exclusively in the context of EGF repeats, whereas the fucose-specific beta1,3-glucosyltransferase modifies O-fucose exclusively in the context of TSR. These results add further support to the contention that GDP-fucose is the only common factor between the O-fucosylation pathways for modification of EGF repeats and TSRs.


Figure 7
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  		FIGURE 7.
Two distinct pathways for O-fucosylation of EGF repeats and TSRs. The flowchart shows biosynthesis pathways of O-linked fucose containing saccharide structures on EGF repeats and TSRs. Details can be found under "Discussion." Adapted from Ref. 40.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated the existence of two distinct O-fucosylation pathways (Fig. 7). One modifies EGF repeats and can extend to the tetrasaccharide NeuAc-{alpha}2,3/6-Gal-beta1,4-GlcNAc-beta1,3-Fuc, whereas the other modifies TSRs in CHO cells and can extend to the disaccharide Glc-beta1,3-Fuc. We have also shown that a novel protein O-fucosyltransferase activity is responsible for addition of O-fucose to TSRs. Despite several similar characteristics between this enzymatic activity and POFUT1, we found that the TSR O-fucosyltransferase is not POFUT1. Finally, we determined that there is no cross-talk between the two O-fucosylation pathways. Each of the Fringe enzymes adds GlcNAc to O-fucose on EGF repeats but not to O-fucose on TSRs. Similarly, the fucose-specific beta1,3-glucosyltransferase modifies O-fucose on TSRs but not EGF repeats. This confirms our hypothesis that EGF-specific and TSR-specific glycosylation pathways exist (40). These results indicate that all of these enzymes (POFUT1, the Fringe enzymes, TSR O-fucosyltransferase, and fucose-specific beta1,3-glucosyltransferase) have the ability to specifically recognize either EGF repeats or TSRs.

Although a great deal is known about the function of O-fucose on EGF repeats, little is known about O-fucose on TSRs. To date only a few proteins have been shown to contain O-fucosylated TSRs. By comparing the protein sequence surrounding O-fucose sites, Hofsteenge and co-workers (23) have suggested a putative consensus sequence for TSR O-fucosylation: WX5C1X2-3S/TC2X2G (O-fucose site is underlined, C and C2 are the first and second conserved cysteine of the TSR, and X represents any amino acid) (23). Using this pattern as a query, a search of the Swiss-Prot/TrEMBL data base revealed a number of proteins with potential TSR O-fucose consensus sequences from various species including human, mouse, rat, Drosophila, Caenorhabditis elegans, and malaria parasite Plasmodium falciparum (several examples from mouse and human genomes are listed in Table 1). These results suggest that O-fucose may occur on TSR repeats from a variety of proteins across many species.


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  		TABLE 1
Examples of proteins containing TSR O-fucosylation Proteins that have been identified to contain TSR O-fucosylation are listed with references. Proteins containing a consensus sequence within a TSR were identified by searching Swiss-Prot and TrEMBL databases at the ScanProsite website using the query pattern: WX(5)CX(2,3)S/TCX(2)G (23). A partial list of the results is shown.

 
Recent studies on the role of O-fucose on EGF repeats may provide insights into the potential function for O-fucose on TSRs. The most striking example of the effects of O-fucose on EGF repeats is the Notch signaling pathway. The extracellular domain of Notch consists mainly of 36 tandem EGF repeats, many of which are modified with O-fucose (3). Recent studies have shown that mutation of highly conserved O-fucose sites at EGF repeats 12, 26, and 27 affect mouse Notch1 activation (19). The fact that EGF repeat 12 is known to function in ligand-binding suggests that the fucose may play a role in interaction with ligands (41). Modification of O-fucose with a GlcNAc by Fringe sensitizes Notch to Delta signaling while desensitizing it to Serrate/Jagged signaling (10, 11). Several recent studies suggest that the modification of O-fucose by Fringe directly affects ligand binding (13, 20, 42, 43). These findings indicate that O-fucose modifications on a small cysteine-knot motif can affect receptor-ligand interactions.

Similar to EGF repeats, TSRs participate in a wide variety of physiological events (24). Their functions have been most extensively studied in the TSP. Thrombospondins are extracellular matrix glycoproteins involved in cell-cell and cell-matrix adhesion (44). Both TSP1 and -2 have three TSRs. The TSRs are known to mediate physical interaction between TSP1 and a variety of receptors, including CD36, heparan sulfate, and fibronectin (24). TSP1 and -2 are known to be inhibitors of tumor growth and angiogenesis (44). Interestingly, the anti-angiogenesis activity appears to be mediated by the interaction of TSRs of TSP1 or -2 with CD36 on endothelial cells (45). This interaction initiates an apoptotic cascade that is responsible for the anti-angiogenic effect (46). Because changes in the structure of O-fucose saccharides on EGF repeats appears to be able to modulate protein-protein interactions in the case of Notch and its ligands (20), it will be very interesting to see if O-fucose on TSR of TSP1 modulates the interaction with CD36.

An interesting aspect of O-fucosylation on TSR repeats is that a glucose can be added in a beta-linkage to the 3'-hydroxyl group of fucose to form a disaccharide (22). This elongation is reminiscent of the elongation of O-fucose on EGF repeats by Fringe. However, the O-fucose glycan on EGF can be further elongated to a tetrasaccharide, whereas O-fucose glycan on TSR repeats remains a disaccharide. We previously characterized an UDP-glucose: O-linked fucose beta1,3-glucosyltransferase activity capable of forming this unique linkage (33). It is a soluble enzyme present in extracts of cultured cells from a variety of species. Intriguingly, this glucosyltransferase activity displayed activity only toward TSR-O-fucose and not toward EGF-O-fucose (Fig. 6B), indicating that this activity can differentiate the underlying protein structure. Because Fringe modulates Notch signaling, it will be interesting to see how modification by this novel glucosyltransferase affects the functions of proteins with O-fucosylated TSRs.


   FOOTNOTES
 
* This work was supported by a grant from the Mizutani Foundation for Glycoscience and National Institutes of Health Grant GM 61126. 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 To whom correspondence should be addressed. Tel.: 631-632-7336; Fax: 631-632-8575; E-mail: rhaltiwanger{at}ms.cc.sunysb.edu.

2 The abbreviations used are: EGF, epidermal growth factor-like; TSR, thrombospondin type 1 repeat; TSP, thrombospondin; POFUT1, human protein O-fucosyltransferase-1; OFUT1, Drosophila protein O-fucosyltransferase-1 (Drosophila); CHO, Chinese hamster ovary; Gal, galactose; GlcNAc, N-acetylglucosamine; NeuAc, N-acetylneuraminic acid; HPLC, high performance liquid chromatography; Ni-NTA, nickel-nitrilotriacetic acid; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Deane Mosher (University of Wisconsin) for provision of the plasmid encoding TSR1-3 from human thrombospondin 1, Dr. Vlad Panin (Texas A&M) and Dr. Pamela Stanley (Albert Einstein College of Medicine) for helpful discussions, and members of the Haltiwanger laboratory for critically reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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