The C-terminal N-glycosylation sites of the human {alpha}1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V and -VI) are necessary for the expression of full enzyme activity

Lise Lotte Christensen, Uffe Birk Jensen2, Peter Bross3 and Torben Falck Ørntoft1

Laboratory of Molecular Diagnostics, Department of Clinical Biochemistry, Skejby University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, Denmark, 2Institute of Human Genetics, University of Aarhus, 8000 Aarhus C, Denmark, and 3Research Unit for Molecular Medicine, University of Aarhus, Skejby University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, Denmark

Received on February 4, 2000; revised on March 12, 2000; accepted on March 18, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 

The {alpha}1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human {alpha}1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to ~40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Key words: fucosyltransferases/glycoprotein/N-glycosylation/site-directed mutagenesis


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fucosyltransferases are involved in the final step in the synthesis of a wide spectrum of fucosylated cell surface glycoconjugates. Fucosylated glycoconjugates play important roles in embryogenesis, lymphocyte trafficking, inflammation, cancer metastasis and the synthesis of blood group antigens (Mollicone et al., 1992Go; McEver et al., 1995Go; Maly et al., 1996Go; McEver, 1997Go; Kim and Varki, 1997Go; Watkins, 1980Go) .

The {alpha}1,3/4-fucosyltransferases (FucTs) constitute a group of fucosyltransferases with a high degree of sequence similarity. The FucTs catalyze the transfer of fucose from GDP-fucose to sub-terminal and/or internal N-acetylglucosamine residues on glycoproteins and glycolipids. Six human FucTs (hFucTs) have been cloned (Goelz et al., 1990Go; Kukowska et al., 1990Go; Kumar et al., 1991Go; Koszdin and Bowen, 1992Go; Weston et al., 1992aGo,b; Natsuka et al., 1994Go; Sasaki et al., 1994Go; Kaneko et al., 1999Go). They are all type II membrane glycoproteins sharing the same general domain structure as other glycosyltransferases, i.e., a short amino-terminal cytoplasmic tail, a transmembrane domain, a luminal stem region, and a long luminal C-terminal catalytic domain. In addition all the hFucTs have potential N-glycosylation sites within their catalytic domains.

Human {alpha}1,3/4-fucosyltransferase III (hFucTIII), also named the Lewis transferase, is responsible mainly for the synthesis of the Lea and Leb antigens and the E-selectin ligand sLea. Human FucTIII is composed of 361 amino acid residues. The catalytic domain has two potential N-glycosylation sites at Asn154 and Asn185. The two N-glycosylation sites of hFucTIII are conserved in human {alpha}1,3-fucosyltransferase V (hFucTV) (Asn167 and Asn198) and human {alpha}1,3-fucosyltransferase VI (hFucTVI) (Asn153 and Asn184), the two fucosyltransferases showing the highest sequence similarity to hFucTIII (hFucTV 89% and hFucTVI 84%). Both hFucTV and -VI contain two additional potential N-glycosylation sites compared to hFucTIII, which are both situated closer to the N-terminal of the proteins (Figure 1).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Schematic presentation of the hFucTIII, -V and -VI proteins. All three fucosyltransferases contain a short amino terminal cytoplasmic tail, a single transmembrane domain (solid bars), a luminal stem region and a luminal C-terminal catalytic domain. Potential N-linked glycosylation sites in the catalytic domains are indicated with asterisks and their respective amino acid positions are given below.

 
The role of N-glycosylation sites and their glycosylation has been studied in several glycosyltransferases and it seems that their role vary from protein to protein. As for other glycoproteins it has been shown that N-linked glycans are required for full biological activity of some glycosyltransferases (e.g., {alpha}2,6-sialyltransferase [ST6Gal I], rat UDP-N-acetylglucosamine: ß-D-mannoside ß-1,4N-acetylglucosaminyltransferase III [GnTIII], human core 2 ß-1,6-N-acetyl-glucosaminyltransferase [C2GnT] and chicken GD3synthase [ST8Sia I]) (Fast et al., 1993Go; Nagai et al., 1997Go; Toki et al., 1997Go; Martina et al., 1998Go). However, in other glycosyltransferases site-directed mutagenesis of the N-glycosylation sites, had no apparent effect on catalytic activity (e.g., human ß-1,4-galactosyltransferase [Gal-T] and bovine UDP-N-acetylglucosamine:{alpha}1,3-D-mannoside ß1,4-N-acetylglucosaminyltransferase [GnT-IV]) (Malissard et al., 1996Go; Minowa et al., 1998Go). Furthermore, the N-linked glycans are involved in correct protein folding, prevention of proteolytic degradation and intracellular transport (Haraguchi et al., 1995Go; Malissard et al., 1996Go; Nagai et al., 1997Go; Toki et al., 1997Go; Martina et al., 1998Go).

The role of the potential N-glycosylation sites in the group of hFucTs has only been evaluated for some of the sites in hFucTV and -VI. Elimination of potential N-glycosylation sites in hFucTV (N105H) and hFucTVI (N46S and N91H) had no influence on acceptor substrate specificity (Nguyen et al., 1998Go; Legault et al., 1995Go). Furthermore, Kukowska et al. (1990)Go have shown that hFucTIII expressed in an in vitro translation system is glycosylated at both N-glycosylation sites.

In the present study full-length hFucTIII, -V, and -VI were expressed in COS-7 cells. The role of core glycosylation in hFucTIII was examined using the N-glycosylation inhibitor, tunicamycin (TM) and a series of mutants that lack the potential N-glycosylation sites. Furthermore, trimming of the glucose residues of the N-linked glycans in hFucTIII was inhibited using castanospermine (CS). We showed that N-linked glycosylation at both glycosylation sites and subsequent glucose trimming of the N-glycans are necessary for expression of full catalytic activity of hFucTIII. In addition, it was shown that the two N-glycosylation sites in hFucTV and -VI corresponding to the two sites in hFucTIII were necessary for expression of full catalytic activity of these fucosyltransferases. We conclude that the two conserved C-terminal N-glcosylation sites of the human {alpha}1,3/4-fucosyltransferases III, -V, and -VI are necessary for the expression of full catalytic activity.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effect of tunicamycin and castanospermine on hFucTIII wt expressed in COS-7 cells
Tunicamycin is an inhibitor of N-linked glycosylation (Elbein, 1984Go) and castanospermine, an inhibitor of glucosidase I and II, inhibits the initial trimming of the glucose residues on N-glycans (Elbein, 1991Go). COS-7 cells transfected with hFucTIII wt were treated with tunicamycin or castanospermine to study the role of core glycosylation and the subsequent glucose trimming of the N-glycans in hFucTIII. Western blotting of hFucTIII in COS-7 cells grown with or without tunicamycin/castanospermine was performed using OLI antiserum (Figure 2), a rabbit polyclonal antiserum against recombinant hFucTVI which crossreacts with hFucTIII as described by Borsig et al. (1998)Go. The wt hFucTIII migrated with an apparent molecular mass of ~42 kDa (Figure 2, lane 1), whereas the hFucTIII from tunicamycin treated cells migrated faster at ~36 kDa (Figure 2, lane 2) due to the lack of N-linked glycans. The hFucTIII from the castanospermine treated cells migrated as a polypeptide of ~43 kDa (Figure 2, lane 3), a little above the enzyme from the untreated cells due to the inhibition of glucose trimming. The steady state levels of hFucTIII in treated and untreated cells are comparable. There is a discrepancy between the observed molecular mass of the wt (~42 kDa) and the one predicted from the amino acid sequence plus two oligosaccharide chains of approximately 2–3 kDa each (~46–48 kDa). This difference between the observed molecular mass and the predicted one has also been described by Kukowska et al. (1990)Go; and Borsig et al. (1998)Go. It may be explained by the observation that membrane-spanning proteins can migrate more rapidly through SDS–polyacrylamide gels than soluble protein molecular mass markers (Mueckler et al., 1985Go; James et al., 1989Go).



View larger version (85K):
[in this window]
[in a new window]
 
Fig. 2. Western blot analysis of hFucTIII wt expression in transfected COS-7 cells with or without tunicamycin (TM) or castanospermine (CS). Tunicamycin (1 µg/ml) or castanospermine (50 µg/ml) was added to the COS-7 cells 6 h after transfection of the cells. The cells were harvested 16 h later. Lysates of transfected COS–7 cells (2 µg/lane) were separated on a 10% NuPAGE gel. The separated proteins were transferred to a PVDF membrane and immunoblotting was performed with antiserum against recombinant hFucTVI (OLI antiserum) (see Materials and methods for further details). Lane M, ECL protein molecular mass markers; lane 1, hFucTIII wt in COS-7 in the absence of inhibitor; lane 2, hFucTIII wt in COS-7 cells treated with TM; lane 3, hFucTIII wt in COS-7 cells treated with CS; and lane 4, mock-transfected COS-7 cells.

 
The {alpha}1,3/4-fucosyltransferase activity in COS-7 cells treated with tunicamycin or castanospermine is presented in Figure 3. The hFucTIII activity was completely abolished by the tunicamycin treatment. In addition, the hFucTIII activity in the castanospermine treated cells was diminished to ~40% of the activity in untreated cells. These results indicate that expression of hFucTIII enzyme activity requires core glycosylation and that trimming of the glucose residues are required for the expression of full enzyme activity.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. The {alpha}1,3/4-fucosyltransferase activity in lysates of hFucTIII wt COS-7 cells treated with tunicamycin (TM) or castanospermine (CS). Tunicamycin (1 µg/ml) or castanospermine (50 µg/ml) was added to the COS-7 cells 6 h after transfection of the cells. The cells were harvested 16 h later and the {alpha}1,3/4-fucosyltransferase activity in the cell lysates was measured. Assays were performed as described under Materials and methods. Lacto-N-biose I (LNB) and Lacto-N-fucopentaose I (LNF) were used as oligosaccharide acceptors.

 
Site-directed mutagenesis
Site-directed mutagenesis of hFUT3, hFUT5, and hFUT6 wt was carried out to study the roles of individual N-glycosylation sites. In hFucTIII two asparagine residues at amino acid 154 and 185 are potential glycosylation sites (Asn-X-Thr) (Figure 4A). Three types of hFucTIII mutants (N154Q, N185Q, and N154Q/N185Q) were generated from wt hFUT3 by site-directed mutagenesis replacing the asparagine residues in the N-glycosylation sites with glutamine (Figure 4B). Human FucTV and -VI contain four potential N-glycosylation sites (Figure 4A). The two C-terminal sites, Asn167 and Asn198 of hFucTV and Asn153 and Asn184 of hFucTVI, are homologous to the two sites in hFucTIII. Three types of hFucTV mutants (N167Q, N198Q and N167Q/N198Q) and three types of hFucTVI mutants (N153Q, N184Q and N153Q/N184Q) were generated form wt hFUT5 and hFUT6 by site-directed mutagenesis (Figure 4C and D). Sequencing of the hFUT3, hFUT5, and hFUT6 wt and mutants revealed that the mutations present in the target mutagenic primers were the only ones introduced (data not shown).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Structure of hFucTIII, -V and –VI wt and mutant constructs. The potential N-glycosylation sites of hFucTIII (Asn154 and –185), -V (Asn60, –105, –167 and –198) and -VI (Asn46, –91, –153 and –184) are localized in the catalytic domain (A). Mutant hFucTs generated in this study by site-directed mutagenesis: hFucTIII (B), hFucTV (C) and hFucTVI (D). TM, Transmembrane domain.

 
Northern blot analysis
The Northern blot analysis (Figure 5) showed that similar amounts of FUT3 transcripts (~1.3 kb) were present in COS-7 cells expressing either hFucTIII wt, N154Q, N185Q, or N154Q/N185Q (Figure 5A, lanes 1–4). The FUT3 transcript includes 5' and 3' untranslated regions from the expression vector leading to a larger transcript than the one predicted from the FUT3 insert. Mock-transfected COS-7 cells contained no FUT3 transcript (Figure 5A, lane 5). The GAPDH transcript (~1.2 kb.) was present in equal amounts in all the transfected COS-7 cells (Figure 5B, lanes 1–5). The expression of equal amounts of wt and mutant hFUT5 and hFUT6 transcripts in the COS-7 cells transfected with these constructs was also verified by Northern blotting in a similar way (data not shown).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5. Northern blot analysis of total RNA (10 µg/lane) from COS-7 cells expressing either hFucTIII wt or the mutants. The blot was probed with 32[P]-labeled FUT3 (A) and GAPDH (B) probes. Lane 1, N154Q/N185Q cells; lane 2, N185Q cells; lane 3, N154Q cells; lane 4, hFucTIII wt cells; and lane 5, mock-transfected cells. The sizes of the transcripts are shown on the right.

 
The fucosyltransferase expression level and activity of the mutant hFucTIIIs, hFucTVs, and hFucTVIs
The presence of hFucTIII translation product in COS-7 cells transfected with the wt and the three different mutants was verified by Western blotting using the OLI antiserum (Figure 6). A comparable amount of hFucTIII translation products were detected in all of the transfected COS-7 cells independent of which construct had been used for transfection (Figure 6, lanes 1–4). No translation product was detected in mock-transfected COS-7 cells (Figure 6, lane 5). The hFucTIII mutants migrated differently from the wt on SDS–PAGE, according to the number of oligosaccharide chains attached. The wt migrated at ~42 kDa (Figure 6, lane 1) whereas removal of the N-glycosylation sites resulted in a decrease in molecular mass of 2–3 kDa for the single mutants (Figure 6, lanes 2–3) and 4–6 kDa for the double mutant (Figure 6, lane 4). As expected the double mutant migrated with a mass comparable to the hFucTIII enzyme from the tunicamycin treated cells (Figure 2, lane 2; Figure 6, lane 4). In all of the COS-7 cells transfected with hFucTV and –VI wt and mutants comparable amounts of translation product was detected as was a decrease in the molecular mass of the mutants compared to the wt (data not shown). The {alpha}1,3/4-fucosyltransferase activity of the hFucTIII wt and the mutants, measured with four different substrates, is shown in Figure 7. The N154Q/N185Q double mutant exhibited no enzyme activity with any of the four oligosaccharide acceptors, corresponding to the case of the tunicamycin treated hFucTIII wt described above. The enzyme activity was also completely abolished in the N154Q transfectants whereas the N185Q transfectants exhibited ~27% of the activity of the hFucTIII wt transfectants using LNB as oligosaccharide acceptor. A similar reduction in the activity of the N185Q mutant was seen using the other three oligosaccharide acceptors. No {alpha}1,3/4-fucosyltransferase activity was detectable in mock-transfected COS-7 cells (data not shown). Furthermore, no {alpha}1,3/4-fucosyltransferase activity could be detected in either the N154Q or the N154Q/N185Q transfectants when concentrations up to 50 mM of the oligosaccharide acceptors were used (data not shown). These results demonstrate that N-glycosylation at any of the two sites in hFucTIII contributes to the expression of normal activity. However N-glycosylation at Asn154 is more essential for enzyme activity than N-glycosylation at Asn185.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 6. SDS–PAGE and Western blotting analysis of hFucTIII wt and mutants. Lysates of transfected COS–7 cells (2 µg/lane) were separated on a 10% NuPAGE gel. The separated proteins were transferred to a PVDF membrane and immunoblotting was performed with OLI antiserum (see Materials and methods for further details). Lane M, ECL protein molecular mass markers; lane 1, hFucTIII wt cells; lane 2, N154Q cells; lane 3, N185Q cells; lane 4, N154Q/N185Q cells; and lane 5, mock transfected COS-7 cells.

 


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 7. The {alpha}1,3/4-fucosyltransferase activity in the lysate of COS-7 cells expressing either hFucTIII wt or the mutants. Assays were done as described under Materials and methods. Four different oligosaccharide acceptors were used: Lacto-N-biose I (LNB), Lacto-N-fucopentaose I (LNF), N-acetyllactosamine (NAL) and 2"-Fucosyllactose (FL). The average of three independent experiments is presented for LNB and for LNF, NAL, and FL the average of two independent experiments is presented.

 
The {alpha}1,3-fucosyltransferase activity of the hFucTV and –VI wt and mutants is shown in Figure 8. The enzyme activity was completely abolished in the hFucTV N167Q and the N167Q/N198Q transfectants whereas the hFucTV N198Q transfectants exhibited ~32% of the activity of the hFucTV wt transfectants. Cells expressing the hFucTVI N153Q/N184Q mutant exhibited a very low level of enzyme activity (~5% of the hFucTVI wt activity) compared to cells expressing the hFucTVI wt. In addition, enzyme activity was detected both in cells expressing the N153Q mutant (~15% of the hFucTVI wt activity) and in cells expressing the N184Q mutant (~38% of the hFucTVI wt activity). In both cases cells expressing the hFucTVI single mutants exhibited lower enzyme activity than cells expressing hFucTVI wt and the enzyme activity in the hFucTVI N153Q transfectants was lower than in the N184Q transfectants. No {alpha}1,3-fucosyltransferase activity was detectable in mock-transfected COS-7 cells (data not shown). These results show that N-glycosylation at any of the two examined sites in hFucTV and -VI contributes to the expression of a normal level of activity. Furthermore, as in hFucTIII, N-glycosylation at Asn167 (hFucTV) and Asn153 (hFucTVI) is more essential for enzyme activity than N-glycosylation at Asn198 (hFucTV) and Asn184 (FucTVI). However, the activity in the hFucTVI N153Q transfectants was not completely abolished as in the hFucTIII N154Q transfectants and the hFucTV N167Q transfectants.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8. The {alpha}1,3-fucosyltransferase activity in the lysate of COS-7 cells expressing either hFucTV or -VI wt, or mutants. Assays were done as described under Materials and methods. N-acetyllactosamine was used as oligosaccharide acceptor. The average of three independent experiments is presented.

 
Kinetic parameters of the hFucTIII N185Q mutant
In order to examine the effect of the N185Q mutation on hFucTIII enzyme activity in detail, kinetic analysis of the hFucTIII wt and the N185Q mutant was performed. As shown in Figure 9A,B the apparent Km values of the wild type and the mutant are comparable to each other, both for the oligosaccharide acceptor (Lacto-N-biose) (A) and the nucleotide sugar donor (GDP-fucose) (B). The Km was 16 mM (wt) and 17 mM (N185Q) for Lacto-N-Biose I and 20 µM (wt) and 30 µM (N185Q) for the GDP-fucose. These results demonstrate that the N185Q mutation and hence lack of glycosylation at this site in hFucTIII has no significant effect on the affinity towards the oligosaccharide acceptor or the nucleotide sugar donor.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 9. Kinetic analysis of hFucTIII wt and the N185Q mutant. The {alpha}1,3/4-fucosyltransferase activity in the lysates of transfected COS-7 cells was measured with various concentrations of oligosaccharide acceptor (Lacto-N-biose I) (A) or nucleotide sugar donor (GDP-fucose) (B). Assays were performed as described under Materials and methods. The data are given as a Lineweaver-Burk plot. Wild type (square) and N185Q (circle).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
As a novel finding, the present study demonstrates that hFucTIII, -V and -VI requires N-glycosylation of the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

The roles of the potential N-glycosylation sites and their glycosylation in the glycosyltransferases have been analyzed in several cases (Fast et al., 1993Go; Haraguchi et al., 1995Go; Malissard et al., 1996Go; Nagai et al., 1997Go; Toki et al., 1997Go; Martina et al., 1998Go; Minowa et al., 1998Go). To investigate the role of N-glycosylation in the hFucTs we initially blocked N-glycosylation of hFucTIII with tunicamycin. Tunicamycin has been shown to inhibit the enzyme activity of other glycosyltransferases. In the present study tunicamycin treatment of COS-7 cells transfected with wt hFucTIII resulted in a decrease in the molecular mass of the enzyme corresponding to the size of two core glycosylations (4–6 kDa). The {alpha}1,3/4-fucosyltransferase activity was completely lost in the tunicamycin treated COS-7 demonstrating that hFucTIII requires N-glycosylation for expression of its activity. In addition hFucTIII wt transfected COS-7 cells were treated with castanospermine to study the role of glucose trimming in hFucTIII activity. The hFucTIII in the castanospermine treated cells migrated a little above the wt enzyme in untreated cells due to the inhibition of glucose trimming. The hFucTIII enzyme activity in the castanospermine treated cells was reduced to ~40% of the activity in untreated cells. Although required for full activity of hFucTIII, trimming of the glucose residues from the N-linked glycans is not essential for enzyme activity. This is in accordance with studies on lipoprotein lipase (LPL) in which it has been shown, using castanospermine, that glucose trimming is required only for the expression of full catalytic activity (Ben-Zeev et al., 1992Go; Masuno et al., 1992Go). Furthermore, Molinari and Helenius have shown that castanospermine strongly perturb the folding of Semlik Forest Virus (SFV) glycoproteins by blocking the binding of these glycoproteins to the ER chaperones calnexin and calreticulin. However, a fraction of the SFV glycoproteins (20%) were able to fold in the presence of castanospermine due the assistance of other ER chaperones (Molinari and Helenius, 1999Go).

The role of the potential N-glycosylation sites has only been studied poorly in the fucosyltransferases. Kukowska et al. (1990)Go have demonstrated that hFucTIII expressed in an in vitro translation system in the presence of microsomes is glycosylated at both of its potential N-glycosylation sites and that the carbohydrate residues are endoglycosidase H-sensitive (Kukowska et al., 1990Go). Legault et al., 1995Go, made a domain swap study of hFucTVI and -III to analyze the role of different sub-domains in acceptor substrate specificity (Legault et al., 1995Go). In one of the constructs hFucTVI was only altered by a single amino acid (N91H) (see Figure 1). In another construct several amino acids were altered including the potential N-glycosylation site at Asn46 (N46S) (see Figure 1). Neither of these changes in hFucTVI did affect acceptor substrate specificity. Potential N-glycosylation sites corresponding to Asn46 and Asn91 in hFucTVI are also present in hFucTV (Asn60 and Asn105) (see Figure 1). The role of Asn105 has been analyzed by Nguyen et al. (1998)Go. The hFucTV THRKT mutant, in that study, included the substitution of the potential N-glycosylation site at Asn105 with a His residue. The acceptor substrate specificity of the THRKT mutant was not altered compared to wt hFucTV. Furthermore, there was no indication that Asn105 was glycosylated when expressed in COS cells. Human FucTV and -VI contain two other N-glycosylation sites in addition to the two sites mentioned above (hFucTV; Asn167 and Asn198 and hFucTVI; Asn153 and 184). These sites are situated in the C-terminal part of the catalytic domain and are homologous to the two potential N-glycosylation sites present in hFucTIII (see Figure 1).

To evaluate the contribution of individual N-linked oligosaccharides to the expression of enzyme activity in the hFucTs, N-glycosylation mutants of hFucTIII, -V, and -VI were generated using site-directed mutagenesis. Northern and Western blotting analysis showed that both transcripts and translation products of the wt and the mutants were present in equal amounts in transfected COS-7 cells. In addition the mutants migrated differently from the wt, according to the number of oligosaccharide chains attached. Elimination of any one of the N-linked glycosylation sites in hFucTIII lead to decrease in the enzyme activity compared to the wt. However, lack of glycosylation at individual sites had different effects. Mutagenesis of hFucTIII at Asn154 and on both Asn154 and Asn185 resulted in complete loss of enzyme activity corresponding to the loss of activity in the tunicamycin treated cells and hence N-linked carbohydrates at Asn154 are indispensable for the production of a catalytically active hFucTIII. Asn167 of hFucTV and Asn153 of hFucTVI, corresponding to Asn154 of hFucTIII, are also essential for the expression of catalytic activity of these enzymes. However, a low level of activity was detected in the hFucTVI N153Q and the N153Q/N184Q transfectants. Human FucTIII share the greatest amino acid sequence similarity with hFucTV and -VI in the C-terminal region of the catalytic domain containing both of the conserved N-glycosylation sites. The common feature of all fucosyltransferases is the use of the same nucleotide sugar and hence the homologous region is likely involved in binding of the nucleotide sugar. Hydrophobic Cluster Analysis (HCA) alignment of FucTs from different species has shown that the Asn154 (hFucTIII), Asn167 (hFucTV), and Asn153 (hFucTVI) are situated in a highly homologous region, which is speculated to be a part of the nucleotide sugar binding domain of {alpha}1,3/4-fucosyltransferases (Breton et al., 1998Go). From the results in the present study one might speculate that the lack or very low level of enzyme activity in the cells transfected with the N154Q, the N167Q, the N153Q and the corresponding double mutants may be caused by their inability or highly reduced ability to bind the nucleotide sugar.

Lack of N-glycosylation at Asn185 (hFucTIII), Asn198 (hFucTV), and Asn184 (hFucTVI) resulted in a decrease in enzyme activity compared to the activity of wt transfected cells. Kinetic analysis of the hFucTIII N185Q mutant revealed that the Km values for both the oligosaccharide acceptor and the nucleotide sugar donor were similar to the 2Km values obtained with the wt. These results demonstrate that the affinity toward the acceptor and the nucleotide sugar donor is not significantly affected by the elimination of Asn185 and hence N-linked carbohydrates at this particular site may not be involved in the interaction of the enzyme with either of its two substrates. However, the N-linked carbohydrates at this site in both hFucTIII, -V, and -VI do seem to be important for the expression of full enzyme activity.

Most glycoproteins need their N-linked carbohydrates during folding in the ER, whereas a few display only partial misfolding in the absence of N-linked carbohydrates. In the latter case, a fraction of the deglycosylated proteins folds correctly and is transported normally out of the ER, whereas the rest misfolds and remains in the ER (Helenius, 1994Go; Ben-Zeev et al., 1994Go). In some glycoproteins it has been found that glycosylation at certain sites proves more important for folding than others (Roberts et al., 1993Go; Newrzella and Stoffel, 1996Go). From the results in the present study it may be hypothesized that the N-linked carbohydrates in hFucTIII and also in hFucTV and -VI are involved in the folding of the enzymes. One might speculate that N-linked glycosylation at Asn154, Asn167, and Asn153 are more important for proper folding into a catalytically active enzyme than that at Asn185, Asn198 and Asn184. In that case the hFucTIII N185Q, the hFucTV N198Q and the hFucTVI N184Q mutants only displays partial misfolding and the reduced activity in the COS-7 cells expressing these mutant may be due to activity of a minor amount of properly folded enzyme. This hypothesis is supported by the fact that the Km values of the hFucTIII N185Q mutant and the wt are the same. The hFucTIII N185Q mutant seems to be properly, or almost properly, folded since major changes in the folding of the enzyme would lead to changes in the active sites and hence change in the Km values of the enzyme. Incompletely folded proteins are often retained in the ER and degraded. In the present study there was no sign of degradation of the translation products of the mutants. However misfolded forms of some proteins e.g. ß-hexosaminidase A, form insoluble aggregates which are retained in the ER without any apparent sign of degradation (Weitz and Proia, 1992Go).

In conclusion, we demonstrate that the two conserved C-terminal N-glycosylation sites of the human {alpha}1,3/4-fucosyltransferases III, -V, and -VI are necessary for the expression of full catalytic activity. We show that N-linked glycosylation at both glycosylation sites in hFucTIII and subsequent glucose trimming of the N-glycans are necessary for expression of full catalytic activity of hFucTIII. In addition, we demonstrate that the absence of N-linked carbohydrates at Asn185 in hFucTIII did not affect the affinity towards either of the enzyme substrates. Therefore, N-linked carbohydrates at this site are not essential for the binding of either of the enzyme substrates.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Unique Site Elimination (U.S.E.) Mutagenesis Kit, ProbeQuant G-50 columns, ECL + Plus Western Blotting Detecting System and ECL protein molecular mass markers were purchased from Amersham Pharmacia Biotech. Radiochemicals [{alpha}-32P]dATP and GDP-[U-14C]fucose were from Amersham Pharmacia Biotech. The pCRII, pCR2.1-TOPO (TA cloning vectors) and the pCDNA3.1 (mammalian expression vector) were from Invitrogen. BigDye terminator cycle sequencing kit with AmpliTag polymerase FS was purchased from Perkin Elmer. Prime-It II Random Primer Labeling Kit was from Stratagene. Lacto-N-biose I (Galß1–3GlcNAc), Lacto-N-fucopentaose I (Fuc{alpha}1–2Galß1–3GlcNAcß1–3Galß1–4Glc), N-acetyllactosamine (Galß1–4GlcNAc) and 2"-Fucosyllactose (Fuc{alpha}1–2Galß1–4Glc) were obtained from Dextra Laboratories. Tunicamycin, castanospermine and FUGENE 6 Transfection Reagent were from Roche Molecular Biochemicals. The RNAzol RNA isolation system was purchased from WAK-Chemie Medical GMBH. The Zeta-Probe nylon membrane and the AG 4-X4 anion exchange resin were delivered from Bio-Rad. NuPAGE gels (10%) were purchased from NOVEX. The PVDF plus transfer membrane was from Micron separations inc. Horseradish peroxidase (HRP)-conjugated swine anti-rabbit IgG was from DAKO. The rabbit polyclonal antiserum to recombinant hFucTVI (OLI-antiserum) was kindly provided by Prof. Eric G.Berger, Institute of Physiology, University of Zürich, Switzerland. The selection primer and the target mutagenic primers were synthesized by Hobolt DNA Syntese.

Site-directed mutagenesis and cloning of the hFUT3, -5, or -6 wt and mutants
Amplification of hFUT3 ORF (open reading frame) and subsequent ligation of the hFUT3 ORF into pCRII have been described previously (Orntoft et al., 1996Go). The hFUT5 and hFUT6 ORFs were amplified by PCR from human genomic DNA and cloned into the pCR2.1-TOPO vector. The hFUT5 and hFUT6 wt were cut out of the pCR2.1 TOPO vector with EcoRI and ligated into pCDNA3.1 (a eukaryotic expression vector containing a CMV promoter) prior to the generation of the site-directed mutations. Site directed mutagenesis was performed using U.S.E. Mutagenesis Kit. The kit is based on the unique site elimination procedure developed by Deng and Nickoloff (1992)Go, which uses a two-primer system to generate site-specific mutations. The selection primer eliminates a unique non-essential restriction site in the plasmid, which subsequently serves as selection of mutated plasmids. The target mutagenic primer(s) introduces the desired mutation(s) in the gene of interest. The target mutagenic primers used to replace Asn by Gln in hFucTIII were as follows: 5'-GACAGATACTTCCAGCTCACCATGTCCTAC-3' for N154Q and 5'-GCCCACCCACCGCTCCAGCTCTCGGCCAAGAC-3' for N185Q. The selection primer used to eliminate a unique nonessential restriction site (HindIII) in the pCRII vector (hFUT3) was as follows: 5'-GAGCTCGGTACCAAGCGCGATGCATAGCTTG-3'. The target mutagenic primers used to replace Asn by Gln in hFucTV and -VI were as follows: 5'-CGGATACTTCCAGCTCACCATGTCC-3' for N167Q, 5'-CCCACCGCTCCAGCTCTCGGCCAAG-3' for N198Q, 5'-GATACTTCCAGCTCACCATGTCCTAC-3' for N153Q and 5'-CCCACCGCTCCAGCTCTCGGCCAAG-3' for N184Q. The selection primer used to eliminate a unique non-essential restriction site (Mfe1) in the pCDNA3.1 vector (hFUT5 and hFUT6) was as follows: 5'-GCAAGGCTTGACCGACGCGCGCATGAAGAATCTGC-3'. The mutations introduced by the primers are underlined. The target mutagenic primers and the selection primer were all phosphorylated at the 5' end and purified by HPLC. Inserts containing either hFUT3 wt or the mutants were cut out of the pCRII vector with EcoRI and ligated into pCDNA3.1 after generation of the site-directed mutations. To verify the presence of the mutations introduced by the U.S.E mutagenesis kit and to exclude other mutations, the different hFUT3, hFUT5, and hFUT6 inserts were sequenced in both directions using an ABI PRISM 377 DNA sequencer and the BigDye terminator cycle sequencing kit with AmpliTag polymerase FS.

Transient expression of hFucTIII, -V, and -VI wt and mutants in COS-7 cells and tunicamycin and castanospermine treatments of hFucTIII
Transfection and subsequent transient expression of hFucTIII wt and the mutants in COS-7 cells were performed using the calcium phosphate-DNA coprecipitation method described by Graham and van der Eb (Graham and van der Eb, 1973Go). Transfection and subsequent expression of the hFucTV and -VI wt and mutants were performed using FUGENE 6 Transfection Reagent. Cells were harvested approximately 40 h after transfection. The tunicamycin or castanospermine treated COS-7 cells were transfected using FUGENE 6 Transfection Reagent. Tunicamycin (1 µg/ml) or castanospermine (50 µg/ml) was added to the cells 6 h after transfection. After additional 16 h the cells were harvested.

Northern blot analysis
Total RNA was isolated from transfected COS-7 cells using a modified phenol-chloroform method, the RNAzol B method. Ten micrograms (per lane) of total RNA was subjected to electrophoresis in a 1% agarose formaldehyde gel according to standard procedure (Gething and Sambrook, 1992Go) and blotted to a nylon membrane (Zeta-Probe). A FUT3 specific probe (1125 bp) was amplified by PCR. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific probe (cDNA position 53–417, 364 bp) amplified by RT-PCR was used as control (Ercolani et al., 1988Go). Probes were labeled with [{alpha}-32P]dATP by random priming using Prime-It II Random Primer Labeling Kit and purified with a ProbeQuant G-50 column. Hybridization was carried out at 42°C for 16–20 h. Blots were subsequently washed once in 2 x SSPE, 0.05% SDS for 20 min at 25°C, once in 1 x SSPE, 0.1% SDS for 20 min at 50°C, once in 1 x SSPE, 0.1% SDS for 20 min at 60°C and once in 0.3 x SSPE, 0.1% SDS for 20 min at 65°C. Autoradiography was performed with intensifying screen at –80°C for 6 h (FUT3 probe) or 36 h (GAPDH probe).

Measurement of {alpha}1,3/4-fucosyltransferase activity
The {alpha}1,3/4-fucosyltransferase activity of hFucTIII, -V and -VI wt and mutants in transfected COS-7 cells was measured using a modification of a procedure previously published (Goelz et al., 1994Go). COS-7 cells were lysed in PBS containing 1% Triton X-100. Twenty microliters of the lysed cells was added to 80 µl of a reaction mixture containing 25 mM Tris–HCl (pH 7.2), 0.5% Triton X-100, 10 mM MnCl2, 5 mM ATP, 2,5 µM GDP-[U-14C]fucose (~150.000 c.p.m.) and 5 mM Lacto-N-biose I or N-acetyllactosamine or 1 mM Lacto-N-fucopentaose I or 2'-Fucosyllactose (oligosaccharide acceptors). The reaction mixture was incubated at 37°C for 30 min. To stop the reaction 1 ml of cold water was added and the entire solution was added to a 2 ml column packed with anion exchange resin (AG 4-X4). The column was washed with 5 ml of water and incorporation of [14C]fucose was determined by liquid scintillation counting of the flow-through fraction. Enzyme kinetics was determined by measuring initial velocity (10 min) for both Lacto-N-biose I and GDP-fucose.

SDS–PAGE and Western blot analysis
Cell lysates (~2 µg of protein) were heated to 70°C in SDS-sample buffer with 5% mercaptoethanol and electrophoresed on a 10% NuPAGE gel. Subsequently the separated proteins were electrophoretically transferred to a PVDF-plus membrane at 200 mA for 1 h. Nonspecific binding sites on the PVDF membrane were blocked with 2% BSA and 0.1% Tween in PBS (phosphate-buffered saline). Human FucTIII was detected using the OLI antiserum (1:300 dilution) (Borsig et al., 1998Go) followed by HRP-conjugated swine anti-rabbit IgG (1:10,000 dilution). The immunoreactive bands were visualized with ECL + Plus Western Blotting Detection System.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are especially grateful to Susanne Bruun and Anette Thomsen for skillful technical assistance and to Bjarne Jochimsen for helpful discussions concerning the kinetic analysis. We thank Prof. Eric G.Berger for the generous gift of OLI antiserum. This work was supported by grants from the Research Initiative of Aarhus Council, the University of Aarhus, Frits, Georg og Marie Cecilie Glud’s Legat, MC og JK Moltums fond and Købmand M.Kristjan Kjær og Hustru Margrethe Kjær født La Cour-Holmens Fond.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FucTs, {alpha}1,3/4-fucosyltransferases; hFucTs, human {alpha}1,3/4-fucosyltransferases; hFucTIII, human {alpha}1,3/4-fucosyltransferase III; hFucTV, human {alpha}1,3-fucosyltransferase V; hFucTVI, human {alpha}1,3-fucosyltransferase VI; TM, tunicamycin; CS, castanospermine; wt, wild type; HRP, horseradish peroxidase; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ben-Zeev,O., Doolittle,M.H., Davis,R.C., Elovson,J. and Schotz,M.C. (1992) Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment. J. Biol. Chem., 267, 6219–6227.[Abstract/Free Full Text]

Ben-Zeev,O., Stahnke,G., Liu,G., Davis,R.C. and Doolittle,M.H. (1994) Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme. J. Lipid Res., 35, 1511–1523.[Abstract]

Borsig,L., Katopodis,A.G., Bowen,B.R. and Berger,E.G. (1998) Trafficking and localization studies of recombinant {alpha}1,3-fucosyltransferase VI stably expressed in CHO cells. Glycobiology, 8, 259–268.[Abstract/Free Full Text]

Breton,C., Oriol,R. and Imberty,A. (1998) Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology, 8, 87–94.[Abstract/Free Full Text]

Deng,W.P. and Nickoloff,J.A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem., 200, 81–88.[ISI][Medline]

Elbein,A.D. (1984) Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. CRC Crit. Rev. Biochem., 16, 21–49.[ISI][Medline]

Elbein,A.D. (1991) Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing. FASEB J., 5, 3055–3063.[Abstract/Free Full Text]

Ercolani,L., Florence,B., Denaro,M. and Alexander,M. (1988) Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J. Biol. Chem., 263, 15335–15341.[Abstract/Free Full Text]

Fast,D.G., Jamieson,J.C. and McCaffrey,G. (1993) The role of the carbohydrate chains of Gal ß-1,4-GlcNAc {alpha}2,6-sialyltransferase for enzyme activity. Biochim. Biophys. Acta, 1202, 325–330.[ISI][Medline]

Gething,M.J. and Sambrook,J. (1992) Protein folding in the cell. Nature, 355, 33–45.[ISI][Medline]

Goelz,S.E., Hession,C., Goff,D., Griffiths,B., Tizard,R., Newman,B., Chi-Rosso,G. and Lobb,R. (1990) ELFT: a gene that directs the expression of an ELAM-1 ligand. Cell, 63, 1349–1356.[ISI][Medline]

Goelz,S.E., Kumar,R., Potvin,B., Sundaram,S., Brickelmaier,M. and Stanley,P. (1994) Differential expression of an E-selectin ligand (SLex) by two Chinese hamster ovary cell lines transfected with the same {alpha} (1,3)-fucosyltransferase gene (ELFT). J. Biol. Chem., 269, 1033–1040.[Abstract/Free Full Text]

Graham,F.L. and van der Eb,A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology, 52, 456–467.[ISI][Medline]

Haraguchi,M., Yamashiro,S., Furukawa,K., Takamiya,K. and Shiku,H. (1995) The effects of the site-directed removal of N-glycosylation sites from ß-1,4-N-acetylgalactosaminyltransferase on its function. Biochem. J., 312, 273–280.[ISI][Medline]

Helenius,A. (1994) How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol. Biol. Cell, 5, 253–265.[ISI][Medline]

James,D.E., Strube,M. and Mueckler,M. (1989) Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature, 338, 83–87.[ISI][Medline]

Kaneko,M., Kudo,T., Iwasaki,H., Ikehara,Y., Nishihara,S., Nakagawa,S., Sasaki,K., Shiina,T., Inoko,H., Saitou,N. and Narimatsu,H. (1999) {alpha}1,3-Fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX. FEBS Lett., 452, 237–242.[ISI][Medline]

Kim,Y.J. and Varki,A. (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj. J., 14, 569–576.[ISI][Medline]

Koszdin,K.L. and Bowen,B.R. (1992) The cloning and expression of a human {alpha}-1,3 fucosyltransferase capable of forming the E-selectin ligand. Biochem. Biophys. Res. Commun., 187, 152–157.[ISI][Medline]

Kukowska,L.J., Larsen,R.D., Nair,R.P. and Lowe,J.B. (1990) A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group {alpha} (1,3/1,4)fucosyltransferase. Genes Dev., 4, 1288–1303.[Abstract]

Kumar,R., Potvin,B., Muller,W.A. and Stanley,P. (1991) Cloning of a human alpha (1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants. J. Biol. Chem., 266, 21777–21783.[Abstract/Free Full Text]

Legault,D.J., Kelly,R.J., Natsuka,Y. and Lowe,J.B. (1995) Human {alpha} (1,3/1,4)-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J. Biol. Chem., 270, 20987–20996.[Abstract/Free Full Text]

Malissard,M., Borsig,L., Di Marco,S., Grutter,M.G., Kragl,U., Wandrey,C. and Berger,E.G. (1996) Recombinant soluble {alpha}-1,4-galactosyltransferases expressed in Saccharomyces cerevisiae. Purification, characterization and comparison with human enzyme. Eur. J. Biochem., 239, 340–348.[Abstract]

Maly,P., Thall,A., Petryniak,B., Rogers,C.E., Smith,P.L., Marks,R.M., Kelly,R.J., Gersten,K.M., Cheng,G., Saunders,T.L. and others. (1996) The {alpha} (1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis. Cell, 86, 643–653.[ISI][Medline]

Martina,J.A., Daniotti,J.L. and Maccioni,H.J. (1998) Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase. J. Biol. Chem., 273, 3725–3731.[Abstract/Free Full Text]

Masuno,H., Blanchette-Mackie,E.J., Schultz,C.J., Spaeth,A.E., Scow,R.O. and Okuda,H. (1992) Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine. J. Lipid Res., 33, 1343–1349.[Abstract]

McEver,R.P. (1997) Selectin–carbohydrate interactions during inflammation and metastasis. Glycoconj. J., 14, 585–591.[ISI][Medline]

McEver,R.P., Moore,K.L. and Cummings,R.D. (1995) Leukocyte trafficking mediated by selectin–carbohydrate interactions. J. Biol. Chem., 270, 11025–11028.[Abstract/Free Full Text]

Minowa,M.T., Oguri,S., Yoshida,A., Hara,T., Iwamatsu,A., Ikenaga,H. and Takeuchi,M. (1998) cDNA cloning and expression of bovine UDP-N-acetylglucosamine: {alpha}1,3-D-mannoside ß1,4-N-acetylglucosaminyltransferase IV. J. Biol. Chem., 273, 11556–11562.[Abstract/Free Full Text]

Molinari,M. and Helenius,A. (1999) Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells [see comments]. Nature, 402, 90–93.[ISI][Medline]

Mollicone,R., Candelier,J.J., Mennesson,B., Couillin,P., Venot,A.P. and Oriol,R. (1992) Five specificity patterns of (1,3)-{alpha}-L-fucosyltransferase activity defined by use of synthetic oligosaccharide acceptors. Differential expression of the enzymes during human embryonic development and in adult tissues. Carbohydr. Res., 228, 265–276.[ISI][Medline]

Mueckler,M., Caruso,C., Baldwin,S.A., Panico,M., Blench,I., Morris,H.R., Allard,W.J., Lienhard,G.E. and Lodish,H.F. (1985) Sequence and structure of a human glucose transporter. Science, 229, 941–945.[ISI][Medline]

Nagai,K., Ihara,Y., Wada,Y. and Taniguchi,N. (1997) N-glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology, 7, 769–776.[Abstract]

Natsuka,S., Gersten,K.M., Zenita,K., Kannagi,R. and Lowe,J.B. (1994) Molecular cloning of a cDNA encoding a novel human leukocyte {alpha}-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x determinant [published erratum appears in J. Biol. Chem., 1994, 269 (32): 20806]. J. Biol. Chem., 269, 16789–16794.[Abstract/Free Full Text]

Newrzella,D. and Stoffel,W. (1996) Functional analysis of the glycosylation of murine acid sphingomyelinase. J. Biol. Chem., 271, 32089–32095.[Abstract/Free Full Text]

Nguyen,A.T., Holmes,E.H., Whitaker,J.M., Ho,S., Shetterly,S. and Macher,B.A. (1998) Human {alpha}1,3/4-Fucosyltransferases. I. identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis. J. Biol. Chem., 273, 25244–25249.[Abstract/Free Full Text]

Orntoft,T.F., Vestergaard,E.M., Holmes,E., Jakobsen,J.S., Grunnet,N., Mortensen,M., Johnson,P., Bross,P., Gregersen,N., Skorstengaard,K. and others. (1996) Influence of Lewis {alpha}1-3/4-L-fucosyltransferase (FUT3) gene mutations on enzyme activity, erythrocyte phenotyping and circulating tumor marker sialyl-Lewis a levels. J. Biol. Chem., 271, 32260–32268.[Abstract/Free Full Text]

Roberts,P.C., Garten,W. and Klenk,H.D. (1993) Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. J. Virol., 67, 3048–3060.[Abstract]

Sasaki,K., Kurata,K., Funayama,K., Nagata,M., Watanabe,E., Ohta,S., Hanai,N. and Nishi,T. (1994) Expression cloning of a novel {alpha}1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes. J. Biol. Chem., 269, 14730–14737.[Abstract/Free Full Text]

Toki,D., Sarkar,M., Yip,B., Reck,F., Joziasse,D., Fukuda,M., Schachter,H. and Brockhausen,I. (1997) Expression of stable human O-glycan core 2 ß-1,6-N- acetylglucosaminyltransferase in Sf9 insect cells. Biochem. J., 325, 63–69.[ISI][Medline]

Watkins,W.M. (1980) Biochemistry and Genetics of the ABO, Lewis and P blood group systems. Adv. Hum. Genet., 10, 1–116.

Weitz,G. and Proia,R.L. (1992) Analysis of the glycosylation and phosphorylation of the {alpha}-subunit of the lysosomal enzyme, ß-hexosaminidase A, by site-directed mutagenesis. J. Biol. Chem., 267, 10039–10044.[Abstract/Free Full Text]

Weston,B.W., Nair,R.P., Larsen,R.D. and Lowe,J.B. (1992a) Isolation of a novel human {alpha} (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group {alpha} (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities. J. Biol. Chem., 267, 4152–4160.[Abstract/Free Full Text]

Weston,B.W., Smith,P.L., Kelly,R.J. and Lowe,J.B. (1992b) Molecular cloning of a fourth member of a human {alpha} (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x and difucosyl sialyl Lewis x epitopes [published erratum appears in J. Biol. Chem., 1993, 268 (24): 18398]. J. Biol. Chem., 267, 24575–24584.