Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation

Assou El-Battari3, Maëlle Prorok3, Kiyohiko Angata4, Sylvie Mathieu3, Mourad Zerfaoui3, Edgar Ong1,4, Misa Suzuki3, Dominique Lombardo3 and Minoru Fukuda2,3

3 INSERM U-559/UEA-3289 Université de la Méditerranée, 27 Bd. J. Moulin, 13385 Marseille Cedex 5, France; and 4 Glycobiology Program, Cancer Research Center, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037

Received on June 25, 2003; revised on August 25, 2003; accepted on August 28, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 ß1,3-N-acetylglucosaminyltransferase (C1-ß3GnT) and core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, {alpha}1,2-fucosyltransferase-I (FucT-I) and {alpha}1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, {alpha}2,3-sialyltransferase-I (ST3Gal-I) and {alpha}2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-ß3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.

Key words: autoglycosylation / disufide bonds / Golgi retention / proteolysis / secretion


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The Golgi glycosyltransferases are type II transmembrane enzymes that catalyze the transfer of monosaccharides to proteins and lipids and share some domain features, such as a single transmembrane domain flanked by a short amino-terminal domain (the so-called cytoplasmic tail) and a large carboxy-terminal catalytic domain oriented to the lumen of the Golgi apparatus (Paulson and Colley, 1989Go). Extensive studies of the N- and O-glycosylation pathways in mammalian cells have revealed that glycans are synthesized by an ordered series of sugar transfer reactions, so that the final structure of the oligosaccharide is a result of a narrow acceptor specificity. Synthesis of such specific oligosaccharide structures depends not only on the expression level of a given glycosyltransferase but also on how glycosyltransferases segregate into distinct compartments of the Golgi complex. In this regard, glycosyltransferases are thought to be compartmentalized in the same order in which they act, such that enzymes acting early in glycan biosynthesis are localized to cis and medial compartments, whereas enzymes acting later in the biosynthetic pathway tend to colocalize in the trans-Golgi cisternae and the trans-Golgi network (reviewed in Fukuda, 1994Go).

There is a limited number of X-ray crystal structures of glycosyltransferases; therefore, little information on the three-dimensional conformation of these proteins is available (reviewed in Unligil and Rini, 2000Go). Despite this fact, several biochemical studies have pointed to a possible relationship between disulfide-mediated oligomerization, proteolytic cleavage, and Golgi retention of these enzymes (Weisz et al., 1993Go; Nilsson et al., 1994Go; Yamaguchi and Fukuda, 1995Go; Kitazume-Kawaguchi et al., 1999Go; Chen et al., 2000Go; Sasai et al., 2001Go). Using rat {alpha}2,6-sialyltransferase (ST6Gal-I), Colley's group demonstrated that one Cys residue in the transmembrane domain is involved in dimerization, and the Cys residues of the catalytic domain are required for trafficking and catalytic activity of the enzyme (Ma and Colley, 1996Go). Moreover, work from Taniguchi's group showed that the portion between the transmembrane and the catalytic domain region, the so-called stem region, of the N-acetylglucosaminyltransferase V (GnT-V) directly influences the Golgi retention through disulfide bond oligomerization (Sasai et al., 2001Go). The stem region is also susceptible to one or more proteolytic cleavage events, and some glycosyltransferase activities could be found as a soluble form in secretions and body fluids. In this regard, studies on the secretion of GalNAcT-I, ST6Gal-I, and ß4GalT-I have revealed that the soluble forms were the result of proteolytic cleavage at different positions from the transmembrane domain, suggesting that different proteases are involved in the release of these enzymes (Weinstein et al., 1987Go; Masri et al., 1988Go; Homa et al., 1993Go). As another feature of glycosyltransferase secretion, GnT-V secreted from human colon carcinoma WiDr cells is directly involved in tumor angiogenesis in a glycosylation-independent manner, providing a biological significance for the secretion of this glycosyltransferase (Saito et al., 2002Go).

In addition to these posttranslational modifications, many Golgi glycosyltransferases have one or more consensus N-glycosylation sites as well as serine and threonine residues that could be modified by O-glycosylation. However, few studies have been devoted to the study of the importance of these events, especially N-glycosylation, which in some instances has been shown to be required for proper folding and/or enzyme activity (Toki et al., 1997Go; Barbier et al., 2000Go; Christensen et al., 2000Go) and limited studies indicate that glycosyltransferases could also be subject to "autoglycosylation" (Mühlenhoff et al., 1996Go; Close and Colley, 1998Go; Angata et al., 2000Go).

So far, most of the studies on structure/function relationship in the glycosyltransferase field have been carried out on one particular enzyme using approaches and cellular systems different from one laboratory to the next. This in turn hampers the extension of data obtained in a given laboratory to the others. Therefore, a comparative study where several enzymes are investigated is desirable to understand the biological importance of the posttranslational events for their functions. In this regard, we have recently reported on the Golgi-targeting amino acid sequences of glycosyltransferases by comparing five different enzymes of the O- and N-glycosylation pathways, including glycosyltransferases including core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT-I), {alpha}1,2-fucosyltransferase-I (FucT-I), GalNAcT-I, {alpha}1,3-fucosyltransferase-VII (FucT-VII), and ST6Gal-I. We have shown that all these proteins share the same minimal Golgi targeting determinant comprising the cytoplasmic tail and the transmembrane domain, but not the stem region (Zerfaoui et al., 2000Go). We also showed that the minimal Golgi targeting determinant of C2GnT-I is able to displace FucT-VII to the C2GnT-I compartment, that is the cis-medial Golgi (Zerfaoui et al., 2002Go).

In the present study, we chose six different glycosyltransferases to investigate how these enzymes are processed for disulfide dimerization, secretion, and autoglycosylation. We found that except for FucT-VII, all glycosyltransferases tested are secreted to the culture medium. Moreover, we found that all of the glycosyltransferases except {alpha}2,3- sialyltransferase-I (ST3Gal-I) showed a different degree of dimerization, and dimeric forms of FucT-I and core 1 ß1,3-N-acetylglucosaminyltransferase (C1-ß3GnT) are secreted from the cells. Interestingly, we also found that a dimeric form of ST6Gal-I and FucT-I is fully active and autoglycosylates itself. These results strongly suggest that disulfide-bonded dimerization and secretion differentially play a role in the processing and function of glycosyltransferases in the Golgi apparatus.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
EGFP tagging does not interfere with function or Golgi compartmentalization of glycosyltransferases
The main goal of this work was to study such features as secretion, oligomerization, and glycosylation of six different glycosyltransferases in relation to their intracellular distribution and regulation of enzymatic activities. To allow examination of these proteins in living cells, the enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of the enzymes. Before undertaking further studies, we wanted to rule out the possibility that tagging with EGFP could interfere with in vivo catalytic properties or intracellular distribution of glycosyltransferases in vivo. To this end, to assay for in vivo enzyme activity of C2GnT-I-EGFP, we used Chinese hamster ovary (CHO)/CD43 cells stably expressing human leukosialin (CD43) and T305 monoclonal antibody that exclusively reacts with the sialylated core 2-branched CD43 hexasaccharide isoform (Piller et al., 1991Go; Bierhuizen and Fukuda, 1992Go). To evaluate the impact of EGFP tagging on FucT-I and ST6Gal-I, we used staining with the lectins Ulex europaeus agglutinin-I (UEA-1) and Sambucco nigra agglutinin-I (SNA-I), which reacts with {alpha}1,2-linked fucose and {alpha}2,6-linked sialic acid, respectively. For FucT-VII, cell surface sialyl-Lewis x expression was assessed by staining with CSLEX-I antibody. CHO cells were chosen as a reporter cell line because CHO cells do not express C2GnT-I (Bierhuizen and Fukuda, 1992Go), ST6Gal-I, FucT-I (Lee et al., 1989Go), or FucT-VII (Larsen et al., 1990Go).

As shown in Figure 1A, those cells transfected by EGFP-conjugated C2GnT-I, FucT-VII, FucT-I, or ST6Gal-I express the expected glycotopes (shown in red), that are T305 and CSLEX-I immunoreactivities (generated by C2GnT-I and FucT-VII, respectively) and UEA-I and SNA-I lectin staining (generated by FucT-I and ST6Gal-I, respectively). These results indicate that ligating EGFP to the carboxyl-terminus of glycosyltransferases did not affect their enzyme activities in vivo, as seen previously on ST8Sia-IV (Angata et al., 2001Go).



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Fig. 1. Intracellular distribution and in vivo function of glycosyltransferase-(GTs-)EGFP. (A) Simultaneous imaging of intracellular distribution of glycosyltransferase-EGFPs and cell surface expression of carbohydrate structures. Cells were stained by rhodamine-conjugated lectins or antibodies followed by rhodamine-conjugated secondary antibody and examined by confocal microscopy as indicated under Materials and methods (red: rhodamine staining; green: EGFP fluorescence). (B) Coexpression of EGFP-conjugated C2GnT-I and ST6Gal-I with FLAG-tagged enzymes. CHO/C2GnT-I-EGFP or CHO/ST6Gal-I-EGFP was transiently transfected with C2GnT-I-FLAG or ST6Gal-I-FLAG, respectively. Twenty-four hours later, cells were permeabilized, stained with the anti-FLAG M2 antibody followed by rhodamine-conjugated secondary antibodies, and examined by confocal microscopy. EGFP fluorescence (green), anti-FLAG staining (red), and merged images (yellow) are presented.

 
We then asked whether EGFP could interfere with the intracellular localization of glycosyltransferases. To address this question, C2GnT-I and ST6Gal-I were selected because these enzymes were reported to localize at different Golgi compartments. ST6Gal-I has been repeatedly shown to reside in the trans-Golgi compartment (Roth et al., 1985Go; Paulson and Colley, 1989Go), whereas C2GnT-I has been shown to localize to cis/medial-Golgi (Skrincosky et al., 1997Go). Localization studies were performed by transfecting EGFP-conjugated C2GnT-I and ST6Gal-I constructs in CHO cells that stably express a FLAG-tagged version of the enzymes. FLAG tag is short enough so that no significant effect on Golgi localization was expected. Indeed, preliminary experiments showed that the staining by antibodies specific to C2GnT-I (Skrincosky et al., 1997Go) overlapped well with the staining of FLAG tag when both intact and FLAG-tagged C2GnT-I were coexpressed in CHO cells (data not shown). After transfection, cells were stained with M2 anti-FLAG monoclonal antibody, followed by tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody and examined by confocal microscopy. As shown in Figure 1B, when FLAG-tagged (red) and EGFP-tagged (green) enzymes are coexpressed in the same cell, they completely overlap in their Golgi distribution (yellow). Taken as a whole, the data from functional and localization studies indicate that the EGFP-tagged enzymes, like their intact counterparts, reach the compartments where their activities are normally expressed.

Most glycosyltransferases are released in culture media
Many glycosyltransferases are found as soluble enzymes in body fluids, but only a few have been studied with regard to secretion mechanisms; among them ST6Gal-I is probably the best documented (Ma and Colley, 1996Go; Kitazume-Kawaguchi et al., 1999Go; Chen et al., 2000Go). The capability of detecting EGFP spectrofluorimetrically, together with the high immunoreactivity of the anti-EGFP monoclonal antibody JL-8, gave us a strong impetus to seek for GTs-EGFP released into culture media and determine their molecular characteristics in comparison with their membrane-bound counterparts. EGFP-tagged enzymes in both media (M) and cells (C) were thus subjected to western blot analysis.

As shown in Figure 2, ST6Gal-I has by far the highest ratio—about 2.5-fold fluorescence in the medium versus cells, followed in order by C1-ß3GnT, FucT-I, ST3Gal-I, and C2GnT-I. Surprisingly no detectable amount of FucT-VII-EGFP could be found in the medium, indicating that this enzyme is not secreted from CHO cells. This result was confirmed by examining the proteins under reducing conditions and immunoblotting analyses. Similarly, FucT-VII-EGFP was not secreted from ovarian cancer cells either (Porok and El-Battari, unpublished data). Moreover, extracts of the transfected CHO cells contained 0.39 pmols/mg protein/h of FucT-VII activity, whereas no activity of FucT-VII was detected in the medium. These results indicate that FucT-VII-EGFP is properly folded and transported to Golgi apparatus but not secreted to the medium.



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Fig. 2. Secretion of GTs-EGFP as quantitated by EGFP fluorescence (Medium/cell ratio, upper panel) and analyzed by immunoblotting (lower panel). Culture media of CHO/glycosyltransferases-EGFP cells (about 80% confluency) were replaced with serum-free Opti-MEM and collected after 8 h incubation at 37°C and assayed for EGFP fluorescence. Media were then concentrated and compared to cell extracts by SDS–PAGE under reducing conditions and immunoblotting was performed as described under Materials and methods.

 
As shown in Figure 2 (lower panel) except for FucT-VII-EGFP, all the enzymes tested exist as both membrane and soluble species. According to their electrophoretic mobilities, most glycosyltransferase-EGFPs have apparent molecular weights around 75 kDa. Provided that the EGFP contribution likely accounts for 30 ± 2 kDa, the molecular masses of glycosyltransferases, deduced from at least 12 independent determinations under reducing and nonreducing conditions, are consistent with those based on their amino acid sequences and some glycosylation. Similarly, the ratio of in vitro enzyme activities in the cells and media of C2GnT-I, FucT-I, and ST6Gal-I was calculated and summarized in Table I. These values are consistent with the range of fluorescence ratios (compare data from Figure 2 and Table I), indicating that the released fluorescence reflects the amount of the enzymes in the media. These results clearly indicate that (1) with the exception of FucT-VII, protein secretion seems to be a common feature of glycosyltransferases; and (2) the level of protein secretion, expressed as medium/cell ratio, is different from one glycosyltransferase to another; the order being: ST6Gal-I>C1-ß3GnT>FucT-I>ST3Gal-I>C2GnT-I>>FucT-VII.


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Table I. GTs-EGFP enzyme activities in cell extracts, media, and fractions from UEA-I and SNA-I lectin chromatography

 
To gain insight into the kinetics of glycosyltransferase secretion, we chose ST6Gal-I because it has the highest M/C ratio, and followed the appearance of the enzyme in the medium at increasing incubation periods. As shown in Figure 3, fluorescence becomes detectable 6 h after the medium was replaced, followed by a linear increase of M/C ratio up to 2.3 after 18 h incubation (Figure 3B). We noticed that no plateau of M/C ratio could be obtained even after 48 h incubation (data not shown), indicating that secretion is a slow and nonsaturable process.



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Fig. 3. Kinetic of ST6Gal-I-EGFP secretion. Culture media were replaced with serum-free Opti-MEM and collected at indicated times. The amounts of secreted and cell-bound ST6Gal-I-EGFP were determined as described in Figure 2. Results are presented as EGFP fluorescence (A) or as the ratio between the fluorescence in media (Med) and cells (Cell) (B).

 
Secretion versus oligomerization of glycosyltransferases
It has been suggested that disulfide-mediated oligomerization of glycosyltransferases could represent a critical step for the retention of these proteins in the Golgi by preventing them from entering the secretory pathway (Ma and Colley, 1996Go; Chen et al., 2000Go). To address this hypothesis, media and cell extracts were prepared as described except that 2-mercaptoethanol was omitted and iodoacetamide was added to prevent disulfide bond formation. The samples were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by immunoblotting. As shown in Figure 4, all of the cell-associated C1-ß3GnT, C2GnT-I, FucT-I, FucT-VII, and ST6Gal-I form dimers (see lanes "Cell"), and ST3Gal-I is the only glycosyltransferase that does not form oligomers. Regarding secreted glycosyltransferases (see lanes "Med"), C2GnT-I and ST6Gal-I, two enzymes that form oligomers in the cell, provide only monomeric species in the media. The result with ST6Gal-I is consistent with the previously reported data for this enzyme (Ma and Colley, 1996Go; Chen et al., 2000Go). However, the secreted forms of C1-ß3GnT and FucT-I consist of dimers and monomers, with FucT-I being predominantly dimeric. It is noteworthy that both monomers and dimers of the secreted C1-ß3GnT have molecular weights higher than their membrane-bound counterparts (see later discussion).



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Fig. 4. Oligomerization and secretion of EGFP-conjugated glycosyltransferases. Cells (Cell) and Media (Med) were prepared as in Figure 2 except that 2-mercaptoethanol was omitted. The samples were then analyzed by SDS–PAGE and immunoblotting using the anti-EGFP antibody.

 
To rule out the possibility of EGFP-mediated oligomerization, cell extracts and concentrated media from CHOs expressing intact C2GnT-I were prepared under nonreducing conditions as described and compared to the samples from CHO/C2GnT-I-EGFP cells. The anti-C2GnT-I polyclonal antibody (Skrincosky et al., 1997Go) was used to probe intact C2GnT-I, whereas the chimeric protein (C2GnT-I-EGFP) was detected by anti-EGFP antibody. Figure 5 shows that the cellular intact C2GnT-I also forms a dimer while the soluble protein appears almost exclusively as a monomer, just as in the case of the EGFP-conjugated enzyme (compare samples from C2GnT-I-EGFP and intact C2GnT-I, in lanes Cell and Med).



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Fig. 5. Comparison of dimerization and secretion between C2GnT-I-EGFP and intact C2GnT-I. Culture media (Med) and cells (Cell) were collected and probed for C2GnT-I as in Figure 4 using anti-EGFP antibody (left) and anti-C2GnT-I antibodies (right). Both C2GnT-I-EGFP chimeric protein and intact C2GnT-I form dimers and are secreted.

 
Given that ST3Gal-I does not oligomerize but is fused to EGFP (see Figure 4) and according to the result with intact C2GnT-I, our data demonstrate that (1) the observed oligomerization is due to disulfide bonds formed between glycosyltransferase monomers rather than between EGFP molecules, (2) oligomerization does not occur with all glycosyltransferases, and (3) disulfide bond–mediated dimerization does not prevent glycosyltransferases from being secreted, as seen in C1-ß3GnT and FucT-I.

Secretion and oligomerization of glycosyltransferases in other cell lines
We then established three tumor cell lines stably expressing FucTI-EGFP. These include hepatocarcinoma HepG2, pancreatic BxPC3, and colonic HT29 tumor cells. Using these cell lines, we investigated whether secretion and oligomerization of FucT-I are cell-dependent phenomena. Cell extracts and concentrated media were prepared, and the samples were analyzed under nonreducing conditions by immunoblotting using the anti-EGFP antibody. Figure 6 shows that FucT-I, like in CHO cells (see Figure 4), forms monomers and dimers; the latter being predominantly present regardless of the tumor cell lines tested. Concerning the secretion, the enzyme was released from HepG2 and HT29, but soluble FucT-I was undetectable in BxPC3 medium. The secreted FucT-I-EGFP appears as monomers and dimers, both having molecular weights slightly lower than that of the cellular forms (Figure 6, lanes Med). This result indicates that unlike oligomerization, the secretion of FucT-I is different from one cell to another.



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Fig. 6. Differential dimerization and secretion of FucT-I from different cell lines. Cell extracts and media from HepG2, BxPC3, and HT-29 cells, which were stably expressing FucT-I-EGFP, were analyzed by SDS–PAGE under nonreducing conditions and immunoblotting with anti-EGFP antibody. Note that BxPC3 cells do not secrete the enzyme and that in all cases FucT-I dimers are predominantly represented both in cells and media.

 
Secreted and cell-associated glycosyltransferases are differently glycosylated
The data presented in Figures 2, 4, and 5 showed that the size of secreted C1-ß3GnT is larger than their cell-associated counterparts. We therefore hypothesized that the increased size of secreted C1-ß3GnT might be due to a negligible loss in the protein portion largely compensated by an increase of the carbohydrate part. To address this hypothesis, we compared the N-glycosylation status of cellular and secreted forms of C1-ß3GnT and extended the study to the other glycosyltransferases.

As illustrated in Figure 7A, PNGase-F, which cleaves N-linked oligosaccharides of glycoproteins (Tarentino et al., 1985Go), converts both the cellular and secreted forms of either C1-ß3GnT, C2GnT-I, or FucT-I into proteins having virtually the same molecular weights, whereas, as expected, the size of de-N-glycosylated sialyltransferases ST3Gal-I in the medium is slightly smaller. These results combined indicate that C1-ß3GnT is more heavily N-glycosylated than their cell-associated counterparts.



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Fig. 7. Analysis of glycosyltransferase-EGFP dimeric proteins before or after removal of N-glycans or sialic acid. Cell extracts and media were prepared as described and treated either with PNGase-F (A) or neuraminidase (Neur) (B) as indicated under Materials and methods and analyzed as shown in Figure 4. Anti-EGFP antibody was used to detect glycosyltransferases-EGFP. Note that the neuraminidase-treated samples (+) were put on adjacent lanes to facilitate the comparison between the treated cell-associated and soluble forms of the glycosyltransferases.

 
It has been shown that in CHO cells a bulk of sialyltransferases reside in the late Golgi compartments (Chege and Pfeffer, 1990Go). Although resident glycosyltransferases may likely shuttle between adjacent cisternae of the Golgi, the analysis of N-glycans of N-glycoproteins can provide us with valuable information on whether they entered the late Golgi compartments. We therefore asked whether the cellular and the secreted forms of a given glycosyltransferase have similar or different neuraminidase sensitivities. The results presented in Figure 7B show that the soluble form of C1-ß3GnT carries a significant amount of sialic acids compared to the cell-associated enzyme, which is consistent with the results shown in Figure 7A. Neither the soluble nor the membrane-bound C2GnT-I is neuraminidase sensitive. On the other hand, despite the fact that the cellular and the secreted FucT-I-EGFP appeared as doublets in this experiment, only the soluble species showed a small neuraminidase effect. Interestingly, sialyltransferases ST3Gal-I and ST6Gal-I are by far the most sialylated enzymes. In fact, both the cell-bound and the secreted forms of ST3Gal-I carry a large amount of sialic acid. The ST6Gal-I, however, unlike the ST3Gal-I, seems to be significantly sialylated only on its soluble form (Figure 7B).

For FucT-I and ST6Gal-I, bands of smaller molecular weights were produced more in Figure 7B than in Figure 7A. This is probably due to the presence of protease(s) active at acidic pH during the neuraminidase treatment. Apparently those proteases were not completely inhibited by the protease inhibitor cocktail used. Taken as a whole, these data indicate that the difference in size between cellular and secreted forms of C1-ß3GnT is due to the difference in N-glycosylation and the soluble forms of glycosyltransferases are generally more sialylated than their membrane-bound counterparts. The latter result strongly suggests that proteolytic cleavage of these glycosyltransferases takes place before trans Golgi.

Dimers of glycosyltransferases exhibit interglycosyltransferase glycosylation
It is possible that glycosyltransferases may use themselves as acceptors, resulting in increased glycosylation under certain conditions. To our knowledge, only polysialyltransferases have been reported to be able to perform such a kind of reaction, the so-called autopolysialylation (Muhlenhoff et al., 1996Go; Close and Colley, 1998Go; Angata et al., 2000Go). To determine if other glycosyltransferases can also use themselves as acceptors, we chose two chain-terminating enzymes, FucT-I and ST6Gal-I, because the glycoconjugates they produce can be easily and specifically isolated by lectin chromatography. We used agarose-immobilized UEA-I to isolate {alpha}1,2-fucosylated glycoconjugates and SNA-I-agarose to isolate {alpha}2,6-sialylated N-linked glycoproteins. In this experiment we used cell extracts prepared under nonreducing conditions to investigate which monomers or dimers carry the sugar transferred by the enzyme. As shown in Figure 8, after mixing the cell extract of CHO/FucT-I-EGFP with UEA-agarose, unbound fractions (flow-through) contain almost exclusively FucT-I monomers, whereas the material specifically eluted with 0.1 M fucose (eluate) is predominantly composed of dimers. Similar results were obtained with SNA-I lectin chromatography of CHO/ST6Gal-I-EGFP (Figure 8B). Thus dimers but not monomers of FucT-I and ST6Gal-I carry carbohydrate chains having terminal {alpha}1,2-linked fucose and {alpha}2,6-linked sialic acid, respectively. These results argue for an interglycosyltransferase glycosylation that takes place between two molecules under homodimer situation (one molecule using its partner as a sugar acceptor).



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Fig. 8. Homodimers of FucT-I and ST6Gal-I carry {alpha}1,2-linked fucose and {alpha}2,6-linked sialic acids, respectively. (A) Cell extracts were prepared from CHO/FucT-I-EGFP or CHO/ST6Gal-I-EGFP under nonreducing conditions as indicated in Materials and methods. The samples were then mixed with either UEA-I-Agarose (FucT-I) or SNA-I-Agarose (ST6Gal-I) and incubated batchwise for 2 h at 4°C. Effluent was monitored for fluorescence to measure the amount of EGFP. The rinse fractions (flow-through) and the eluates were collected in fractions (500 µl/fraction). (B) The effluent was analyzed by SDS–PAGE followed by immunoblotting with anti-EGFP monoclonal antibody JL-8.

 
It has been reported previously that the disulfide-formed homodimers of ST6Gal-I are catalytically inactive while retaining the galactose-binding property (Ma and Colley, 1996Go). Because lectin chromatography allows us to separate (at least mostly) dimers from monomers, we investigated the in vitro enzyme activities of flowthrough and eluate fractions. Table II shows that the Km of FucT-I, which was bound and eluted from UEA-1, was almost a half of Km of FucT-I which was not bound, while Vmax was the same for both fractions. In contrast, no difference in Km and Vmax was observed for ST6Gal-I between those bound and unbound to SNA-I. As shown in Figure 7A, dimeric forms of FucT-I and ST6Gal-I can be dissociated into their monomeric forms after reducing disulfide bonds. These results indicate that FucT-I dimers formed by disulfide bond(s) is more active than its monomer, whereas ST6Gal-I dimer is as active as its monomeric form.


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Table II. Cellular FucT-I and ST6Gal-I fractionated by UEA-I and SNA-I lectin chromatography

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this article, we examined proteolytic processing, dimer formation, and autoglycosylation of six different human glycosyltransferases, including C1-ß3GnT, C2GnT-I, FucT-I, FucT-VII, ST3Gal-I, and ST6Gal-I. Our choice was based on the following considerations; (1) these enzymes cover a large panel of glycosyltransferases based on sugar donor specificities, that is, C2GnT-1 and C1-ß3GnT use UDP-GlcNAc, FucT-I and FucT-VII use GDP-fucose and ST3Gal-I, and ST6Gal-I use CMP-NeuAc; and (2) these enzymes contribute to synthesize or attenuate the formation of tumor-associated antigens (Fukuda, 1996Go; Hakomori, 2002Go), and therefore it is important to analyze the molecular signals underlying their posttranslational modification.

Our results show that with the exception of FucT-VII, all the enzymes tested are secreted into the culture medium at different levels as measured by the medium-to-cell fluorescence ratios (M/C ratio). The highest secretion was observed with ST6Gal-I, followed by C1-ß3GnT, FucT-I, ST3Gal-I, and C2GnT-I (see Figure 2). Among all, the ST6Gal-I and FucT-VII are the only enzymes that have been studied previously with respect to secretion, and our results are consistent with the previously reported data for ST6Gal-I (Chen et al., 2000Go; Kitazume-Kawaguchi et al., 1999Go; Qian et al., 2001Go). De Vries and colleagues have shown that FucT-VII, which is not naturally secreted, becomes secretable after replacing its amino-terminal domain, including a portion of its stem region, with the first 64 amino-acid sequence of FucT-VI (amino acids 1–63) (de Vries et al., 2001Go). Analyses of the amino-terminal sequences of several truncated glycosyltransferases revealed that there is no unique consensus proteolytic cleavage sequences or sites on the stem region (Kitazume-Kawaguchi et al., 1999Go, and references therein). Likewise, examining sequences of the putative stem regions of 6 enzymes tested in the present study (up to 50 amino acids from the transmembrane domain) in comparison with the reported sequences of secreted glycosyltransferases from ST6Gal-I (Weinstein et al., 1997; Kitazume-Kawaguchi et al., 1999Go), GalNAcT-I (Homa et al., 1993Go), GM2 synthase (Jaskiewicz et al., 1996Go), {alpha}1,3-galactosyltransferase (Taylor et al., 2002Go), or ß4GalT-I (Masri et al., 1988Go) did not allow us to delineate any common proteolytic sequence (data not shown).

What, then, controls the activity of proteases that cleave glycosyltransferases and what could be their biological significance? The diversity of processed sequences within the stem region suggests that either (1) there could be as many proteases as cleavable sequences, or alternatively (2) the glycosyltransferases-acting proteases might be specific for a three-dimensional conformation of the stem region rather than of a amino acid sequence, and/or (3) these proteases might be associated with glycosyltransferases in the same Golgi compartment. In regard to the latter case, Kitazume and colleagues reported that ST6Gal-I is processed by the beta-secretase BACE1 that is responsible for the release in the brain, of the amyloid beta-peptide associated with the development of Alzheimer's disease (Kitazume et al., 2001Go). Interestingly, these researchers found that ST6Gal-I colocalized with BACE1 in the Golgi apparatus, suggesting that BACE1 acts on ST6Gal-I within the same intracellular compartment. It is thus tempting to hypothesize that glycosyltransferase-acting proteases might be compartmentalized in the Golgi, just like glycosyltransferases themselves. It has been shown that C2GnT-I, ST3Gal-I, and FucT-I reside at Golgi compartments close to each other (Whitehouse et al., 1997Go; Skrincosky et al., 1999; Priatel et al., 2000Go; Zerfaoui et al., 2000Go). Indeed the amounts of secreted forms of these enzymes, expressed as M/C ratios (see Figure 2, upper panel), are of similar order of magnitude. This is consistent with a hypothesis that a common protease is present at the same Golgi compartment as C2GnT-I, and such an enzyme cleaves these three glycosyltransferases. It is also possible that proteases shuttle between the Golgi apparatus and the endosomes, as proposed to explain the activity of the cathepsin D–like proteases on GM2 synthase (Jaskiewicz et al., 1996Go). On the other hand, it is likely that a protease, which can cleave the stem region of FucT-VII, is absent in the Golgi compartments where FucT-VII resides in CHO cells. It is possible that these processing depends on cell type because FucT-I was secreted from CHO cells (present study) but not from COS-1 cells (Smith et al., 1996Go). This conclusion was confirmed by expressing FucT-I in different cells in the present study, showing FucT-I was secreted from HepG2 and HT29 cells but not from pancreatic BXPC3 cells (Figure 6). Additional experiments are needed to verify these hypotheses.

Another parameter that may regulate proteolysis of glycosyltransferases is the presence or absence of disulfide bonds. In fact, a number of Golgi glycosyltransferases have been shown to form disulfide-bonded dimers including FucT-I (Andrew et al., 2000Go), FucT-VI (Borsig et al., 1998Go), GM2 synthase (Zhu et al., 1997Go), ß4GalT-I (Aoki et al., 1992Go; Yamaguchi and Fukuda, 1995Go), and ST6Gal-I (Chen et al., 2000Go). However, only a limited number of studies were dedicated to investigate a correlation between disulfide-mediated homodimerization and secretion of glycosyltransferases. In this regard, it was reported that mutating the Cys-139 or Cys-403 of ST6Gal-I resulted in a decreased secretion due to a more stable Golgi retention of the enzyme (Chen et al., 2000Go). Work with the GM2 synthase from Young's group has revealed that disulfide bonds are formed in the endoplasmic reticulum (Zhu et al., 1997Go), whereas proteolytic cleavage occurs in the Golgi (Jaskiewicz et al., 1996Go). Therefore one would speculate that dimerization of a given glycosyltransferase may directly influence the yield of retention in the Golgi and secretion in the medium. However, the secreted forms of six different glycosyltransferases tested are either almost exclusively monomer (C2GnT-I, ST3Gal-I, and ST6Gal-I) or almost exclusively dimer (FucT-I) or both monomer and dimer (C1-ß2GnT) (see Figure 4). These results indicate that disulfide-bonded dimerization does not necessarily prevent glycosyltransferases from being secreted.

Our data with human ST6Gal-I are consistent with numerous reports concerning the Golgi retention and secretion of the rat ST6Gal-I, showing that the cellular form is a combination of monomers and dimers and the secreted enzyme is monomeric (Ma and Colley, 1996Go; Kitazume-Kawaguchi et al., 1999Go; Chen et al., 2000Go). Interestingly, Qian and co-workers showed that the dimerization of the rat ST6Gal-I isoform STtyr involved the Cys-24 in the transmembrane domain and therefore provides an explanation for the absence of dimers in the secreted enzyme (Qian et al., 2001Go). Similarly, mutation of the cysteine residue at the transmembrane domain failed to direct ß4GalT-I into Golgi compartments (Aoki et al., 1992Go). Besides ST6Gal-I, C2GnT-I is the only enzyme that dimerizes in the cell and is secreted as a monomer. However, C2GnT-I does not have a cysteine residue within its transmembrane domain. Taken as a whole, our results suggest that the disulfide-mediated dimerization of glycosyltransferases does not seem to influence their further proteolytic processing and secretion.

The sizes of the soluble ST3Gal-I and, to some extent that of FucT-I, are as expected lower than those of their corresponding cell-associated species, suggesting that a proteolytic cleavage may have occurred at the level of their stem regions, just as in the case of the secreted ST6Gal-I. However, the size of the secreted C1-ß3GnT was unexpectedly bigger than its cell-associated form (see Figures 2 and 7), suggesting an increase in the degree of glycosylation in the secreted forms. In fact, the de-N-glycosylation of C1-ß3GnT with N-glycanase (PNGase F) resulted in cell-associated and secreted proteins having very close sizes (see Figure 7A). In addition, except for C2GnT-I, the soluble forms of all enzymes tested are more sialylated than their membrane-bound counterparts (Figure 7B). These results strongly suggest that the cell-associated enzymes may be arrested at their targeted Golgi compartment, whereas the proteolytically cleaved enzymes enter the secretion pathway where their N-glycans are further matured and sialylated.

Given that glycosyltransferases are glycosylated proteins, they can be regarded as potential substrate acceptors for glycosylation including autoglycosylation. So far, only polysialyltransferases have been reported to be able to perform such a kind of reaction, the so-called autopolysialylation (Mühlenhoff et al., 1996Go; Close and Colley, 1998Go; Angata et al., 2000Go). Using FucT-I and ST6Gal-I as model molecules and lectin chromatography to isolate {alpha}1,2-fucosylated or {alpha}2,6-sialylated glycoproteins (with UEA-I or SNA-I, respectively) we found that under nonreducing conditions, the fraction specifically retained on the lectin gel is mainly composed of dimers, whereas the majority of monomers were not retained (Figure 8). Thus FucT-I dimers carry {alpha}1,2-linked fucose and those of ST6Gal-I carry {alpha}2,6-sialylated N-glycans, indicating that dimers are able to interglycosylate (autoglycosylate) each other using their partner as an acceptor. Apparently, monomers are less efficient in performing such a reaction due to lack of close proximity to each other. These results are consistent with findings that the dimers of FucT-I and ST6Gal-I are more active or as active as their monomers (Table II). Further studies will be significant to determine if all glycosyltransferases are able to perform autoglycosylation and the biological significance of such autoglycosylation with regard to catalytic activity, Golgi compartmentalization, and secretion.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Restriction enzymes and Taq DNA polymerase were purchased from Promega (Madison, WI). Lipofectamine Plus reagent, neomycin (G418) and Ham's F12 medium were purchased from Invitrogen (Carlsbad, CA). CSLEX-1 was prepared from the hybridoma HB8580 (American Type Culture Collection, Rockville, MD). Rhodamine-conjugated UEA-I and biotin-conjugated SNA-I were from E-Y laboratories (TCS Biosciences, Buckingham, England). The same lectins immobilized on agarose beads were purchased from Calbiochem (San Diego, CA). Clostridium perfringens neuraminidase (Type X) was from Sigma-Aldrich (St. Louis, MO) and Flavobacterium meningosepticum N-glycosidase F (PNGase F) and endo-ß-galactosidase were from Roche Diagnostics (Meylan, France).

Preparation of plasmid DNAs and cell transfection***
Plasmid DNAs of EGFP-conjugated glycosyltransferases including C2GnT-I (Bierhuizen and Fukuda, 1992Go), FucT-I, and FucT-VII, were constructed in pcDNA3 as described previously (Zerfaoui et al., 2002Go). cDNAs encoding the EGFP-conjugated enzymes C1-ß3GnT (Yeh et al., 2001Go), ST3Gal-I (Zerfaoui et al., 2000Go), and ST6Gal-I (Weinstein et al., 1987Go) were constructed using the same approach as described. Briefly, pcDNA3.1/EGFP was made by excising the EGFP cDNA from pEGFP-N1 (Clontech, BD Biosciences, Palo Alto, CA) with EcoRI and NotI and ligating into EcoRI/NotI-digested pcDNA3.1(+) (Zerfaoui et al., 2002Go). cDNAs encoding glycosyltransferases were generated by polymerase chain reaction, gel purified, digested with BamHI and AgeI, and ligated into BamHI/AgeI digested pcDNA3/EGFP, producing an in-frame C-terminal fusion of glycosyltransferases to EGFP (referred to as GTs-EGFP). FLAG-tagged versions of C2GnT-I and ST6Gal-I were made by fusing the sequence coding for the FLAG peptide YKDDDDK at the 3' ends of the cDNA encoding C2GnT-I or ST6Gal-I by polymerase chain reaction. The resultant cDNAs were subcloned in pcDNA3.1 vector to generate pcDNA3.1/C2GnT-I-FLAG and pcDNA3.1/ST6Gal-I-FLAG, respectively.

CHO-K1 cells (American Type Culture Collection) were grown in Ham's F12 medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected with pcDNA3.1/GTs-EGFP or pcDNA3.1 alone for 3 h using Lipofectamine Plus reagent according to the manufacturer's instruction. After 2–3 weeks in the presence of 1 mg/ml G418, all resistant transfectants were collected to avoid clone-to-clone variation. These cells were then sorted by FACscan flow cytometry on the basis of EGFP fluorescence, and positive cells were maintained in complete HamF-12 medium containing 200 µg/ml G418. Mock-transfected CHO cells (referred to as CHOneo) were used as a negative control throughout this study.

Fluorescence microscopy
The intracellular distribution of EGFP-fusion proteins in living cells was visualized with an Olympus IMT-2 inverted microscope. Simultaneous imaging of intracellular distribution of glycosyltransferases and cell surface expression of their resultant carbohydrate structures was performed by confocal microscopy. To this end, cells were fixed in 1% paraformaldehyde in phosphate buffered saline (PBS) and incubated with the following reagents: rhodamine-conjugated lectin UEA-I was used to detect {alpha}1,2-linked fucose (on FucT-I-transfected cells) and biotin-conjugated SNA-I was used to detect {alpha}2,6-linked sialic acids on ST6Gal-I-expressing cells. The latter was further stained with TRITC-conjugated Extravidin (Sigma). The monoclonal antibody T305 was used to detect core 2 branched O-linked oligosaccharides on CD43-expressing cells (CHO/CD43 cells) (Bierheizen and Fukuda, 1992Go) after transfection with C2GnT-I-EGFP. These cells were counterstained with TRITC-conjugated anti-mouse IgG. Sialyl-Lewis x (sLex) structures generated by FucT-VII were probed with CSLEX-1 monoclonal antibody followed by TRITC-conjugated anti-mouse IgM. All incubations were carried out at 4°C in PBS containing 1% bovine serum albumin. Intracellular and cell surface fluorescence was then examined with a Leica confocal microscope (Wetzlar, Germany). Excitation of EGFP was performed using an argon ion laser at 488 nm, and TRITC fluorescence was excited using a green helium neon laser 543 nm. Images were processed with Metamorph Imaging system version 3.5 and transferred to Adobe Photoshop as TIFF files.

Secretion and oligomerization studies
Cells were cultured in six-well plates until they have reached 80% confluency, washed three times with serum-free Opti-MEM medium (Invitrogen), and incubated overnight in the same medium at 37°C, 5% CO2. Culture media (2 ml/well) were collected and centrifuged at 1000 rpm for 5 min to remove detached cells. Soluble glycosyltransferase-EGFP proteins in the medium were detected by fluorescence spectroscopy using a Perkin Elmer LS3B spectrofluorimeter set at an excitation and emission wavelengths of 473 nm and 506 nm, respectively. Cell monolayers were harvested using the nonenzymatic dissociation solution (Sigma), pelleted at 1000 rpm for 5 min and resuspended in the same volume of Opti-MEM as the media (2 ml) to allow a direct comparison between EGFP fluorescence in cells (referred to as C or Cell) and media (M or Med). Fluorescence intensity ratios (M/C) were determined for each cell line relative to cellular fluorescence. CHOneo cells and their medium were used as control.

Protein characterization was performed by SDS–PAGE under either reducing or nonreducing conditions. To this end, cells were pelleted and resuspended in the SDS sample buffer in the presence (reducing conditions) or the absence (nonreducing conditions) of 1% 2-mercaptoethanol. Under nonreducing conditions, iodoacetamide (100 mM, final concentration) was added in the sample buffer to alkylate free cysteine residues to prevent disulfide bond formation during sample preparation. Media were concentrated and processed as already described. A 10-fold concentrated sample buffer was added to media and cell extracts with respect to reducing or nonreducing conditions, and samples were resolved on SDS–PAGE (the protein contents were routinely between 10 and 50 µg). After electrophoresis, proteins were transferred onto nitrocellulose membranes, stained with Ponceau red, and blocked overnight at 4°C in Tris-buffered saline (150 mM NaCl, 20 mM Tris–HCl, pH 7.5) containing 0.05% Tween 20 and 5% nonfat milk. Membranes were probed with 1/500 anti-EGFP monoclonal antibody JL-8 (Clontech) followed by goat horseradish peroxidase–conjugated anti-mouse antibody. Visualization of bands was achieved using enhanced chemiluminescence detection kit (Amersham Bioscience).

Protein N-glycosylation
To compare N-glycosylation status of cell-associated and secreted forms of glycosyltransferases, cells and media were prepared as before and subjected to PNGase F or neuraminidase treatments. For PNGase F treatment, samples were resuspended in 100 mM Tris–HCl buffer, pH 8.6 (PNGase-F buffer) or 0.1 M sodium acetate buffer, pH 5.0 (neuraminidase buffer), both containing 1% Triton X-100, 1% 2-mercaptoethanol, and ethylenediamine tetra-acetic acid–free protease inhibitor cocktail (Roche). Extracts were then treated with 20 U/ml PNGase-F or 1 U/ml neuraminidase at 37°C for 120 min. Control samples did not receive the enzymes. For treatments of media, a 10-fold concentrated PNGase buffer was added to the samples and incubations were performed as described. After addition of a fivefold concentrated SDS sample buffer, aliquots were boiled for 3 min at 100°C and loaded onto SDS–polyacrylamide gel (7.5%) for electrophoresis.

Lectin chromatography
With this study we intended to investigate whether glycosyltransferases could transfer sugars on themselves (under monomer situation) or on neighboring partners (under oligomer situation). For this purpose, we used UEA-I-agarose to isolate {alpha}1,2-fucosylated FucTI-EGFP and SNA- I-agarose column to isolate {alpha}2,6-sialylated ST6GalI-EGFP. Cells expressing FucTI-EGFP and those expressing ST6GalI-EGFP were solubilized in 20 mM HEPES buffer, pH 8.5 containing 1% Triton X-100, 150 mM NaCl, 1 mM CaCl2 and 1 mM MgCl2 (lectin buffer). Detergent extracts were calibrated to 0.5 mg protein/ml, mixed with 500 µl slurry lectin-agarose resin, and incubated in batches for 2 h at 4°C. Gels were then pelleted, and unbound proteins were removed by several rinses with the lectin buffer. UEA- I-bound material was eluted with 0.1 M fucose, and SNA-I-agarose was eluted with 0.5 M lactose in the lectin buffer. All steps were monitored for EGFP fluorescence by spectrofluorimetry as described and by spectrophotometry at 280 nm.

In vitro enzyme assays
C2GnT-I activity was assayed as described previously, using Galß1,3GalNAc{alpha}-O-p-nitrophenyl (Toronto Research Chemicals, Downsview, Canada) as an acceptor and UDP-[6-3H]-GlcNAc as a donor (Yousefi et al., 1991Go). FucT-I and ST6Gal-I activities were assayed as described previously (Zhu et al., 1998; Goupille et al., 2000Go) using the synthetic oligosaccharide Galß1,4GlcNAcß1-octyl as an acceptor for both enzymes and the sugar donors GDP-[14C]-fucose (for FucT-I) or CMP-[14C]-NeuAc (for ST6Gal-I). For FucT-VII, NeuNAc{alpha}2,3Galß1, 4GlcNAcß1-octyl was used as an acceptor. After 2 h incubation at 37°C, reaction mixtures were diluted to 5 ml and applied to Sep-Pak C18 cartridges (Waters, Milford, MA). Columns were then rinsed with 20 ml water, and bound acceptors were eluted with methanol and counted. Picomoles of GlcNAc, fucose, or NeuAc transferred per h and per mg protein were calculated based on the specific activities of 980 cpm/pmole UDP-[6-3H]-GlcNAc, 880 cpm/pmole GDP-[14C]-fucose, or 990 cpm/pmole CMP-[14C]-NeuAc, respectively.


    Acknowledgements
 
We thank Drs. Rafael Oriol and John Lowe for providing cDNAs encoding FucT-I and FucT-VII, respectively, and Thu Gruenberg 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 (ICRETT) to A.E.B., and grants R37 CA33000 and R01 CA48737 by the National Cancer Institute to M.F.


    Footnotes
 
1 Present address: Corvas International Inc., San Diego, CA 92121 Back

2 To whom correspondence should be addressed; e-mail: minoru{at}burnham.org Back


    Abbreviations
 
C1-ß3GnT, core 1 extension ß1,3-N-acetylglucosaminyltransferase; C2GnT-I, core 2 ß1,6-N-acetylglucosaminyltransferase; CHO, Chinese hamster ovary cells; EGFP, enhanced green fluorescent protein; FucT-I, (H-enzyme) {alpha}1,2- fucosyltransferase-I; FucT-VII, {alpha}1,3-fucosyltransferase-VII; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SNA-I, Sambucco nigra agglutinin-I; ST6Gal-I, {alpha}2,6-sialyltransferase-I; ST3Gal-I: {alpha}2,3-sialyltransferase-I; TRITC, tetramethyl rhodamine isothiocyanate; UEA-I, Ulex europaeus agglutinin-I


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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