2Institut National de la Santé et de la Recherche Médicale (INSERM) U-559 Faculté de Médecine, 27 Bd. J. Moulin, 13385 Marseille Cedex 5, France; 3The Burnham Institute, La Jolla, CA 92037, USA; and 4Centre dImmunologie de Marseille-Luminy (CIML), case 906, 13288, Marseille, France
Received on April 19, 2001; revised on July 29, 2001; accepted on August 15, 2001.
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Abstract |
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Key words: (1,2)-fucosyltransferase/
(1,3)-fucosyltransferase/ß1,6 N-acetylglucosaminyltransferase/Golgi targeting/P-selectin
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Introduction |
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The human O-glycan core2 ß(1,6)N-acetylglucosaminyltransferase (C2GnT) is a 5760-kDa type II integral membrane protein that has been shown to localize to the cis to medial/Golgi, first by confocal microscopy (Skrincosky et al., 1997) and very recently by electron microscopy (Dalziel et al., 2001
). What could be the mechanism by which the C2GnT is retained in this particular compartment? Perhaps if a specific Golgi-retention signal for C2GnT is present, it may be in or near the TM domain of the enzyme. To determine which of the cytoplasmic tail, the TM domain, or the stem region plays a critical role in targeting and retaining C2GnT in the Golgi, we constructed hybrid cDNAs encoding different N-terminal domains of the enzyme fused to enhanced green fluorescent protein (EGFP). We found that the cytoplasmic tail and the transmembrane domain (but not the stem region) represent the minimal peptide sequence responsible for the membrane binding and targeting of the enzyme. We extended this study to other glycosyltransferases; here also, the stem region was found to be not necessary for the retention of the enzymes in the Golgi apparatus.
We have recently shown that when the H-type (1,2)-fucosyltransferase (FucTI) and the
(1,3)-fucosyltransferase-VII (FucTVII) are coexpressed, the sLex precursor (Galß1,4GlcNAcß1R) is preferentially converted by FucTI to the histo-blood group H structure (Fuc
1,2Galß1,4GlcNAcß1R), which cannot be further
(1,3)-fucosylated by FucTVII, leading to a dramatic decrease in sLex expression and E-selectin adhesion (Zerfaoui et al., 2000
). Inasmuch as synthesis of functional E-selectin ligand(s) by FucTVII requires the presence of
(2,3)-linked sialic acid, those data suggested that FucTI might take precedence over the sLex-priming
(2,3)-sialyltransferases (ST3). Thus the final structure of sialyl-Lewis antigens might be influenced not only by the level of expression of the glycosyltransferases involved but also by their position within the Golgi apparatus. In support to this assumption, FucTI, ST3, and FucTVII may distribute within the Golgi in the following order: FucTI, then ST3, then FucTVII. We herein addressed the question of what would be the consequence on sLex synthesis, if FucTVII was placed in an earlier temporal compartment? To answer this question, we fused its catalytic domain to the cytosolic tail and transmembrane domain (CTd) of C2GnT and compared its intracellular distribution and in vivo function with those of FucTI.
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Results |
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Confocal laser microscopy was used to compare the intracellular distribution of the EGFP-fused proteins with that of C2GnT in CHO/C2P1 cells (Figures 5 and 6). As shown in Figure 5, FucTVII-EGFP exhibits a strikingly broad distribution, unusual for a Golgi glycosyltransferase (Figure 5A) and different from the pattern observed with the FucTVII-derived CTd (see Figure 3D). The distribution of FucTVII-EGFP is clearly different from that of C2GnT (Figure 5B and C for merged images) and from that of -mannosidase-II (Figure 5E and F for merged images). In contrast, the chimeric C2/FucTVII-EGFP shows a predominant typical Golgi staining and a complete colocalization with both C2GnT (Figure 5H and I for merged images) and
-mannosidase-II (Figure 5K and L for merged images). This result indicates that FucTVII and C2GnT have distinct Golgi distribution and that the CTd of C2GnT is able to efficiently mislocalize FucTVII to the same location as C2GnT (i.e., medial/Golgi). Figure 6 shows some overlap of FucTI-EGFP (Figure 6A) with C2GnT (Figure 6B and C for merged images) and a high overlapping degree with
-mannosidase-II (Figure 6E and F for merged images), suggesting that FucTI may reside in the medial/Golgi, but not exactly at the same place as C2GnT. Interestingly, the chimeric C2/FucTI (Figure 6G) distribute similarly to C2GnT (Figure 6H and I for merged images) and to
-mannosidase-II (Figure 6K and L for merged images) indicating that the latter is localized to the same compartment as the C2GnT (i.e., medial/Golgi). Taken together, these data demonstrate that the C2GnT-derived CTd function as a (cis)/medial-Golgi determinant because it can target to this compartment proteins other than EGFP. In fact, although this peptide has only a limited effectif any at allon the cis/Golgi enzyme FucTI (Hartel-Schenk et al., 1991
), it dramatically alters the intracellular distribution of a glycosyltransferase not normally present in this compartment, such as the
(1,3)-fucosyltransferase FucTVII. Other important findings raise from these experiments, including the unique distribution pattern of FucTVII and the confirmation of previous data regarding the medial/Golgi localization of C2GnT (Skrincosky et al., 1997
; Dalziel et al., 2001
) and FucTI (Hartel-Schenk et al., 1991
).
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Discussion |
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Other glycosyltransferases were tested with respect to the efficiency of their CTd to target EGFP to the Golgi in living cells. These comprise one Golgi early-acting enzyme, the polypeptide GalNAc-T1, and three Golgi late-acting glycosyltransferases, ST6Gal-1, FucTI, and FucTVII. These enzymes do not possess an obvious sequence homology that would suggest a common Golgi retention signal. GalNAcT-1 was first localized to the cis/Golgi of the submaxillary gland (Roth et al., 1994) but a recent study by Röttger et al. (1998)
has shown that all the three GalNAcT (T1, T2, and T3) are present throughout the Golgi stack of HeLa cells, which suggests that initiation of O-glycosylation may not be restricted to cis-Golgi, but occur at multiple sites within the Golgi apparatus. Still, there is no information available regarding the Golgi retention mechanism of this enzyme. ST6Gal-I has been repeatedly reported to topologically and/or functionally reside in the trans/Golgi and the TGN. Along with the ß(1,4)-galactosyltransferase (Aoki et al., 1992
; Hartel-Schenk et al., 1991
), ST6Gal-I is perhaps the most studied glycosyltransferase in terms of Golgi retention mechanism (Munro, 1991
; Colley et al., 1992
; Wong et al., 1992
; Dahdal and Colley, 1993
; Rabouille et al., 1995
). Depending on the experimental system used, the TM domain alone or combined with the cytosolic tail, with or without the stem region have been shown to target a reporter protein to the Golgi (reviewed in Colley, 1997
and references therein). On the other hand, Colley et al. (1992)
showed that replacement of a four-to five-amino-acid sequence had no effect on Golgi localization of this enzyme and Munro (1995)
demonstrated that the TM domain of native ST6Gal-I can be totally replaced by a polyleucine sequence of similar length without affecting its Golgi retention.
Regarding FucTI, very little is known on its localization; the only data available comes from cell fractionation experiments using preparative free-flow electrophoresis. The authors showed that fucosyltransferase activity distributed in two peaks, one corresponding to cis cisternae and the other to trans cisternae. The (1,2)fucosyltransferase was found concentrated in the peak corresponding to the cis cisternae of the Golgi (Hartel-Schenk et al., 1991
). So far, FucTVII has not been yet topologically assigned to a particular compartment. However, because this enzyme acts on glycans after sialyltransferases (Maly et al., 1996
), data from our group (Zerfaoui et al., 2000
) and Conradts one (Grabenhorst and Conradt, 1999
) suggest a post-Golgi pathway for this enzyme. Consistent with this is the study from Bergers group on another
(1,3)-fucosyltransferase the FucT-VI, showing that this enzyme codistributes with the ß(1,4)-galactosyltransferase-I in hepatocarcinoma cells HepG2 (Borsig et al., 1999
). We herein demonstrate that, regardless of their topology and function, these five glycosyltransferases share homologous domains (i.e., the CTd) for their targeting and retention in the Golgi apparatus.
We then addressed the question of whether exchanging the CTd of C2GnT with other glycosyltransferases, functionally and topologically different from C2GnT such as FucTVII and FucTI, would alter their intracellular distribution and function. Using confocal microscopy we show that both FucTI and its C2GnT chimera (C2/FucTI) colocalize with C2GnT in medial/Golgi compartment (Figure 6). This data is in line with results from flow cytometric studies of anti-H immunoreactivity, showing that fusing the catalytic domain of FucTI to the CTd of C2GnT has no consequence on H glycotope synthesis (see also Figure 7). This is partly consistent with data from cell fractionation studies reported by Hartel-Schenk and co-workers who assigned the (1,2)-fucosylation activity to the cis/Golgi (Hartel-Schenk et al., 1991
).
As discussed above, FucTVII acts terminally on sialylated glycans to form the sLex structure (NeuAc2-3Galß1-4[Fuc
1-3]GlcNAc) (see Figure 7) and its sulfated variants (reviewed in Fukuda et al., 1999
). Besides their central role in inflammation, these compounds are carried by mucin-like O-glycans (reviewed in Varki, 1994
) and are regarded as tumor-associated antigens, the expression of which is up-regulated in many cancers and seems to be related to the metastasising capacity of cancer cells (reviewed in Hakomori, 1991
; Turner and Catteral, 1996
). We herein used sLex synthesis and P-selectin binding as functional assays to investigate the behavior of FucTVII on fusion of its luminal portion with the C2GnT-derived CTd. Cells expressing FucTVII-EGFP exhibit a perinuclear, but diffused staining (Figure 5, panels A and D and Figure 8B, panel a) different from the staining obtained when its CTd alone is fused to EGFP (Figure 3D). Interestingly, despite this unusual distribution, the enzyme is fully active based on its ability to complete sLex synthesis (Figure 8B, panel b) and to procure P-selectin binding to transfected cells (Figure 8A, panel a). The reason for the difference in the staining pattern between the full-length FucTVII-EGFP and the FucTVII(134)-EGFP fusion peptide is not immediately apparent and the lack of data from the literature regarding the intracellular distribution of FucTVII, makes it difficult to draw a conclusion. Current efforts using immunostaining approaches are aimed at studying the subcellular distribution of this particular fucosyltransferase.
Most important is the finding that the CTd of C2GnT is able to efficiently mislocalize FucTVII to C2GnT compartment, leading to a dramatic decrease in sLex expression and P-selectin binding. We provide evidence that this alteration is a consequence of a mislocalization of FucTVII rather than a change in the catalytic activity of the enzyme. It is likely that within the C2GnT compartment the chimeric C2/FucTVII-EGFP may not encounter the sugar nucleotide donor GDP-fucose and/or the (2,3)-sialylated acceptors (Figure 7). Yet the fact that FucTI and its chimeric form C2/FucTI were shown to localize to the same compartment as C2GnT where they are fully active precludes the possibility of a scarcity in GDP-fucose donor. It is likely, however, that because
(1,3)-fucosylation requires
(2,3)-sialylated acceptors, glycoproteins (including the PSGL-1) may enter the C2GnT subcompartment (where C2/FucTVII-EGFP has moved) under unsialylated forms and therefore, could not be fucosylated by the chimeric enzyme.
In a previous work, we have shown that the conversion of C2GnT into a trans-Golgi enzyme by replacing its N-terminal portion with the corresponding part of ST6Gal-I resulted in a substantial decrease of C2GnT- branched oligosaccharides (Skrincosky et al., 1997). On the other hand, we have recently reported that transfection of FucTVII-expressing cells with FucTI inhibits the synthesis of sLex structures and the subsequent E-selectin adhesion, presumably by intercepting the sLex precursors before being sialylated (Zerfaoui et al., 2000
). Although variations in the distribution of a given enzyme may occur among different cell types (Roth et al., 1985
; Velasco et al., 1993
), it is generally accepted that glycosyltransferases are organized throughout the Golgi cisternae in the same order in which they sequentially add sugar residues to the growing oligosaccharide chains (reviewed in Colley, 1997
). Our previous and present data are consistent with this assumption. In addition, we herein demonstrate that the combination of the CTd of C2GnT can act as a cis to medial/Golgi determinant to bring to this early compartment of the Golgi, a late-acting glycosyltransferase, such as FucTVII. From a pathological point of view, it would be of significance to use this approach to tentatively "deviate" the aberrant glycosylation often associated with tumorogenicity toward a normal (or closer) glycosylation pathway.
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Materials and methods |
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Preparation of plasmid DNAs and cell transfection
The vector pcDNA3/EGFP was constructed by digesting pEGFP-N1 with EcoR I and Not I and ligating the excised EGFP cDNA into EcoR I /Not Idigested pCDNA 3.1(+). DNA fragments encoding different peptides of the amino-terminal portion of C2GnT (Figure 1A) were generated by polymerase chain reaction (PCR) using the sense and antisense primers described in Table I and pcDNA1/C2GnT as a template (Bierhuizen and Fukuda, 1992). DNAs were gel purified, restricted by BamH I and Age1, and ligated into BamH I/Age1digested pcDNA3/EGFP. Other glycosyltransferases, including FucTVII, GalNAcT-I, FucTI, and ST6Gal-I, were tested with respect to Golgi-targeting efficiency of their CTds. For this purpose, DNA fragments encoding amino acids 134 of FucTVII (FucTVII[134]-EGFP fragment), 128 of GalNAcT-I (GalNAcT[128] fragment), 125 of FucTI (FucTI[125] fragment), or 126 of ST6Gal-I (ST6[126] fragment) were generated by PCR using the primers presented in Table I and cloned in pcDNA3/EGFP between BamH1 and Age 1 sites as described above.
To construct chimeric fucosyltransferases containing the CTd of C2GnT and the catalytic domains of either FucTI or FucTVII, DNAs coding for the luminal portions of the enzymes, respectively Lum-FucTI and Lum-FucTVII, were amplified by PCR using the appropriate primers listed in Table I and cloned into the unique Age 1 site of C2(132)-EGFP (Figure 1B and C). The native counterparts FucTI-EGFP and FucTVII-EGFP were constructed by cloning the same DNA fragments (Lum-FucTI or Lum-FucTVII) into the Age 1 site of FucTI(125)-EGFP or FucTVII(134)-EGFP fragments, respectively (Figure 1B and C). All the constructs listed above were verified by automated DNA sequencing.
CHO-K1 cells were grown in Hams F12 medium containing 10% fetal calf serum, 2 mM L-glutamine, and 100 µg/ml of each penicillin and streptomycine. Unless otherwise specified, cells were transfected with 1 µg plasmid DNAs for 3 h using Lipofectamine PlusTM reagent according to the manufacturers instructions. For colocalization studies and P-selectin binding assay, a stable cell line expressing both C2GnT and PSGL-1 was produced by transfecting CHO-K1 cells with 1 µg pZeoSV/PSGL-1 and 5 µg of pcDNA1/C2GnT. Clonal cell lines were derived from within the zeocin-resistant transfectants, and several clones expressing both C2GnT and PSGL were obtained after 3 weeks of selection in the presence of 500 µg /ml zeocin. One clone (referred to as CHO/C2P1) was further transfected with 1 µg DNA of either EGFP-conjugated glycosyltransferases or their derived CTds. After another 3-week period in the presence of 200 µg /ml zeocin and 1 mg/ml G418, stable transfectants expressing either FucTI-EGFP, FucTVII-EGFP, or their chimeric counterparts were collected. To avoid clone-to-clone variations in enzyme expression, clones were sorted by FACscan flow cytometry (see below) on the basis of EGFP fluorescence.
Fluorescence microscopy and flow cytometry
Twenty four hours after transfection, cells were trypsinized, reseeded, and cultured for an additional 24 h. The intracellular distribution of EGFP-fusion proteins in living cells was visualized by direct fluorescence microscopy with an Olympus IMT-2 microscope (Olympus Optical, Tokyo). For colocalization studies, 48 h after transfection, cells were fixed in 3.5% paraformaldehyde and permeabilized with 0.5% TritonX100 in phosphate buffered saline containing 1% fetal calf serum (PFT medium). The rabbit polyclonal antibodies 1719.39, raised against the catalytic domain of C2GnT (Skrincosky et al., 1997) and anti-
-mannosidase-II (Velasco et al., 1993
) were used to map the intracellular distribution of C2GnT and
-mannosidase-II, respectively. After permeabilization, cells were incubated at room temperature for 1 h with the antibodies (1:1000 dilution in PFT medium), followed by rhodamine-conjugated goat anti-rabbit IgG for an additional h. Confocal microscopy was performed on a Leica instrument (Uniblitz Sutter Instrument). Images were processed with Metamorph Imaging system version 3.5, and volumes were originally retraced as a 24-bit TrueColor images and transferred to Adobe Photoshop as 24-bit RGB TIFF files.
The consequence of fusing the catalytic domain of FucTVII to the CTd of C2GnT on fucosylation, was assessed by in vitro (1,3)-fucosyltransferase activity, fluorescence microscopy, flow cytometry, and P-selectin binding assay. P-selectin interaction with its ligand (PSGL-1) requires C2GnT-branching of PSGL-1 O-glycans, which are further
(1,3)-fucosylated by FucTVII (Li et al., 1996
). Therefore, the effect of fusing FucTVII to the CTd of C2GnT was evaluated by measuring the P-selectin binding to CHO/C2P1 cells after transfection with FucTVII or the chimeric C2/FucTVII. CHO/C2P1 cells were stably transfected either with pcDNA3/FucTVII-EGFP or pcDNA3/C2/FucTVII-EGFP, and resistant transfectants were collected and sorted by FACscan flow cytometry on the basis of EGFP fluorescence as indicated above. The EGFP-positive cells were then assayed for sLex expression and P-selectin binding using CSLEX-1 immunoreactivity and binding of recombinant P-selectin-IgG chimera, respectively. CSLEX-1 immunoreactivity was assessed by fluorescence microscopy (Zerfaoui et al., 2000
) and bound P-selectin-IgG was stained with Cy5-conjugated anti-human IgG and analyzed on a FACScan flow cytometer, essentially as described by others (Tsuboi et al., 2000
). All incubations were carried out at 4°C in PBS containing 1 mM Ca2+ and Mg2+ and1% bovine serum albumin. Fluorescence was analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) by measuring the fluorescence of 10,000 cells and displayed on a four-decade log scale.
To evaluate the effect of fusing the C2GnT-derived CTd on FucTI-catalyzed fucosylation, CHO-K1 cells were transfected with pcDNA3/FucTI-EGFP or pcDNA3/C2/FucTI-EGFP as described above and selected for 3 weeks in the presence of 1 mg/ml G418. For the same reasons as above, all resistant transfectants were collected and sorted by FACscan flow cytometry with respect to EGFP fluorescence (data not shown). The EGFP-positive cells were then incubated with 10 µg/ml anti-H mAb for 30 min followed by incubation with Cy5-labeled goat anti-mouse IgM. All incubations were carried out at 4°C in phosphate buffered saline containing 1% bovine serum albumin and fluorescence was analyzed as described above.
(1,3)-Fucosyltransferase activity
Cells were harvested, washed three times in phosphate buffered saline, and hand homogenized in 50 mM HEPES buffer, pH 7.5, containing 0.25 M sucrose and EDTA-free protease inhibitor cocktail (Roche Molecular Biochemical, Meylan, France). FucTVII activity in cell homogenates were measured essentially as previously described (Zerfaoui et al., 2000), using the 3'-
-sialyl-N-acetyllactosamine as acceptor (Toronto Research Chemicals, Ontario, Canada). Briefly, 50 µg of protein extract (10 mg/ml) were assayed for fucosyltransferase activity in 40 µl of 50 mM HEPES buffer, pH 7.5, containing 5 mM MnCl2, 7 mM ATP, 3 mM NaN3, 5 mM GDP-fucose, 0.07 µCi of GDP-[5,6-3H]fucose (Amersham, Les Ulis, France) and 3 mM 3'-
-sialyl-N-acetyllactosamine. The acceptor substrate was omitted in control samples. After 1 h incubation at 37°C, the mixture was diluted with 1 ml water and applied to a Dowex-1-Cl column. The column was washed three times with 1 ml of water and 500 µl of combined fractions was counted in 5 ml of scintillant (PCS, Amersham). The [3H]-fucosylated sialylated acceptors were eluted with 3 ml of 0.2 M NaCl, and 500 µl were counted. To obtain values solely due to fucosylation of the acceptor substrate, total counts of samples without acceptor (control samples) were subtracted from total counts of samples with acceptor. The fucosyltransferase activity was calculated as pmol · min1 mg1.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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