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.
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Abstract |
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The 1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human
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
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Introduction |
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The 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., 1990
; Kukowska et al., 1990
; Kumar et al., 1991
; Koszdin and Bowen, 1992
; Weston et al., 1992a
,b; Natsuka et al., 1994
; Sasaki et al., 1994
; Kaneko et al., 1999
). 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 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
1,3-fucosyltransferase V (hFucTV) (Asn167 and Asn198) and human
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).
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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., 1998; Legault et al., 1995
). Furthermore, Kukowska et al. (1990)
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 1,3/4-fucosyltransferases III, -V, and -VI are necessary for the expression of full catalytic activity.
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Results |
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Discussion |
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The roles of the potential N-glycosylation sites and their glycosylation in the glycosyltransferases have been analyzed in several cases (Fast et al., 1993; Haraguchi et al., 1995
; Malissard et al., 1996
; Nagai et al., 1997
; Toki et al., 1997
; Martina et al., 1998
; Minowa et al., 1998
). 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 (46 kDa). The
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., 1992
; Masuno et al., 1992
). 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, 1999
).
The role of the potential N-glycosylation sites has only been studied poorly in the fucosyltransferases. Kukowska et al. (1990) 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., 1990
). Legault et al., 1995
, made a domain swap study of hFucTVI and -III to analyze the role of different sub-domains in acceptor substrate specificity (Legault et al., 1995
). 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)
. 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 1,3/4-fucosyltransferases (Breton et al., 1998
). 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, 1994; Ben-Zeev et al., 1994
). In some glycoproteins it has been found that glycosylation at certain sites proves more important for folding than others (Roberts et al., 1993
; Newrzella and Stoffel, 1996
). 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, 1992
).
In conclusion, we demonstrate that the two conserved C-terminal N-glycosylation sites of the human 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.
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Materials and methods |
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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., 1996). 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)
, 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, 1973). 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, 1992) 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 53417, 364 bp) amplified by RT-PCR was used as control (Ercolani et al., 1988
). Probes were labeled with [
-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 1620 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 1,3/4-fucosyltransferase activity
The 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., 1994
). 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 TrisHCl (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.
SDSPAGE 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., 1998
) followed by HRP-conjugated swine anti-rabbit IgG (1:10,000 dilution). The immunoreactive bands were visualized with ECL + Plus Western Blotting Detection System.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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