3 Department of Cell Biology and Anatomy, Finch University of Health Sciences/the Chicago Medical School, North Chicago, IL 60064, USA
4 Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, College of Medicine, 1819 W. Polk Street, M/C 536, Chicago, IL 60612, USA
Received on June 14, 2002; revised on August 21, 2002; accepted on September 17, 2002
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Abstract |
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Key words: catalytic activity / cellular localization / Golgi / isoform / sialyltransferase
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Introduction |
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The ST6Gal I isoforms also exhibit trafficking and processing differences (Ma et al., 1997). STtyr is transiently localized in the Golgi and subsequently cleaved and secreted, whereas STcys is halted in the Golgi and remains intracellular. The different trafficking behaviors of the two isoforms might suggest a difference in their respective intra-Golgi localization, with a restriction of STcys to early and middle regions of the Golgi, and STtyr to trans-most regions of the Golgi for cleavage by proteases active in these low-pH compartments.
Other glycosyltransferase isoforms with very few differences in their overall amino acid sequences have been shown to differ in their activities and/or cellular trafficking. Most notable are the galactosyl- and N-acetylgalactosaminyl-transferases encoded by the ABO blood group locus, which differ by a total of only seven amino acids (Yamamoto et al., 1990). Of these variant amino acids, four have been shown to direct enzyme specificity for UDP-Gal (B blood group) and UDP-GalNAc (A blood group) sugar nucleotide donors. Other glycosylation enzymes are characterized by naturally occurring isoforms as well (Rajput et al., 1994
; Schneikert and Herscovics, 1995
). For example, Schneikert and Herscovics (1995)
found two naturally occurring
-mannosidase IB isoforms that differ in three amino acid residues. Analysis of recombinant soluble forms of these isoforms revealed that one of the amino acid changes (Phe to Ser at position 592) abolished activity, and the other two changes appeared to slow secretion.
The goals of this study were twofold. First, we wanted to determine whether the ST6Gal I isoforms demonstrate any differences in activity and substrate choice. Second, we aimed to determine whether these isoforms exhibit significant differences in intra-Golgi localization. Using purified recombinant STtyr and STcys proteins and in vitro analyses, we found that STcys is somewhat more active with standard asialoglycoprotein substrates. SNA lectin blotting showed that the ST6Gal I isoforms did not differ in their substrate choice when expressed in CHO cells and suggested that the small differences observed in catalytic activity in vitro may not be of consequence in vivo. Immunolocalization of the isoforms by light and electron microscopy demonstrated that STtyr was restricted to the Golgi in most transiently expressing cells, whereas STcys was found in the endoplasmic reticulum (ER), as well as in the Golgi. Interestingly, despite the differences in Golgi residence time indicated in our previous work (Ma et al., 1997), the isoforms showed no obvious differences in intra-Golgi localization and were found predominantly in the medial-trans but not in the trans-most regions of this compartment.
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Results |
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To determine more rigorously whether the two ST6Gal I isoforms differ in their activity toward the standard sialyltransferase substrates, asialo-1-acid glycoprotein, asialo-transferrin, and asialo-fetuin, we used purified recombinant soluble forms of each isoform. Recombinant baculoviruses expressing soluble catalytic domains of STtyr (consisting of amino acids 97403) and STcys (consisting of amino acids 89403) fused to a cleavable signal peptide were constructed and used to infect High Five insect cells. We found that although a shorter STcys protein consisting of amino acids 97403 was catalytically active, an additional eight amino terminal residues (8996) were required for efficient secretion. The secreted STtyr and STcys proteins were purified from cell medium using a combination of SP-Sepharose and CDP-gel chromatography. The purified recombinant enzymes were then used in sialyltransferase assays employing CMP-[14C]NeuAc and asialoglycoprotein substrates (Paulson et al., 1978
).
Surprisingly, the recombinant STcys isoform appeared to be somewhat more active than the recombinant STtyr isoform with all three substrates (Table I). Most notable was the threefold higher activity of STcys with asialo- transferrin, a glycoprotein substrate with complex type biantennary N-linked oligosaccharides that has been used in the past as a ST6Gal I-specific acceptor (Dall'Olio et al., 1992; Paulson et al., 1978
). To further explore this difference in activity with asialo-transferrin, we performed a kinetic analysis and calculated the Km and Vmax of each isoform with this substrate. We found that STtyr and STcys had comparable Km values of 147 µM and 163 µM, respectively, but that they differed in their maximum velocity (Vmax) with this glycoprotein substrate. The Vmax for STcys was 17.4 µM sialic acid transferred per h per mg enzyme, fourfold higher than the Vmax of STtyr (4.4 µM SA transferred per h per mg enzyme). Whether or not the differences in isoform activity observed in vitro were indicative of more general differences in activity and substrate choice in vivo was unclear at this point and led to the next study.
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To determine the basis for ER localization of the tagged STcys in the present studies (e.g., its high level of expression, the presence of the C-terminal epitope tag, and/or the cell type used for expression), we expressed STcys and STtyr in HeLa cells under the control of the weaker SV40 promoter (pSVL expression vector), and with the epitope tags removed. The tight Golgi immunostaining of STtyr remained high (usually over 90% of expressing cells) with or without the epitope tag (Figure 4A). For cells expressing STcys without the tag, we observed a Golgi-only pattern of localization in 3540% of the cells and prominent Golgi immunostaining combined with weak but discernible ER staining in another 4550% of the cells (Figure 4B). However, when STcys containing the V5 epitope tag was expressed under the control of the pSVL promoter, it was distributed primarily in the ER for 98% of the cells with a weak but detectable Golgi signal in 4060% of these cells (Figure 4C). Because these results indicated that the carboxy-terminal epitope tag affected localization of the STcys isoform, we used untagged isoforms in immunoelectron microscopic localization studies to pursue the question of intra-Golgi localization.
Immunoelectron microscopy confirms the differential ER/Golgi distribution of ST6Gal I isoforms and reveals a similar intra-Golgi localization
To extend the cell compartment analysis to the ultrastructural level, ST6Gal I isoforms were localized in HeLa cells using immunoperoxidase reactions. These studies focused on ST6Gal I proteins expressed under the control of the SV40 promoter and lacking the epitope tags. For the expressing cell shown in Figure 5A, STtyr immunoreactivity was restricted to the Golgi stacks and absent from other cellular structures. Consistent with immunofluorescence results shown in Figure 4, STcys was localized both within Golgi stacks and in the ER (Figure 5B). Significant product was present in the nuclear envelope for STcys but not for STtyr (Figure 5, double arrows).
Differential immunoreactivity across the Golgi stacks was observed in some cells, with some cisternae strongly reactive and others less reactive or apparently nonreactive (Figure 5). The Golgi stacks of HeLa cells characteristically consist of three cisterna, and predictions about orientation can be made by evaluating the position of the Golgi stack relative to the nucleus. Often, we observed that the end cisterna on one face of the Golgi, likely to be the trans cisterna (asterisk, Figure 5), was less immunoreactive with ST6Gal I antibodies than the medial cisterna. Sometimes part of the region at the entry, or cis face, was also less immunoreactive. To clarify the Golgi organization and orientation and better define the regions containing the ST6Gal I isoforms, we returned to immunofluorescence localization and determined the relationship of localized compartment-specific markers to the intra-Golgi distribution of STtyr and STcys in optically sectioned HeLa cells. As shown in Figure 6AC, the cis, or forming face of the Golgi, identified by immunostaining of the ERGolgiintermediate compartment (ERGIC) protein ERGIC-53 (Schweitzer et al., 1988; Klumperman et al., 1998
), was localized in a peripheral vesicular tubular pattern that encircled the more tubular STtyr-containing structures. STtyr exhibited some colocalization on the opposite face of the Golgi with TGN46, a marker protein characteristic of the trans-Golgi network (TGN) (Figure 6DF) (Ponnambalam et al., 1984
; Prescott et al., 1997
). Significant colocalization (not shown) was observed between STtyr and the predominantly medial Golgi enzyme
-mannosidase II (Velasco et al., 1993
). Similar relationships were observed between the Golgi-localized STcys and compartment- specific markers (data not shown). These consistent relationships revealed the overall cis-to-trans orientation of Golgi stacks. In consideration of this orientation, the ultrastructural analysis indicates that STtyr and STcys are distributed in the medial and trans Golgi but are relatively less abundant in the cis-most and trans-most regions of the Golgi (Figure 5).
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Discussion |
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Earlier in vitro analysis of ST6Gal I isoform activities using solubilized membranes suggested that STcys was five- to sixfold less active than STtyr using asialo-1 acid glycoprotein as a substrate (Ma et al., 1997
). In the present study, the in vitro comparison of the catalytic activities of purified, soluble, recombinant STtyr and STcys proteins demonstrated that STcys was slightly more active with asialo-
1 acid glycoprotein, as well as with two other sialyltransferase substrates, asialo-fetuin and asialo- transferrin (Table I).
How do we explain the differences in activity between the soluble recombinant STcys and the full-length isoform found in solubilized membranes? One possibility is that the enhanced ability of the full-length STcys protein to oligomerize may have compromised its activity in solubilized membranes. Previous work has demonstrated that nearly 100% of the STcys is recovered as Triton X-100 insoluble oligomers when Golgi membranes are solubilized at pH 6.4, the pH of the late Golgi (Chen et al., 2000). In contrast, only 13% of STtyr is recovered as insoluble oligomers under the same conditions. Interestingly, this oligomerization is pH-dependent because neither isoform is recovered as oligomers when Golgi membranes are solubilized at pH 8.0. These and other experiments suggest that the conformation of the STcys catalytic domain differs from that of STtyr in such a way as to enhance its ability to oligomerize. In our previous experiments, aberrant oligomerization of the detergent solubilized full-length STcys under the conditions of the assay system may have led to decreased substrate access and a lower apparent catalytic activity. Taking this possibility into consideration, we would suggest that the relative activities of the purified, soluble isoforms in this work more closely match the in vivo situation.
Our lectin blotting analysis demonstrated no detectable differences in the substrate choice of the enzymes expressed in CHO cells, again suggesting that the small activity differences observed in vitro may not be significant in vivo. However, we cannot exclude the possibility that the isoforms exhibit differences for other substrates not tested in our experiments. It may be that CHO cells are not optimal for substrate analysis because the ST6Gal I and many of its substrates are most highly expressed in liver (Kitagawa and Paulson, 1994). However, CHO cells are the only known cell line to be devoid of ST6Gal I and other enzymes that synthesize
2,6-sialylated structures recognized by SNA (Ma et al., 1997
; Smith et al., 1990
) and so were best for this analysis.
In the localization studies reported here, we characterize ST6Gal I isoform-containing Golgi regions after expression in cultured cells. The lack of significant STtyr in the earliest Golgi compartments is suggested by minimal overlap with ERGIC-53 and by the electron microscopic observation of nonreactive cis Golgi regions after immunoperoxidase staining (Figures 5 and 6). Representation of STtyr and STcys in the medial Golgi is supported by considerable overlap with -mannosidase II (data not shown), and in the trans Golgi, by partial overlap with TGN46 (Figure 6). Although endogenous ST6Gal I has been reported in the medial and trans Golgi cisternae as well as in the TGN of hepatocytes (Roth et al., 1985
) and intestinal cells (Roth et al., 1986
; Taatjes et al., 1988
), we do not find either isoform in the trans-most/TGN Golgi sites of transiently expressing HeLa cells. Our inability to detect the ST6Gal I isoforms in these Golgi regions was somewhat surprising in light of the observation by Rabouille et al. (1995)
that both ß1,4-galactosyltransferase and ST6Gal I (human STtyr) are localized in the trans cisternae and the TGN of stably expressing HeLa cells.
The similarity in late Golgi localization patterns for both the STtyr and STcys isoforms is intriguing in view of differences observed in their subsequent fates. As we showed in previous studies (Ma et al., 1997), STtyr is cleaved and secreted, but STcys is retained intracellularly. One possibility that would reconcile the observed localization patterns and isoform behaviors is that, for STtyr, cleavage is accomplished in the TGN, followed so rapidly by secretion of the soluble portion of the protein that the isoform is essentially undetectable in the TGN. Using this scenario, we would predict that the STcys never enters the TGN and is instead halted in the trans Golgi by a retention mechanism or by an efficient ongoing retrograde transport mechanism, as predicted by the cisternal maturation model discussed later (Cole et al., 1998
; Fullekrug and Nilsson, 1998
; Glick et al., 1997
; Glick and Malhotra, 1998
). Another possibility is that STtyr is slowly cleaved in the trans Golgi, allowing the enzyme to be detected in this compartment, whereas STcys cannot be cleaved due to conformational changes that block the cleavage site or because of extensive oligomerization.
In support of the first idea, we have observed that STcys is cleaved and secreted in some CHO cell clones. This finding suggests that STcys may be cleaved if it comes into contact with the appropriate proteolytic enzymes, but that in most cell types, the lack of cleavage may be due to the inability of STcys to move into compartments containing the proteases responsible for cleavage in the stem region. Perhaps the difference in localization discussed between stably expressed human STtyr reported by Rabouille et al. (1995) and rat STtyr described in our study reflects a difference in the site or rate of cleavage for the human versus the rat enzyme.
In general, high levels of expression favor increased ER localization for ST6Gal I isoforms and other Golgi proteins (Ma et al., 1997; Munro, 1991
; Teasdale et al., 1992
). Presumably this is due to an overwhelming of the protein folding machinery in the ER. However, we observed that the STcys isoform was more prevalent in the ER than STtyr, and its representation in the ER was increased by the presence of a C-terminal epitope tag. Why does STcys exhibit an increased ER localization relative to STtyr in transiently expressing cells? One possibility is that it requires additional time to fold, has more extensive interactions with chaperones, and as a result is retained in the ER for longer times than the STtyr. Our previous work (in which we analyzed the endoglycosidase H sensitivity of the N-linked oligosaccharides of both transiently expressed isoforms in COS-1 cells after a 1-h pulse labeling and 6 h chase period) suggested that similar amounts of both untagged isoforms had reached or passed through the medial Golgi at this time point and further suggested that differences in isoform ER exit time, if they existed, were subtle (Ma et al., 1997
). However, the observed differences in the ability of STtyr and STcys to form oligomers imply that the isoforms have localized conformation differences in their catalytic domains (Chen et al., 2000
). This feature may explain the more severe effect of the C-terminal V5 epitope tag on the intracellular distribution of STcys. In the case of tagged STcys, it is likely that the epitope tag complicates folding and could be the basis for increased ER localization of the tagged protein as a result of prolonged interaction with chaperones.
Another possibility is that retrograde transport contributes to the increased presence of untagged STcys in the ER. Work by Cole et al. (1998) demonstrated that significant quantities of resident Golgi enzymes recycle back to the ER. Studies by the Nilsson group (Lanoix et al., 1999
, 2001
) and others (Bonfanti et al., 1998
; Martinez-Menarguez et al., 2001
) support the cisternal maturation model of protein transport through the Golgi, which suggests that resident Golgi enzymes are maintained in cisternae via continuous retrograde transport in COPI-coated vesicles rather than by retention. We find that STcys is more stably localized in the Golgi, and STtyr is only transiently localized in this compartment and is ultimately cleaved and secreted (Ma et al., 1997
). In the context of the cisternal maturation model, we would expect that STcys is more readily incorporated into retrograde COPI vesicles than the STtyr. As a consequence, when the STcys is highly expressed, one might predict that it would be found in increasing concentrations in the ER as a result of its increased retrograde transport.
The results presented in this article suggest that the differential expression of the two ST6Gal I isoforms is unlikely to lead to dramatic differences in glycoprotein sialylation in various cell and tissue types. We have found that the two isoforms do not exhibit the significant differences in their catalytic activity or substrate choice that might have been expected because of the single amino acid difference in their catalytic domains. Furthermore, although STcys has a greater tendency to be found in the ER in transiently expressing cells, within the Golgi the two isoforms are localized in similar medial-trans regions. Nonetheless, it is clear that the Golgi residence time of the two ST6Gal I isoforms differs considerably and is likely to reflect the efficiency with which each is "retained" in Golgi cisternae. In future work we will evaluate the relative contributions of protein retention and active COPI-mediated retrograde transport to ST6Gal I isoform Golgi localization and determine whether differences in the ability of the isoforms to oligomerize affect the efficiency with which they use one or both of these localization mechanisms.
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Materials and methods |
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Recombinant baculoviruses were generated, amplified, and titered according to the Pharmingen Baculovirus Expression Vector System Instruction Manual. Briefly, Sf9 cells plated on 60-mm tissue culture dishes and maintained in TNM-FH insect medium (Pharmingen) were cotransfected with either STtyr or STcys coding sequences in the pAcGP67-A transfer vector (5 µg) and the Baculovirus Gold DNA (0.5 µg) using the supplied transfection buffers. After 5 days, supernatants were collected, viruses were amplified in Sf9 cells, and viral titers were determined by plaque assays.
Recombinant rat sialyltransferase protein expression and protein purification
High Five insect (Invitrogen, Carlsbad, CA) cells were plated in 100-mm tissue culture plates (0.8x107 cells per plate) and allowed to grow overnight in Ultimate Insect Serum-free Medium (Invitrogen) at 27°C. The next day, the cells (1.6x107 cells per plate) were infected with a recombinant virus stock at a multiplicity of infection of 10. Cell media were collected after 4 days of infection, and protease inhibitors were added (100 µg/ml leupeptin, 100 µg/ml aprotinin, 100 µg/ml pepstatin A in methanol, and 10 µg/ml freshly made phenylmethylsulfonyl fluoride in isopropanol). For each protein preparation the medium from 63x100 mm plates of infected cells was used (
630 ml). Collected media were clarified by centrifugation at 50,000 rpm in a Ti70 rotor for 1 h at 4°C. Supernatant was collected and sonicated three times for 15 s each with 30-s intervals between each sonication. The supernatant was then filtered using a 0.22 µ polyethersulfone bottle filter (Corning, Corning, NY).
Sialyltransferase protein was purified using a two-step procedure involving ion exchange chromatography on SP-Sepharose FF and affinity chromatography on CDP-gel. A 90 ml SP-Sepharose FF column was equilibrated using 200 ml 50 mM sodium cacodylate, pH 6.5, 0.15 M NaCl at 4°C. Clarified medium (at 4°C) was loaded, and the column was extensively washed with equilibration buffer (550 ml). Sialyltransferase proteins were then eluted using a linear 0.150.75 M NaCl gradient in 50 mM sodium cacodylate, pH 6.5. A total of 300 ml elution buffer is typically used, and 6-ml fractions are collected. The elution position of ST6Gal I isoforms was determined by immunoblotting with specific anti-ST6Gal I antibodies as described previously (Ma and Colley, 1996
), and the purity of the eluted protein was determined by SDSPAGE and staining with Coomassie brilliant blue (staining reagent, 0.1% Coomassie brilliant blue, 25% isopropanol, 10% methanol; destaining reagent, 25% isopropanol, 10% methanol). The ST6Gal I proteins typically eluted between 0.4 and 0. 55 M NaCl. These fractions were pooled and dialyzed in 500 ml 50 mM sodium cacodylate, pH 6.5, 0.3 M NaCl for 23 h at 4°C, and then in 500 ml 50 mM sodium cacodylate, pH 6.5, 0.15 M NaCl overnight at 4°C.
A 5-ml CDP-gel (Calbiochem, La Jolla, CA) column was equilibrated at 4°C in 50 mM sodium cacodylate, pH 6.5, 0.15 M NaCl (Paulson et al., 1977). The dialyzed ST6Gal I protein eluted from the SP-Sepharose FF column was loaded onto the CDP-gel column. The column was extensively washed with 50 mM sodium cacodylate, pH 6.5, 0.3 M NaCl (
400 ml). The ST6Gal I protein was then eluted using a salt gradient from 0.31 M NaCl in 50 mm sodium cacodylate, pH 6.5. Typically, a total volume of 70 ml of elution buffer was used, and fractions of 2 ml were collected.
Elution was monitored by optical density at =280 nm and SDSPAGE followed by Coomassie brilliant blue staining or immunoblotting. Molecular masses were calibrated on gels using marker proteins (BioRad Laboratories, Hercules, CA). STtyr eluted as a broad peak from 0.45 M NaCl to the end of the gradient. STcys also eluted as a broad peak from 0.3 M NaCl to the end of the gradient. Fractions containing the highest and most pure enzyme were dialyzed into and stored in 25 mM sodium cacodylate, pH 6.5, 0.3 M NaCl, 0.1% Triton CF-54, 50% glycerol at -20°C. Yields of STtyr were typically up to 10-fold higher than those of STcys.
Sialyltransferase activity assays
Sialyltransferase assays were performed as previously described (Ma and Colley, 1996). In each assay, 20 pmole of purified recombinant enzyme and 200300 µg of asialoglycoprotein substrate was used. CMP-[14C]NeuAc (249 mCi/mmol) was purchased from New England Nuclear (Boston, MA). Micromoles of sialic acid transferred per h per mg ST6Gal I protein were calculated based on a specific activity of 6607 cpm/nmole CMP-NeuAc.
Isolation and lectin precipitation of 2,6-sialylated glycoproteins from STtyr- and STcys-expressing CHO cells
EcR-CHO cells (Invitrogen) stably expressing either STtyr or STcys were plated on 100-mm dishes to reach 70% confluence after overnight incubation. The next day, the expression of the two ST6Gal I isoforms was induced by the addition of 1 µM (final concentration) of muristerone A (Invitrogen) in 5 ml of Minimal Essential Medium, 10% fetal bovine serum (FBS) for 16 h. Following induction, media were collected and cells washed with 10 ml phosphate buffered saline (PBS). Cells were scraped from plates in 1 ml PBS and pelleted at 14,000 rpm for 2 min. Cell pellets were lysed for 30 min on ice in 250 µl Tris-buffered saline (TBS), pH 8.0, 1% Triton X-100 containing 40 µg/ml each of aprotinin and leupeptin. Lysates were cleared by centrifugation at 14,000 rpm for 3 min. Glycoproteins from STtyr- and STcys-expressing cells were recovered from cell lysates and medium fractions using SNA-agarose (EY Laboratories, San Mateo, CA). Briefly, cell lysates and media were incubated with 50 µl of a 50% slurry of Sepharose CL-4B in PBS by end-over-end rotation for 1 h at 4°C to minimize nonspecific binding to the lectin-agarose. After a brief centrifugation, the supernatants were transferred to new tubes, 60 µl of a 50% slurry of SNA-agarose in PBS was added, and the samples again subjected to end-over-end rotation at 4°C for 1.5 h. The lectin/protein complexes were pelleted and washed three times with 0.01 M phosphate, pH 7.3, 0.15 M NaCl.
Electrophoresis and lectin blotting of STtyr and STcys
The lectin beads with bound protein were resuspended in 50 ml of Laemmli sample buffer (Laemmli, 1970) containing 5% ß-mercaptoethanol and separated on a 10% separating/5% stacking SDSPAGE gel by directly loading the beads into the gel wells. Following electrophoresis, total proteins were electrophoretically transferred to nitrocellulose membranes for 2 h at 500 mA. Lectin blotting was carried out using a previously described protocol by Young and colleagues (Zhu et al., 1998
). Nitrocellulose membranes were incubated for 1 h at room temperature with 30 µg digoxigenin-labeled SNA (Roche, Indianapolis, IN) in 30 ml TBS containing 1 mM MnCl2, 1 mM MgCl2, and 1 mM CaCl2. The membrane was then washed twice for 10 min with 50 ml TBS, 0.1% Tween-20, twice for 10 min with western blocking reagent (Roche), and then incubated with a 1:25,000 dilution of anti- digoxigenin-horseradish peroxidase in western blocking reagent for 30 min at room temperature. After incubation, the membrane was washed four times for 15 min with TBS, 0.1% Tween-20. Proteins were visualized using the BM Chemiluminescence Blotting Kit (Roche) per the manufacturer's protocol.
Cell culture and transfection for localization studies
CHO cells were cultured in Ham's F12 medium containing 10% FBS (Atlanta Biologicals, Norcross, GA) and 1% antibiotic-antimycotic mixture (Life Technologies, Rockville, MD) at 37°C in a humidified atmosphere of 5% CO2. HeLa cells were cultured under the same conditions, but using Dulbecco's Modified Eagle's High Glucose Pyruvate Medium (Irvine Scientific, Santa Ana, CA) in 10% CO2. For transfection and subsequent immunofluorescence studies, cells were grown on coverslips in tissue culture dishes for 40 h to reach 80% confluency. Transfection of CHO cells was performed with the Qiagen SuperFect transfection reagent (Qiagen, Valencia, CA), using 5 µg DNA with 10 µl SuperFect for each coverslip. The cells were incubated in DNA-SuperFect complex for 2 h before being transferred into fresh medium. HeLa cells were transfected using LIPOFECTIN transfection reagent, with 24 µg DNA and 5 µl LIPOFECTIN for each coverslip. The cells were incubated in DNALIPOFECTIN complex for 6 h before being transferred into fresh medium. After continued culture for the desired times (usually 1648 h), the transfected cells were fixed in methanol at 20°C for immunofluorescence staining.
Immunofluorescence localization and microscopy
Fixed cells were reequilibrated in PBS and blocked with 15% normal goat serum in PBS for 15 min prior to antibody incubations. Primary antibody incubations were performed at 37°C for 12 h followed by incubating in fluorophore-conjugated secondary antibody IgGs (Jackson ImmunoResearch, West Grove, PA) for 1 h at 37°C. Expressed proteins were detected with anti-ST6Gal I rabbit polyclonal antibodies (Ma and Colley, 1996). To evaluate compartment-specific distributions of ST6Gal I isoforms within the Golgi, localization of the isoforms was followed by incubations with antibodies to ERGIC-53,
-mannosidase II, and TGN46 (Serotec, Oxford, UK) for cis, medial, and trans-Golgi, respectively, and visualized by fluorophore-coupled secondary antibody IgGs. Antibodies to ERGIC-53 and to
-mannosidase II were generously provided by Dr. H.-P. Hauri (Schweizer et al., 1988
) and by Drs. K.W. Moremen and M.G. Farquhar (Velasco et al., 1993
). The specimens were observed using a Leica DMR microscope equipped with epifluorescence and phase optics. Images were captured by a Hamamatsu CCD camera and Openlab imaging progam (Improvision, Coventry, England). Optical sections were generated by deconvolving a z-series of 0.5-µM intervals using the nearest neighbor DCi module of the Openlab imaging program.
Immunoperoxidase localization and electron microscopy
Procedures were modifications of those used previously (Brown and Farquhar, 1984; Vertel et al., 1993
). Cells in monolayer were rinsed briefly with PBS; fixed for 45 min in 2% paraformaldehyde/75 mM lysine/10 mM NaIO4/37.5 mM Na phosphate, pH 6.2, according to McLean and Nakane (1974)
; and permeabilized by 0.05% saponin/PBS (buffer A) for 20 min at room temperature. Cells were incubated with anti-ST6Gal I rabbit polyclonal antibodies (diluted 1:300 in buffer A containing 5% goat serum) at 37°C for 1 h, followed by repeated washes in buffer A. Subsequently, cells were incubated in horseradish peroxidase-coupled goat anti-rabbit IgG Fab fragments (Organon Teknika, West Chester, PA) (diluted 1:75 in buffer A with 5% goat serum) for 1 h at 37°C and washed extensively in buffer A and 0.1 M sodium cacodylate buffer, pH 7.4, containing 4% sucrose (buffer B). Cells were fixed in 2.5% glutaraldehyde/buffer B for 30 min and washed in buffer B and 0.05 M TrisHCl, pH 7.4. Horseradish peroxidaselinked antibody products were visualized by incubation in 0.2% diaminobenzidine/0.05 M TrisHCl, pH 7.4, for 10 min following the addition of 0.03% H2O2.
After rinsing in 0.05 M TrisHCl, pH 7.4, and 0.1 M sodium cacodylate buffer, pH 7.4, containing 6% sucrose (buffer C), cells were postfixed in the cold for 1 h in 1% OsO4/1.5% KFe4(CN)6 in buffer C. After rinsing in buffer C, cells were further fixed with 1% tannic acid in buffer C for 15 min followed by wash with buffer C. Dehydration through a series of ethanol solutions was followed by passage through hydroxypropylmethacrylate (Electron Microscopy Sciences, Port Washington, PA) and tEpon solutions and embedment in tEpon (Tousimis, Rockville, MD). Sections were cut parallel to the plane of the cell monolayers, usually counterstained with lead citrate for 2 min, and observed in a Zeiss EM10C transmission electron microscope.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: karenc{at}uic.edu
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Abbreviations |
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References |
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