The cytosolic and transmembrane domains of the ß1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant

Mourad Zerfaoui2, Minoru Fukuda3, Claire Langlet4, Sylvie Mathieu2, Misa Suzuki3, Dominique Lombardo2 and Assou El-Battari1,2

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 d’Immunologie de Marseille-Luminy (CIML), case 906, 13288, Marseille, France

Received on April 19, 2001; revised on July 29, 2001; accepted on August 15, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The ß1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type {alpha}(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the {alpha}(1,3)-fucosyltransferase VII (FucTVII), and the {alpha}(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.

Key words: {alpha}(1,2)-fucosyltransferase/{alpha}(1,3)-fucosyltransferase/ß1,6 N-acetylglucosaminyltransferase/Golgi targeting/P-selectin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The majority of glycosyltransferases cloned to date are type II transmembrane proteins (single transmembrane [TM] domain with its NH2 terminus in the cytosol and the COOH terminus in the lumen of the Golgi apparatus). The lack of homology within TM domains of the Golgi glycosyltransferases and throughout their entire sequence makes it difficult to pinpoint any common retention signal. Yet two main mechanisms were put forward to explain targeting and maintenance of proteins in specific Golgi compartment: the lipid-based retention (Munro, 1995Go, 1998; Bretcher and Munro, 1993Go) and the "kin recognition" model (Nilsson et al., 1994Go). According to the first model, the length (rather than the sequence per se) of the hydrophobic TM domain (15–17 residues for Golgi proteins, compared to 20 or more for plasma membrane proteins), would result in the segregation of Golgi proteins into cholesterol-poor domains while plasma membrane proteins would be incorporated into cholesterol-rich domains and transport vesicles (Munro, 1991Go). According to the kin recognition model, two medial-Golgi enzymes the N-acetylglucosaminyltransferase (GnT-I) and the {alpha}-mannosidase-II ({alpha}Man-II) are retained in the medial-Golgi by interacting laterally through their catalytic domains, to form aggregates too large to be incorporated into vesicles destined for the plasma membrane (Nilsson et al., 1994Go).

The human O-glycan core2 ß(1,6)N-acetylglucosaminyltransferase (C2GnT) is a 57–60-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., 1997Go) and very recently by electron microscopy (Dalziel et al., 2001Go). 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 {alpha}(1,2)-fucosyltransferase (FucTI) and the {alpha}(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{alpha}1,2Galß1,4GlcNAcß1R), which cannot be further {alpha}(1,3)-fucosylated by FucTVII, leading to a dramatic decrease in sLex expression and E-selectin adhesion (Zerfaoui et al., 2000Go). Inasmuch as synthesis of functional E-selectin ligand(s) by FucTVII requires the presence of {alpha}(2,3)-linked sialic acid, those data suggested that FucTI might take precedence over the sLex-priming {alpha}(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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The first 32 amino acids determine the Golgi localization of C2GnT
In the present work, we studied the minimal peptide sequence sufficient to target C2GnT to the Golgi. For this purpose, we took advantage from the autofluorescence of EGFP from Aequoria victoria (Chalfie et al., 1994Go) fused to the full-length enzyme or its deletion mutants (Figure 1A). The fluorescence of the resulting fusion proteins was visualized in living Chinese hamster ovary (CHO) cells. Figure 2 shows that the C2GnT-derived peptides 1–112 (Figure 2B), 1–53 (Figure 2C), or 1–32 (Figure 2D) are all capable of targeting the reporter protein to the perinuclear region of the cell; comparable to the pattern exhibited by the full-length construct C2(fl)-EGFP (Figure 2A). However, when the cytosolic amino terminal portion of C2GnT is lacking (see Figure 2A, C2[10–32]-EGFP construct), the majority of cells exhibited a diffused fluorescence (Figure 2E) different from the pattern shown by cells expressing EGFP alone (Figure 2F). Other deletion mutants of C2GnT were tested with respect to their efficiency to target EGFP to the Golgi apparatus. These include the TM domain combined with different lengths of the stem region up to amino acids 53 (construct C2[10–53]-EGFP) and 112 (construct C2[10–112]-EGFP) (Figure 1A, see arrows). Here also, no typical Golgi staining was seen (data not shown). Taken as a whole, these data indicate that the Golgi-targeting sequence of C2GnT can be limited to the first 32 amino with a particularly important role for the cytoplasmic tail.



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Fig. 1. Schematic representation of deletion mutants of C2GnT and other EGFP-fused proteins used in this study. (A) EGFP was fused to different lengths of C2GnT-derived peptides. The numbers and arrows refer to amino acid positions. Sections B and C depict the experimental procedure used to construct normal or chimeric fucosyltransferases. The numbers in parentheses refer to the amino acid residue at the splice junction or the amino acid position at which the protein was truncated. C2GnT-CTd, FucTI-CTd, and FucTVII-CTd correspond to the sequences coding for the cytoplasmic tail and transmembrane domain of C2GnT, FucTI, and FucTVII, respectively. Lum-FucTI and Lum-FucTVII represent the luminal portion of FucTI and FucTVII, respectively

 


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Fig. 2. Visualization of C2GnT-derived peptides fused to EGFP in live cells. CHO-K1 cells were transfected with the full-length C2GnT C2(fl)-EGFP (A) or decreasing lengths of the N-terminal part of the protein: C2(1–112)-EGFP (B), C2(1–53)-EGFP (C), C2(1–32)-EGFP (D), C2(10–32)-EGFP (E), or EGFP alone (F). Twenty four hours later, cells were trypsinized, reseeded, and fluorescent proteins were visualized in live cells after another 24-h incubation period. Bars, 15 µm.

 
The minimal Golgi-targeting sequences of other glycosyltransferases
We then asked whether the case of C2GnT could be generalised to other glycosyltransferases functionally different from C2GnT such as the polypeptide N-acetylgalactosaminyl-transferase-1 (GalNAcT-1), the {alpha}(1,2)-fucosyl-transferase (FucTI), the {alpha}(1,3)-fucosyltransferase (FucTVII) and the {alpha}(2,6)-sialyltransferase (ST6Gal-I). To this end, sequences coding for CTds of these enzymes were fused with EGFP and expressed in CHO cells; their intracellular distribution was analyzed by fluorescence microscopy in living cells. As shown in Figure 3, the fusion proteins GalNAcT(1–28)-EGFP (Figure 3A), FucTI(1–25)-EGFP (Figure 3B), ST6(1–26)-EGFP (Figure 3C), and FucTVII(1–34)-EGFP (Figure 3D), all exhibit a typical Golgi distribution pattern, indicating that CTd of these glycosyltrasferases, contain enough information to target the reporter protein EGFP to the Golgi apparatus. Taken as a whole with data from C2GnT, these findings suggest that the Golgi-targeting determinants of glycosyltransferases could be limited to their cytoplasmic tail and transmembrane domain.



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Fig. 3. CTds of other glycosyltransferases are able to target EGFP to the Golgi apparatus. CHO cells were transfected with the following constructs (see also Table I and Figure 1): (A) FucTI(1–25)-EGFP; (B) ST6(1–26)-EGFP; (C) GalNAc-T(1–28)-EGFP; and (D), FucTVII(1–34)-EGFP. Twenty four hours later, cells were treated as in Figure 2. Bars, 15 µm

 
The C2GnT-derived CTd as a cis/medial-Golgi retention signal
To check whether the CTd of C2GnT could be specific of the compartment where the full-length enzyme is naturally located, the C2(1–32)-EGFP protein was expressed in CHO cells permanently expressing C2GnT and compared by confocal laser scanning microscopy to the distribution of ST6-derived CTd (Table I, construct ST6[1–26]-EGFP, see also Figure 3C) in the same cells. The full-length construct C2(fl)-EGFP was used as a control. As shown in Figure 4, the C2(1–32)-EGFP fusion protein (Figure 4A, green) completely overlap (yellow) with the local C2GnT (Figure 4A, red), similar to the distribution of the full-length C2(fl)-EGFP (Figure 4B, green). However, when EGFP was fused to the CTd of ST6Gal-I (Figure 4C, green), an enzyme known to reside in the trans/Golgi and trans-Golgi network (TGN) (Munro, 1991Go; Colley et al., 1992Go; Wong et al., 1992Go; Dahdal and Colley, 1993Go; Rabouille et al., 1995Go), the reporter protein was not targeted to the same compartment as C2GnT (Figure 4C, red) because no overlapping could be seen between these two proteins. These data indicate that the C2GnT-derived CTd (C2[1–32]-EGFP) is able to target a reporter protein to the same Golgi compartment as the intact C2GnT. Furthermore, these results show that tagging C2GnT with EGFP does not interfere with its Golgi-targeting and indirectly imply that the stem region of C2GnT is not required for this process.


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Table I. Primers and PCR products
 


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Fig. 4. Coexpression of C2GnT with EGFP-tagged proteins. Cells expressing C2GnT (CHO/C2P1 cells) were transfected either with the full-length enzyme C2(fl)-EGFP (A, green) or its derived C2(1–32)-EGFP (B, green) or with the ST6-derived CTd ST6(1–26)-EGFP (C, green). Twenty four hours later, cells were permeabilized, stained with the anti-C2GnT 1719.39, and counterstained by rhodamine-conjugated anti-rabbit antibody (red), as described under Materials and methods. Merged images of double stained cells visualized by confocal microscopy are presented. Magnification: 700x.

 
Fusing the C2GnT-derived CTd to the catalytic domains of FucTVII or FucTI affects {alpha}(1,3)-fucosylation but not {alpha}(1,2)-fucosylation
Having shown that the C2GnT-derived CTd is able to target EGFP to the same location as the intact enzyme, we addressed the question of what would be the consequence on glycosylation and, particularly, fucosylation if FucTI or FucTVII were placed in C2GnT compartment? To answer this question, we fused the catalytic domains of FucTI or FucTVII to the CTd of C2GnT and studied their intracellular distribution and function in CHO cells stably expressing both C2GnT and P-selectin ligand-1 (PSGL-1) (CHO/C2P1 clone). We performed the following two studies: (1) compare the intracellular distribution of the EGFP-fused proteins with that of C2GnT and with the medial-Golgi marker {alpha}-mannosidase-II, using confocal microscopy, and (2) compare the enzymatic activities of intact and chimeric fucosyltransferases using anti-H immunoreactivity (for FucTI and C2/FucTI transfectants) or P-selectin binding (for FucTVII and C2/FucTVII). We chose CHO cells because they are deficient in FucTI activity (Watson et al., 1994Go) and do not bind P-selectin unless PSGL-1, C2GnT, and FucTVII are present (Li et al., 1996Go; Liu et al., 1998Go).

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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 effect—if any at all—on the cis/Golgi enzyme FucTI (Hartel-Schenk et al., 1991Go), it dramatically alters the intracellular distribution of a glycosyltransferase not normally present in this compartment, such as the {alpha}(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., 1997Go; Dalziel et al., 2001Go) and FucTI (Hartel-Schenk et al., 1991Go).



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Fig. 5. Confocal fluorescent micrographs showing the coexpression of EGFP-tagged FucT-VII or C2/FucTVII with C2GnT or {alpha}-mannosidase-II. CHO/C2P1 cells were transfected with constructs coding for FucT-VII or the chimeric C2/FucTVII fused to EGFP (green) and stained by polyclonal antibodies for C2GnT or {alpha}-mannosidase-II (red) and examined by confocal microscopy as described under Materials and methods. The intracellular distribution of FucTVII-EGFP is compared with that of C2GnT (A, B, and C) or {alpha}-mannosidase-II (D, E, and F) and the pattern of C2/FucTVII-EGFP is compared to that of C2GnT (G, H, and I) or {alpha}-mannosidase-II (J, K, and L). The merged images show overlapping red and green pixels in yellow. Magnification: 500x.

 


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Fig. 6. Confocal fluorescent micrographs showing the coexpression of EGFP-tagged FucTI or C2/FucTI with C2GnT or {alpha}-mannosidase-II. A similar experimental procedure as for Figure 5 was used to compare the intracellular distribution of FucTI-EGFP with that of C2GnT (A, B, and C) or {alpha}-mannosidase-II (D, E, and F) and the pattern of C2/FucTI-EGFP with that of C2GnT (G, H, and I) or {alpha}-mannosidase-II (J, K, and L). The merged images show overlapping red and green pixels in yellow. Magnification: 500x.

 
As shown in Figure 7A, FucTI attaches {alpha}(1,2)-fucose to the acceptor Galß1,3/4GlcNAcß1-R, creating the histo-blood group H. According to our confocal microscopic data, this reaction may take place in the cis to medial/Golgi. FucTVII however, requires a sialylated type-II N-acetyllactosaminic structure to make the selectin ligand sialyl-Lewis x, which suggests a post-{alpha}(2,3)sialyltransferase location for this fucosyltransferase (i.e., trans/Golgi or TGN). What then would the consequence on fucosylation if FucTI or FucTVII were "moved" (Figure 7B, arrows) to the C2GnT compartment? To answer this question we compared the enzymatic activities of intact and chimeric fucosyltransferases using flow cytometric analysis of anti-H immunoreactivity (for FucTI and C2/FucTI transfectants) or P-selectin binding (for FucTVII and C2/FucTVII). As shown in Figure 8, cells expressing FucTVII-EGFP (Figure 8A, panel a) on one hand and FucTI-EGFP or the chimeric C2/FucTI-EGFP (Figure 8A, panel b) on the other hand, were virtually homogeneous with respect to P-selectin-IgG binding and the anti-H immunoreactivity, respectively. However, though no difference in anti-H immunoreactivity could be detected between cells expressing the normal and the chimeric FucTI, P-selectin binding to cells expressing C2/FucTVII was dramatically reduced, although a small proportion of cells still significantly bind P-selectin (Figure 8A, panel a). Examination of sLex expression by fluorescence microscopy (Figure 8B), show a complete extinction of this glycotope from the surface of C2/FucTVII transfectants (Figure 8B, compare panels b and d). Because P-selectin binding occurs through sLex structures carried by PSGL-1, which is present on the CHO/C2P1 cells, this result suggests that the chimeric C2/FucTVII enzyme exhibits altered {alpha}(1,3)-fucosyltransferase activity toward the acceptor PSGL-1.



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Fig. 7. Glycosylation pathway involving FucTI and FucTVII. (A) The N-acetyllactosaminic structure Galß1,3/4GlcNAcß1-R is converted to the histo-blood group H by FucTI. Before being {alpha}(1,3)-fucosylated on GlcNAc residue by FucTVII, the acceptor Galß1,4GlcNAcß1-R is first sialylated by {alpha}(2,3) sialyltransferase(s). (B) After fusing the luminal portions of these fucosyltransferases with the C2GnT-derived CTd, no dramatic alteration in subcompartmentation and function of FucTI is observed because this enzyme belongs to the same compartment as C2GnT (see also Figure 6). However, when FucTVII is mislocalized to the medial/Golgi (arrow), it takes precedence over sialyltransferases, and, therefore, the unsialylated acceptors are no longer fucosylated.

 


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Fig. 8. Flow cytometric analysis of P-selectin binding and anti-H immunoreactivity and sLex immunostaining. CHO/C2P1 cells were transfected with constructs coding for the chimeric C2/FucTI or C2/FucTVII enzymes or their normal counterparts fused to EGFP and selected as described in Figure 5. (A) FucTVII and C2/FucTVII-EGFP-positive cells were assayed for P-selectin binding (panel a) and FucTI and C2/FucTI-EGFP-expressing cells were tested for the expression of the blood group H-determinant (panel b) as described under Materials and methods. (B) Effect of mislocalizing FucTVII on sLex expression. Stable cell lines expressing FucTVII-EGFP (a) or C2/FucTVII-EGFP (c) were stained with CSLEX-1 mAb followed by FITC-labeled anti-mouse IgM (compare b and d).

 
Because we used the same DNA fragment coding for the luminal portions of the enzymes cloned into either their own CTds to generate the intact enzymes, or the C2GnT-derived CTd to make their chimeric counterparts (see Figure 1B and C), and because the in vitro {alpha}(1,3)-fucosyltransferase activity determined in cell homogenates from the two cell lines was found to be not significantly different (Table II), we conclude that the alteration of sLex synthesis and P-selectin binding could not be explained at a molecular or catalytic levels, but rather at a Golgi subcompartmentation level.


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Table II. {alpha}1,3-Fucosyltransferase activity in cell homogenates
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We and others have recently localized C2GnT to the cis to medial cisternae of the Golgi (Skrincosky et al., 1997Go; Dalziel et al., 2001Go). The present study was undertaken to study the molecular signals required for targeting C2GnT to this compartment. Our data show that the combination of the cytoplasmic tail and the TM domain (CTd) of C2GnT is highly effective in targeting the reporter protein EGFP to the Golgi apparatus (Figure 2E). In fact, besides the 23 amino acids of the transmembrane domain, we found that the 9 amino acids of the cytoplasmic tail are crucial for Golgi retention of C2GnT. This result is in agreement with the recent finding that the cytoplasmic tail of FucTI contains a sequence for Golgi localization (Milland et al., 2001Go). It is likely that the cytoplasmic tail of C2GnT close interacts with the Golgi phospholipid head-groups through the positively charged sequence 7RRR9 (residues 7–9) thereby contributing to stabilize the protein in the lipid leaflet (Bosshart et al., 1994Go). It has been repeatedly reported that the stem region of glycosyltransferases is a part of their Golgi retention signal (see Colley, 1997Go and references therein). Our data do not support any role for this portion of C2GnT.

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., 1994Go) but a recent study by Röttger et al. (1998)Go 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., 1992Go; Hartel-Schenk et al., 1991Go), ST6Gal-I is perhaps the most studied glycosyltransferase in terms of Golgi retention mechanism (Munro, 1991Go; Colley et al., 1992Go; Wong et al., 1992Go; Dahdal and Colley, 1993Go; Rabouille et al., 1995Go). 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, 1997Go and references therein). On the other hand, Colley et al. (1992)Go showed that replacement of a four-to five-amino-acid sequence had no effect on Golgi localization of this enzyme and Munro (1995)Go 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 {alpha}(1,2)fucosyltransferase was found concentrated in the peak corresponding to the cis cisternae of the Golgi (Hartel-Schenk et al., 1991Go). 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., 1996Go), data from our group (Zerfaoui et al., 2000Go) and Conradt’s one (Grabenhorst and Conradt, 1999Go) suggest a post-Golgi pathway for this enzyme. Consistent with this is the study from Berger’s group on another {alpha}(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., 1999Go). 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 {alpha}(1,2)-fucosylation activity to the cis/Golgi (Hartel-Schenk et al., 1991Go).

As discussed above, FucTVII acts terminally on sialylated glycans to form the sLex structure (NeuAc{alpha}2-3Galß1-4[Fuc{alpha}1-3]GlcNAc) (see Figure 7) and its sulfated variants (reviewed in Fukuda et al., 1999Go). Besides their central role in inflammation, these compounds are carried by mucin-like O-glycans (reviewed in Varki, 1994Go) 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, 1991Go; Turner and Catteral, 1996Go). 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(1–34)-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 {alpha}(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 {alpha}(1,3)-fucosylation requires {alpha}(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., 1997Go). 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., 2000Go). Although variations in the distribution of a given enzyme may occur among different cell types (Roth et al., 1985Go; Velasco et al., 1993Go), 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, 1997Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Restriction enzymes and Taq polymerase were purchased from Promega (Madison, WI). Lipofectamine PlusTM reagent, neomycin (G418) and Ham’s F12 medium were purchased from Gibco BRL (Grand Island, NY). Anti-H mAb (AB5 clone) was from the Institut Mérieux (Lyon, France), and indodicarbocyanine (Cy5)-conjugated second antibodies were from Immunotech (Marseille, France). CSLEX-1 antibody was prepared from the hybridoma HB8580 (American Type Culture Collection, Baltimore, MD) as described (Zhang et al., 1996Go), and the anti-mannosidase II polyclonal antibody (Velasco et al., 1993Go) was a generous gift from Drs. M. G. Farqhuar and K. Moremen (respectively, University of California San Diego, La Jolla CA, and University of Georgia, Athens GA). Zeocin and pcDNA3.1(+) vector were from Invitrogen (Carlsbad, CA), and pEGFP-N1 vector was from Clonetech (Palo Alto, CA). The cDNAs for the human FucTI in pCDM7 (Larsen et al., 1990Go) and FucTVII in pCDM8 (Natsuka et al., 1994Go) were kindly provided by Dr. John Lowe (Howard Hughes Medical Institute, MI). The cDNA for PSGL-1 in pZeoSV (Invitrogen) was kindly provided by Dr. R. P. McEver (University of Oklahoma, OK) and the human polypeptide GalNAcT-I cDNA, (Meurer et al., 1995Go) in pcDNA3 was a generous gift of Dr. V. Piller (Glycobiologie CNRS Orléans, France).

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 I–digested 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, 1992Go). DNAs were gel purified, restricted by BamH I and Age1, and ligated into BamH I/Age1–digested 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 1–34 of FucTVII (FucTVII[1–34]-EGFP fragment), 1–28 of GalNAcT-I (GalNAcT[1–28] fragment), 1–25 of FucTI (FucTI[1–25] fragment), or 1–26 of ST6Gal-I (ST6[1–26] 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(1–32)-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(1–25)-EGFP or FucTVII(1–34)-EGFP fragments, respectively (Figure 1B and C). All the constructs listed above were verified by automated DNA sequencing.

CHO-K1 cells were grown in Ham’s 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 manufacturer’s 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., 1997Go) and anti-{alpha}-mannosidase-II (Velasco et al., 1993Go) were used to map the intracellular distribution of C2GnT and {alpha}-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 {alpha}(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 {alpha}(1,3)-fucosylated by FucTVII (Li et al., 1996Go). 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., 2000Go) 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., 2000Go). 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.

{alpha}(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., 2000Go), using the 3'-{alpha}-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'-{alpha}-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 · min–1 mg–1.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We would like to thank Marc Barrad and Mathieu Maffet (CIML, Marseille, France) for their excellent technical assistance with confocal microscopy; Charles Prévôt (Université de la Méditerranée, Marseille, France) for his assistance with FACscan analyses; and Christian Crotte for organizing the manuscript. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and partly by the International Cancer Technology Transfer to A.E.B., the Association pour la Recherche contre le Cancer, grant 9506, as well as grants CA33000 and CA48737 from the National Cancer Institute.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHO, Chinese hamster ovary; C2GnT, core2-ß(1,6)N-acetylglucosaminyltransferase; CTd, cytosolic tail and transmembrane domain; EGFP, enhanced green fluorescent protein; Fuc, fucose; FucTVII, {alpha}(1,3)-fucosyl-transferase VII; Ga, galactose; GlcNAc, N-acetylglucosamine; FucTI, H-type {alpha}(1,2)-fucosyltransferase; NeuAc, neuraminic acid; PCR, polymerase chain reaction; PFT, phosphate buffered saline containing 1% fetal calf serum; PSGL-1, P-selectin ligand-1; ST6Gal-I, {alpha}(2,6)-sialyltransferase; TGN, trans-Golgi network; TM, transmembrane.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Aoki, D., Lee, N., Yamaguchi, C., Dubois, C., and Fukuda, M.N. (1992) Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain. Proc. Natl Acad. Sci. USA, 89, 4319–4323.[Abstract/Free Full Text]

Bierhuizen, M.F.A. and Fukuda, M. (1992) Expression cloning of a cDNA encoding UDP-GlcNAc:Gal ß 1, 3 GalNAc-R (GlcNAc to GalNAc) ß 1, 6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. Proc. Natl Acad. Sci. USA, 89, 9326–9330.[Abstract]

Borsig, L., Imbach, T., Hochli, M., and Berger, E.G. (1999) Alpha1, 3-Fucosyltransferase VI is expressed in HepG2 cells and codistribute with beta1, 4galactosyltransferase I in the golgi apparatus and monensin-induced swollen vesicles. Glycobiology, 9, 1273–1280.[Abstract/Free Full Text]

Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, P.J., and Bonifacino J.S. (1994) The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system. J. Cell Biol., 126, 1157–1172.[Abstract]

Bretcher, M.S. and Munro, S. (1993) Cholesterol and the Golgi apparatus. Science, 261, 1280–1281.[ISI][Medline]

Chalfie, M., Tu, Y., Euskirshen, G., Ward, W.W., and Prasher, D.C. (1994) Green fluorescent protein as a marker for gene expression. Science, 263, 802–805.[ISI][Medline]

Colley, K.J. (1997). Golgi localization of glycosyltransferases: more questions than answers Glycobiology, 7, 1–13.[Abstract]

Colley, K.J., Lee, E.U., and Paulson, J.C. (1992) The signal anchor and stem regions of the ß-galactoside {alpha} 2, 6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus. J. Biol. Chem., 267, 7784–7793.[Abstract/Free Full Text]

Dahdal, R.Y. and Colley, K.J. (1993) Specific sequences in the signal anchor of the beta-galactoside alpha-2, 6-sialyltransferase are not essential for Golgi localization. Membrane flanking sequences may specify Golgi retention. J. Biol. Chem., 268, 26310–26319.[Abstract/Free Full Text]

Dalziel, M., Whitehouse, C., McFarlane, I., Brockhausen, I., Gschmeissner, S., Schwientek, T., Clausen, H., Burchell, J., and Taylor-Papadimitriou, J. (2001) The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1. J. Biol. Chem., 276, 11007–11015.[Abstract/Free Full Text]

Fukuda, M., Hiraoka, N., and Yeh, J.C. (1999) C-type lectins and sialyl Lewis X oligosaccharides. Versatile roles in cell–cell interaction. J. Cell Biol., 147, 467–470.[Abstract/Free Full Text]

Grabenhorst, E. and Conradt, H.S. (1999) The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi. J. Biol. Chem., 274, 36107–36116.[Abstract/Free Full Text]

Hakomori, S. (1991) Possible functions of tumor-associated carbohydrate antigens. Curr. Opin. Immunol., 3, 646–653.[ISI][Medline]

Hartel-Schenk, S., Minnifield, N., Reutter, W., Hanski, C., Bauer, C., and Morre, D.J. (1991). Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat. Biochim. Biophys. Acta, 1115, 108–122.[ISI][Medline]

Kozak, M. (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem., 266, 19867–19870.[Free Full Text]

Larsen, R.D., Ernst, L.K., Nair, R.P., and Lowe, J.B. (1990). Molecular cloning, sequence and expression of a human GDP-L-fucos:beta-D-galactosiode 2-alpha-L-fucosyltransferase cDNA that can form the H blood group antigen. Proc. Natl Acad. Sci. USA, 87, 6674–6678.[Abstract]

Li, F., Wilkins, P. P., Crawley, S., Weinstein, J., Cummings, R.D., and McEver, R.P. (1996) Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies. J. Biol. Chem., 271, 3255–3264.[Abstract/Free Full Text]

Liu, W.J., Ramachandran, V., Kang, J., Kishimoto, T.K., Cummings, R.D., and McEver, R.P. (1998) Identification of N-terminal residues on P-selectin glycoprotein ligand-1 required for binding to P-selectin. J. Biol. Chem., 273, 7078–7087.[Abstract/Free Full Text]

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]

Meurer, J.A., Naylor, J.M., Baker, C.A., Thomsen, D.R., Homa, F.L., and Elhammer, A.P. (1995) cDNA cloning, expression, and chromosomal localization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase J. Biochem., 118, 568–574.[Abstract]

Milland, J., Taylor, S.G., Dodson, H.C., McKenzie, I.F.C., and Sandrin, M.S. (2001) The cytoplasmic tail of {alpha}1, 2-fucosyltransferase contains a sequence for Golgi localization. J. Biol. Chem., 276, 12012–12018.[Abstract/Free Full Text]

Munro, S. (1991) Sequence within and adjacent to the transmembrane segment of {alpha}-2, 6-sialyltransferase specify Golgi retention. EMBO J., 10, 3577–3588.[Abstract]

Munro, S. (1995) An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J., 14, 4695–4704.[Abstract]

Munro, S. (1998) Localization of proteins to the Golgi apparatus Trends Cell Biol., 8, 11–15.[CrossRef][ISI][Medline]

Natsuka, S., Gertsen, 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. J. Biol. Chem., 269, 20806–20813.[Free Full Text]

Nilsson, T., Hoe, M.H., Sluzarewicz, P., Rabouille, C., Watson, R., Hunte, F., Watzele, G., Berger, E.G., and Warren, G. (1994) Kin recognition between medial Golgi enzymes in HeLa cells. EMBO J., 13, 562–574.[Abstract]

Rabouille, C., Hui, N., Hunte, F., Kieckbusch, R., Berger, E.G., Warren, G. and Nilsson, T. (1995) Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci., 108, 1617–1627.[Abstract/Free Full Text]

Roth, J., Taatjes, D.J., Lucoq, J.M., Weinstein, J., and Paulson, J.C. (1985) Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell, 43, 287–295.[ISI][Medline]

Roth, J., Wang, Y., Eckhardt, A.E., and Hill, R.L. (1994) Subcellular localization of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland. Proc. Natl Acad. Sci. USA, 91, 8935–8939.[Abstract]

Röttger, S., White, J., Wandall, H.H., Olivo, J-C., Stark, A., Bennett, E., Whitehouse, C., Berger, E., Clausen, H., and Nilsson, T. (1998) Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J. Cell Sci., 111, 45–60.[Abstract/Free Full Text]

Skrincosky, D., Kain, R., El Battari, A, Exner, M., Kerjaschki, D., and Fukuda, M. (1997) Altered Golgi localization of Core 2 ß-1, 6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans. J. Biol. Chem., 272, 22695–22702.[Abstract/Free Full Text]

Tsuboi, S., Srivastava, O.P., Palcic, M.M., Hindsgaul, O., and Fukuda, M. (2000) Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface. Arch. Biochem. Biophys., 374, 100–106.[CrossRef][ISI][Medline]

Turner, G.A. and Catterall, J.B. (1996). Surface carbohydrates involved in the adhesive interactions of metastatic cells. Biochem. Soc. Trans, 25, 234–241.[ISI]

Varki, A. (1994) Selectin ligands. Proc. Natl Acad. Sci. USA, 91, 7390–7397.[Abstract]

Velasco, A., Hendricks, L., Moreman, K.W., Tulsiani, D.R.P., Touster, O., and Farquhar, M.G. (1993) Cell-type dependent variations in the subcellular distribution of a-mannosidase I and II. J. Cell Biol., 122, 39–51.[Abstract]

Watson, E., Bhide, A., and van Halbeek, H. (1994) Structure determination of the intact major sialylated oligosacharide chains of recombinant human erythropoetin expressed in CHO cells. Glycobiology, 4, 227–237.[Abstract]

Wong, S.H., Low, S.H., and Hong W. (1992) The 17-residue transmembrane domain of beta-galactoside alpha 2, 6-sialyltransferase is sufficient for Golgi retention. J. Cell Biol., 117, 245–258.[Abstract]

Zerfaoui, M., Fukuda, M., Sbarra, V., Lombardo, D., and El-Battari, A. (2000) Alpha(1, 2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells. Eur. J. Biochem., 267, 53–60.[Abstract/Free Full Text]

Zhang, K., Baeckstrôm, D., Brevinge, H., and Hansson, G., (1996) Secreted MUC1 mucins lacking their cytoplasmic part and carrying sialyl-Lewis a and x epitopes from a tumor cell line and sera of colon carcinoma patients can inhibit HL-60 leukocyte adhesion to E-selectin-expressing endothelial cells. J. Cell. Biochem., 60, 538–549.[CrossRef][ISI][Medline]