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
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Abstract |
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Key words: autoglycosylation / disufide bonds / Golgi retention / proteolysis / secretion
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Introduction |
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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, 2000). 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., 1993
; Nilsson et al., 1994
; Yamaguchi and Fukuda, 1995
; Kitazume-Kawaguchi et al., 1999
; Chen et al., 2000
; Sasai et al., 2001
). Using rat
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, 1996
). 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., 2001
). 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., 1987
; Masri et al., 1988
; Homa et al., 1993
). 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., 2002
).
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., 1997; Barbier et al., 2000
; Christensen et al., 2000
) and limited studies indicate that glycosyltransferases could also be subject to "autoglycosylation" (Mühlenhoff et al., 1996
; Close and Colley, 1998
; Angata et al., 2000
).
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), 1,2-fucosyltransferase-I (FucT-I), GalNAcT-I,
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., 2000
). 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., 2002
).
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 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.
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Results |
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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., 2001).
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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, 1996; Kitazume-Kawaguchi et al., 1999
; Chen et al., 2000
). 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 ratioabout 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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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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As illustrated in Figure 7A, PNGase-F, which cleaves N-linked oligosaccharides of glycoproteins (Tarentino et al., 1985), 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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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., 1996; Close and Colley, 1998
; Angata et al., 2000
). 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
1,2-fucosylated glycoconjugates and SNA-I-agarose to isolate
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
1,2-linked fucose and
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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Discussion |
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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., 2000; Kitazume-Kawaguchi et al., 1999
; Qian et al., 2001
). 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 163) (de Vries et al., 2001
). 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., 1999
, 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., 1999
), GalNAcT-I (Homa et al., 1993
), GM2 synthase (Jaskiewicz et al., 1996
),
1,3-galactosyltransferase (Taylor et al., 2002
), or ß4GalT-I (Masri et al., 1988
) 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., 2001). 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., 1997
; Skrincosky et al., 1999; Priatel et al., 2000
; Zerfaoui et al., 2000
). 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 Dlike proteases on GM2 synthase (Jaskiewicz et al., 1996
). 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., 1996
). 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., 2000), FucT-VI (Borsig et al., 1998
), GM2 synthase (Zhu et al., 1997
), ß4GalT-I (Aoki et al., 1992
; Yamaguchi and Fukuda, 1995
), and ST6Gal-I (Chen et al., 2000
). 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., 2000
). Work with the GM2 synthase from Young's group has revealed that disulfide bonds are formed in the endoplasmic reticulum (Zhu et al., 1997
), whereas proteolytic cleavage occurs in the Golgi (Jaskiewicz et al., 1996
). 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, 1996; Kitazume-Kawaguchi et al., 1999
; Chen et al., 2000
). 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., 2001
). Similarly, mutation of the cysteine residue at the transmembrane domain failed to direct ß4GalT-I into Golgi compartments (Aoki et al., 1992
). 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., 1996; Close and Colley, 1998
; Angata et al., 2000
). Using FucT-I and ST6Gal-I as model molecules and lectin chromatography to isolate
1,2-fucosylated or
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
1,2-linked fucose and those of ST6Gal-I carry
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.
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Materials and methods |
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Preparation of plasmid DNAs and cell transfection***
Plasmid DNAs of EGFP-conjugated glycosyltransferases including C2GnT-I (Bierhuizen and Fukuda, 1992), FucT-I, and FucT-VII, were constructed in pcDNA3 as described previously (Zerfaoui et al., 2002
). cDNAs encoding the EGFP-conjugated enzymes C1-ß3GnT (Yeh et al., 2001
), ST3Gal-I (Zerfaoui et al., 2000
), and ST6Gal-I (Weinstein et al., 1987
) 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., 2002
). 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 23 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 1,2-linked fucose (on FucT-I-transfected cells) and biotin-conjugated SNA-I was used to detect
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, 1992
) 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 SDSPAGE 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 SDSPAGE (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 TrisHCl, 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 peroxidaseconjugated 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 TrisHCl 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 acidfree 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 SDSpolyacrylamide 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 1,2-fucosylated FucTI-EGFP and SNA- I-agarose column to isolate
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-O-p-nitrophenyl (Toronto Research Chemicals, Downsview, Canada) as an acceptor and UDP-[6-3H]-GlcNAc as a donor (Yousefi et al., 1991
). FucT-I and ST6Gal-I activities were assayed as described previously (Zhu et al., 1998; Goupille et al., 2000
) 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
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.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: minoru{at}burnham.org
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Abbreviations |
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References |
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