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Glycobiology Pages 947-953  

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time
Introduction
Results
Discussion
Materials and methods
Acknowledgments
References

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time

Ivan R.Nabi2, James W.Dennis1

Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada H3C 3J7 and 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada

Received on February 23, 1998; revised on March 23, 1998; accepted on March 29, 1998

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of [beta]1-6GlcNAc-TV, [beta]1-3GlcNAc-T(i), [beta]1-2GlcNAc-TI, [beta]1,4Gal-T, [alpha]2-6sialyl-T, and [alpha]2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-[beta]-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20°C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20°C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.

Key words: Golgi apparatus/LAMP/MDCK epithelial cells/polylactosamine

Introduction

Polylactosamine glycosylation consists of repeating Gal[beta]1-4GlcNAc[beta]1-3 disaccharide units preferentially added to [beta]1-6GlcNAc linked antennae attached to the trimannosyl core of complex-type N-linked oligosaccharides. [beta]1-6 branching of complex N-linked oligosaccharides is initiated by [beta]1-6N-acetylglucosaminyltransferase V (GlcNAc-TV) and produces the preferred substrate for [beta]1-3GlcNAc-T(i), the rate-limiting enzyme implicated in polylactosamine elongation (Holmes et al., 1987; Yousefi et al., 1992). [beta]1-6 branching and polylactosamine glycosylation are markers for cellular differentiation and transformation (Fukuda, 1985). Increased expression of [beta]1-6 branching and polylactosamine elongation is associated with tumor cell metastasis and blocking the biosynthesis of these structures by treatment with the mannosidase II inhibitor swainsonine inhibits the increased metastatic ability of the cells (Dennis et al., 1987). Polylactosamine glycosylation presents an interesting paradigm in that extension of the polylactosamine oligosaccharide chain requires the repeated action of two transferases, [beta]1-3GlcNAc-T and [beta]1-4Gal-T, on the same oligosaccharide substrate. In order for the polylactosamine chain to extend beyond a single [GlcNAc-Gal] unit, the oligosaccharide chain must be capable of interacting again with [beta]1-3GlcNAc-T, presumably within the same Golgi cisterna. Galactosyl transferase has been localized to trans Golgi cisternae (Roth and Berger, 1982) and the overlapping distribution of glycosyltransferases over Golgi cisternae has been described previously (Nilsson et al., 1993; Rabouille et al., 1995; Whitehouse et al., 1997). The action of core 2 GlcNAc-T and [beta]1-4Gal-T on Gal[beta]1-3GalNAc-ser/thr initiates a lactosamine branch (i.e., Gal[beta]1-3[Gal[beta]1-4GlcNAc[beta]1-6]GalNAc-) which allows for extension of O-glycans with polylactosamine. Core 2 GlcNAc-T has been localized to cis/medial Golgi cisternae and its Golgi localization proximal to the trans-Golgi location of chain-terminating [alpha]-2,3-sialyltransferase is important for the polylactosamine extension of core-2 O-glycans (Skrincosky et al., 1997; Whitehouse et al., 1997).

Polylactosamine glycosylation is added preferentially to select proteins, including the major lysosomal membrane glycoproteins, LAMPs or lgps, which contain 15-17 N-linked oligosaccharide chains (Carlsson et al., 1988; Heffernan et al., 1989). Increasing the residence time of newly synthesized LAMPs in the Golgi apparatus by incubation of HL-60 cells at 21°C results in the synthesis of extended LAMP polylactosamine chains (Wang et al., 1991). Increased polylactosamine glycosylation of LAMPs at early times following plating has been observed during polarization of CaCo-2 and MDCK epithelial cells (Youakim et al., 1989; Nabi and Rodriguez-Boulan, 1993). In MDCK cells, the degree of polylactosamine glycosylation of endogenous LAMP-2 is inversely proportional to the rate of passage of newly synthesized protein through the Golgi apparatus (Nabi and Rodriguez-Boulan, 1993).

Table I. Glycosyltransferase expression in 1 and 3 day MDCK cells
MDCK GlcNAc-TV GlcNAc-T(i) 6SA-T 3SA-T Gal-T GlcNAc-T1
(1 day) 230 ± 28 319 ± 41 132 ± 22 594 ± 69 20.2 ± 1.2 8.1 ± 0.4
(3 day) 250 ± 50 418 ± 9 126 509 ± 27 18.0 ± 0.6 8.8 ± 1.1
Enzyme activities are presented as pmol/h/mg. The results are the mean ± SD of two to four experiments performed in duplicate.GlcNAc-TV, [beta]1-6GlcNAc-TV; GlcNAc-T(i), [beta]1-3GlcNAc-T; 6SA-T, [alpha]2-6sialyltransferase; 3SA-T, [alpha]2-3sialyltransferase; Gal-T, [beta]1-3 galactosyltransferase; GlcNAc-T1, [beta]1-2GlcNAc transferase 1.

We show here that the differential polylactosamine glycosylation of LAMP-2 in MDCK cells plated for 1 or 3 days is not attributable to glycosyltransferase expression levels and is maintained after nocodazole mediated disruption of gross Golgi architecture and dispersion of Golgi clusters. While previous reports detected no effect of colcemid mediated disruption of the Golgi apparatus on total protein glycosylation (Stults et al., 1989), nocodazole mediated disruption of the Golgi apparatus does influence the terminal N-linked glycosylation of MDCK LAMP-2. Use of a 20°C temperature block to prevent protein exit from the Golgi apparatus (Griffiths et al., 1985) resulted in an equivalent increased molecular size of LAMP-2 from 1 and 3 day MDCK cells in the presence or absence of nocodazole, demonstrating that Golgi residence time determines the extent of polylactosamine glycosylation irrespective of Golgi integrity.

Results

LAMP-2 of MDCK cells plated at high density on polycarbonate filters for 1 day exhibits an increased size relative to LAMP-2 from cells plated under identical conditions for 3 days (Nabi and Rodriguez-Boulan, 1993). The increased size of 1 day LAMP-2 was shown to be due to polylactosamine glycosylation as it could be reduced by treatment with endo-[beta]-galactosidase, specific for polylactosamine extension. One day LAMP-2 also exhibited increased binding to the lectin L-PHA, specific for the [beta]1-6 branching of polylactosamine glycosylation (Nabi and Rodriguez-Boulan, 1993). To assess whether the increased polylactosamine glycosylation of LAMP-2 in MDCK cells plated for 1 day can be attributed to altered glycosyltransferase expression, we measured the activity of [beta]1-6GlcNAc-TV, [beta]1-3GlcNAc-T(i), [beta]1-2GlcNAc-TI, Gal-T, [alpha]2-6sialyl-T, and [alpha]2-3sialyl-T in cell lysates of MDCK cells plated for 1 or 3 days (Table I). [beta]1-6GlcNAc-TV is responsible for the initiation of the [beta]1-6 antennae propitious for polylactosamine chain elongation while [beta]1-3GlcNAc-T and [beta]1-4Gal-T are responsible for chain elongation. [beta]1-6GlcNAc-TV expression does not significantly differ between 1 and 3 day MDCK cells while [beta]1-3GlcNAc-T(i) expression levels are increased by 25% in 3 day cells which exhibit decreased polylactosamine glycosylation. [beta]1-2GlcNAc-TI levels as well as [alpha]2-6 and [alpha]2-3sialyl-T activities, which could conceivably terminally block lactosamine chain elongation, do not show any differences between 1 and 3 day MDCK cells. The increased polylactosamine glycosylation of 1 day MDCK LAMP-2 cannot therefore be attributed to altered glycosyltransferase activity as measured in cell lysates.


Figure 1. Nocodazole disrupts the interconnected Golgi network. 1 day (a, b) and 3 day (c, d) MDCK cells were treated with 20 µM nocodazole for 30 min at 4°C and then for 30 min at 37°C to depolymerize the microtubules of MDCK cells. The cells were immunofluorescently labeled with antibodies to [beta]-COP followed by FITC-conjugated anti-rabbit secondary antibodies and the nuclei labeled with propidium iodide. Serial optical sections encompassing the complete cell height were generated. [beta]-COP labeled sections just apical of the nucleus are presented. An interconnected Golgi apparatus is visualized in untreated 1 day (a) and 3 day (c) MDCK cells. Treatment with nocodazole (b, d) results in the dispersion of the isolated Golgi clusters throughout the cell. Scale bar, 20 µm.

During development of a polarized MDCK epithelial monolayer, Golgi morphology changes from compact at one side of the nucleus in unpolarized cells, to spread around the nucleus at early stages of polarization before reaching its compact apiconuclear localization in polarized epithelial cells (Bacallao et al., 1989). To assess whether changes in Golgi morphology between 1 and 3 day MDCK cells are implicated in LAMP-2 polylactosamine glycosylation, the Golgi apparatus of 1 and 3 day MDCK cells was disrupted by treatment with 20 µM nocodazole for 30 min at 4°C followed by a further 30 min incubation in the presence of nocodazole at 37°C. Golgi morphology was assessed by immunofluorescent labeling with antibodies to [beta]-COP (Figure 1). Confocal microscope sections from the region just apical of the nucleus reveal the presence of an apiconuclear Golgi network in both 1 day and 3 day cultures (Figure 1a,c). Golgi networks are more evident in the 3 day cultures which may reflect a more compact apiconuclear Golgi apparatus in cells cultured for longer times at confluence (Figure 1c). Following nocodazole treatment, the Golgi labeling is dispersed to discrete clusters and Golgi networks are not observed in either 1 day or 3 day MDCK cultures (Figure 1b,d).


Figure 2A. The differential poylactosamine glycosylation of MDCK LAMP-2 is maintained after nocodazole treatment. (A) LAMP-2 was immunoprecipitated from 1 day and 3 day MDCK cell cultures metabolically labeled with 35S-methionine/cysteine for 3 h and chased for 16 h. Nocodazole treated cells were incubated with 20 µM nocodazole at 4°C for 30 min prior to incubation with nocodazole for either 30 min (Noc 30) or 2 h at 37°C (Noc 120), as indicated, prior to metabolic labeling.

Figure 2B. Nocadozole was continually present in the medium of the treated cells. (B) Metabolically labeled LAMP-2 (see above) was immunoprecipitated from 1 day and 3 day MDCK cells, untreated (-) or treated with 20 µM nocodazole at 4°C for 30 min and then at 37°C for 30 min (Noc 30), and then digested with endo-[beta]-galactosidase (E-gal) as indicated. The increased molecular size of 1 day LAMP-2 in both untreated and nocodazole treated cells is reduced by treatment with endo-[beta]-galactosidase, specific for polylactosamine chain extension. Molecular weight markers are as indicated.

Immunoprecipitation of LAMP-2 after metabolic labeling revealed that the differential size of LAMP-2 in 1 day and 3 day MDCK cells was maintained even in the nocodazole treated cells (Figure 2A). The efficiency of metabolic labeling and the rate of maturation of the protein was reduced in the nocodazole treated cells. In order to augment the labeling of mature LAMP-2, cells were labeled for 3 h and chased for 16 h, all in the presence of nocodazole for the treated cells. The number of Golgi clusters formed in HeLa cells has been shown to increase during longer incubations with nocodazole stabilizing after a 2 h period (Cole et al., 1996). Even after preincubation of MDCK cultures with nocodazole for 2 h, the differential migration of 1 day and 3 day LAMP-2 was maintained (Figure 2A). For both untreated cells and cells pretreated with nocodazole for either 30 or 120 min, 1 day LAMP-2 was consistently 4-6 kDa larger than LAMP-2 from 3 day MDCK cells. The increased molecular size of 1 day LAMP-2 could be reduced by treatment with endo-[beta]-galactosidase, demonstrating that the increased size of 1 day LAMP-2 is due to polylactosamine chain extension in both untreated and nocodazole treated cells (Figure 2 B). The increased polylactosamine glycosylation of LAMP-2 in 1 day MDCK cells is therefore maintained following nocodazole mediated disruption of the Golgi apparatus.

Interestingly, LAMP-2 in nocodazole treated cells, cultured for either 1 or 3 days, migrates faster (~5 kDa) than LAMP-2 from the equivalent untreated cells (Figure 2A). The reduced size of LAMP-2 in nocodazole treated cells was maintained after endo-[beta]-galactosidase treatment, indicating that this was not due to a modification of polylactosamine glycosylation (Figure 2B). LAMP-2 from 1 and 3 day MDCK cells acquires endo H resistance with a half-time of 25 min (Nabi and Rodriguez-Boulan, 1993). The high-mannose precursor form of LAMP-2 immunoprecipitated from cells pulsed for 30 min and not chased migrates with an equivalent molecular size from untreated and nocodazole treated cells (Figure 3). The reduced size of mature LAMP-2 from nocodazole treated cells can be seen in the minor fraction of the protein which acquired terminal glycosylation. Nocodazole treatment therefore influences the terminal N-linked glycosylation of LAMP-2, if not its polylactosamine glycosylation.


Figure 3. Nocodazole treatment modifies LAMP-2 glycosylation. LAMP-2 was immunoprecipitated from 3 day MDCK cells untreated (-) or treated with 20 µM nocodazole for 30 min at 4°C and then for 90 min at 37°C (Noc 90) which were pulsed for 30 min with 35S-methionine/cysteine and not chased. The precursor form of LAMP-2 (90 kDa) is indicated by an arrow. Molecular weight markers are as indicated.

Incubation of cells at 21°C, increasing the time of association of newly synthesized proteins with the Golgi apparatus, is associated with increased molecular size of LAMPs due to polylactosamine chain elongation in HL-60 cells (Wang et al., 1991). Similarly, incubation of MDCK cells at 20°C following a 30 min metabolic pulse resulted in the increased molecular size of LAMP-2 (Figure 4). LAMP-2 from both 1 and 3 day cells exhibited a similar molecular size when chased at 20°C for various times and an identical result was obtained in the presence of nocodazole. The increased size of LAMP-2 at 20°C is best seen in Figure 4A; in this experiment, LAMP-2 migrated almost at the gel front such that the differential migration of LAMP-2 at 20°C and 37°C is better visualized. However, the ability to detect the differential migration of LAMP-2 in cells chased at 37°C was reduced. The rate of maturation of LAMP-2 was decreased at 20°C as the precursor form of the protein could be detected at 2.5 and 5 h of chase (Figure 4A). The increased size of mature LAMP-2 could be detected after a 2.5 h chase (Figure 4A) and did not increase following incubations up to 24 h (Figure 4B). The increased size of LAMP-2 following a chase at 20°C is best explained by the retarded transit of the protein through the Golgi stacks and not by retention of the protein in the TGN.


Figure 4. Increased molecular size of LAMP-2 following a 20°C block. MDCK cells plated for 1 or 3 days in the absence or presence of 20 µM nocodazole were pulsed for 30 min with 35S-methionine/cysteine and then chased at either 37°C or 20°C. (A) Cells were chased at 37°C for 2.5 h or at 20°C for either 2.5 or 5 h, as indicated. The precursor form of the of LAMP-2 is present in the samples chased at 20°C. LAMP-2 migrated almost at the front of the gel (arrowhead) and the increased size of mature LAMP-2 at 20°C is best seen in this gel; however, the ability to detect size differences between LAMP-2 chased at 37°C is reduced. (B) Cells were chased at 37°C for 2.5 h or at 20°C for either 7 or 24 h, as indicated. The size of LAMP-2 does not increase with extended incubation at 20°C. Molecular weight markers are as indicated.

Discussion

Polylactosamine chains are added preferentially to [beta]1-6 branched complex oligosaccharides, the result of the action of [beta]1-6 GlcNAc-TV, and chain extension is due to the repeated action of [beta]1-3GlcNAc-T(i) and [beta]1-4Gal-T. [beta]1-4 Gal-T activity is widely expressed and appears to be present in excess in most tissues. Therefore, the rate-limiting step in polylactosamine biosynthesis has been suggested to be either the [beta]1-3GlcNAc-T(i) or GlcNAc-TV catalyzed reactions (Holmes et al., 1987; Yousefi et al., 1992). We could detect no differences in glycosyltransferase activities which could explain the differential polylactosamine glycosylation of 1 day and 3 day LAMP-2 (Table I). The 25% increase in [beta]1-3GlcNAc-T(i) activity detected in 3 day MDCK cells might serve to increase polylactosamine glycosylation while the converse was observed. Transfection of CHO cells with 1,2fucosyltransferase resulted in a decrease in both polylactosamine chain extension and sialylation (Prieto et al., 1997). Since 1,2fucosyltransferase, 2,3 sialyltransferase, and 1,3GlcNAc-T(i) target the same substrate, terminal galactose residues, competition between these enzymes could conceivably affect polylactosamine synthesis. However, sialyltransferase expression levels were similar in 1 day and 3 day MDCK cells and previous reports did not identify significantly increased levels of 3 day LAMP-2 recognized by the fucose-specific lectin UEA-1 (Nabi and Rodriguez-Boulan, 1993). We cannot exclude the possibility that differences in sugar-nucleotide import into the lumen of the Golgi apparatus may regulate glycosyltransferase activity. However, our results do indicate that it is not glycosyltransferase activity levels that are responsible for the differential polylactosamine glycosylation of 1 day and 3 day LAMP-2.

The differential activity of the transferases responsible for polylactosamine chain elongation must therefore be regulated in 1 and 3 day MDCK cells by alternate mechanisms. Changes in cellular physiology may result in altered gene expression or post-translational modification of the enzyme affecting catalytic activity or physical interactions with other enzymes. Regarding gene expression, a second [beta]1-4Gal-T enzyme has been recently cloned (Sato et al., 1997), and shows different but overlapping acceptor specificity compared to that of the known enzyme (Shaper et al., 1986). The GlcNac-T(i) enzyme has recently been cloned (Sato et al., 1997); however, it remains unclear whether other genes exist which encode this activity. In addition to changes in expression of related glycosyltransferase genes, posttranslational modifications of the enzymes could affect polylactosamine chain length. For example, [beta]1-4Gal-T is subject to phosphorylation by p58, a ser/thr-kinase which has been shown to enhance enzyme activity (Bunnell et al., 1990). However, the regulation and physiological significance of Gal-T phosphorylation remains unclear.

A slower rate of passage of newly synthesized LAMP-2 through the Golgi apparatus correlates with the increased polylactosamine glycosylation of 1 day MDCK LAMP-2 (Nabi and Rodriguez-Boulan, 1993). The Golgi apparatus might be more broadly distributed in MDCK cells plated for 1 day prior to acquisition of a more highly polarized epithelial phenotype (Bacallao et al., 1989). Indeed, the presence of compact apiconuclear Golgi networks were more evident in 3 day MDCK cultures (Figure 1). We previously suggested that a more dispersed Golgi apparatus in 1 day MDCK cells might slow the rate of passage of LAMP-2 through the Golgi permitting the prolonged interactions of newly synthesized glycoproteins with [beta]1-3GlcNAc T(i) and [beta]1-4Gal-T required to generate extended polylactosamine chains (Nabi and Rodriguez-Boulan, 1993). If this were the case, disruption of the gross morphology of the Golgi apparatus should eliminate any differences between the rate of Golgi transit of LAMP-2 in 1 and 3 day MDCK cells.

Depolymerization of the microtubule cytoskeleton results in the dispersion of numerous small Golgi clusters throughout the cell which retain the characteristic stacked Golgi morphology (Thyberg and Moskalewski, 1985). Following nocodazole treatment of MDCK cells, the Golgi apparatus is fragmented in both 1 day and 3 day cells and apiconuclear Golgi networks are no longer detected (Figure 1). Nocodazole treatment did reduce the extent of LAMP-2 labeling (Figure 2) as well as slow the rate of maturation of newly synthesized LAMP-2 (not shown) as described previously for VSV G protein (Cole et al., 1996). The apparent molecular weight of LAMP-2 decreased slightly with time in the presence of nocodazole (Figure 2). The smaller size of LAMP-2 in nocodazole treated cells was a consequence of differential terminal glycosylation (other than polylactosamine) as the migration of the high-mannose precursor form of LAMP-2 is not altered following nocodazole treatment (Figure 3). The nocodazole treatment used in these studies therefore induced both morphological and functional alterations of the Golgi apparatus. Colcemid-mediated dispersal of the Golgi apparatus was previously shown not to influence either the processing of N-linked oligosaccharides, including polylactosamines, or surface delivery of total cellular glycoproteins (Stults et al., 1989). Increased expression of terminal GlcNAc residues on glycoproteins of nocodazole treated HT29 cells is associated with mitotic arrest but was not observed at the shorter times of incubation used in this study (Chou and Omary, 1994; Haltiwanger and Philipsberg, 1997). However, it is possible that the altered migration of LAMP-2 following short-term nocodazole treatment reflects a similar increase in structural heterogeneity due to decreased efficiency of the processing pathway that encompasses GlcNAc-branching as well as terminal glycosylation. The detailed structural modification in the N-linked oligosaccharide chain of LAMP-2 following nocodazole-mediated Golgi disruption and whether it is specific to LAMP-2 remain to be determined.

Golgi dispersion following nocodazole treatment does not eliminate the differential polylactosamine glycosylation of LAMP-2 in 1 and 3 day MDCK cells. Increasing the preincubation time with nocodazole to 2 h, previously shown to result in an increased number of Golgi clusters in HeLa cells (Cole et al., 1996), did not affect the differential migration of LAMP-2 in SDS-PAGE. The fidelity of the terminal polylactosamine glycosylation of LAMP-2 is therefore not significantly compromised following disruption of gross Golgi morphology by nocodazole treatment and the formation of numerous clusters of Golgi stacks in both 1 and 3 day cells. Aspects of Golgi morphology are essential for polylactosamine synthesis as the addition of polylactosamine chains to N-glycans is completely inhibited by brefeldin A, which fuses Golgi stacks with the endoplasmic reticulum (Sampath et al., 1992). Our results demonstrate that the Golgi structure and organization that is required for polylactosamine chain extension in 1 day MDCK cells is retained within the numerous, small Golgi clusters generated by nocodazole treatment and localized to ER exit sites (Thyberg and Moskalewski, 1985; Cole et al., 1996).

Extended incubation of HL-60 cells at 21°C, slowing passage of newly synthesized protein through the Golgi apparatus and increasing their residence time in the Golgi apparatus, resulted in increased LAMP polylactosamine glycosylation (Wang et al., 1991). Similarly, incubation of MDCK cells at 20°C results in the increased molecular size and polylactosamine glycosylation of LAMP-2. A 20°C block results in the accumulation of newly synthesized VSV G protein in a swollen trans-Golgi network (Griffiths et al., 1985), distinct from the penultimate trans cisterna to which galactosyl transferase is localized (Roth and Berger, 1982). MDCK LAMP-2 achieved a maximal size after 2.5 h at 20°C indicating that retention of the protein in the trans-Golgi network is not responsible for the increased size of the protein. The ability of 3 day cells to synthesize LAMP-2 of an equivalent size and extent of polylactosamine glycosylation as 1 day cells, albeit at 20°C, clearly indicates that the decreased polylactosamine glycosylation of 3 day LAMP-2 relative to 1 day LAMP-2 at 37°C is not due to the inability of the Golgi apparatus of 3 day cells to synthesize extended polylactosamine chains. The maturation of LAMP-2 occurs more slowly at 20°C (Figure 4), evidence of a slower passage of the protein through the secretory pathway. A slower passage of LAMP-2 through the Golgi apparatus at 20°C would eliminate the differential rates of targeting that exist at 37°C resulting in an equivalent increased polylactosamine glycosylation in 1 and 3 day MDCK cells.

These results corroborate the previously reported correlation between polylactosamine glycosylation and the rate of basolateral delivery of LAMP-2 in MDCK cells and demonstrate that the rate of Golgi transit of LAMP-2 is indeed the critical element determining the extent of polylactosamine glycosylation of LAMP-2 during establishment of the polarized epithelial monolayer of MDCK cells (Wang et al., 1991; Nabi and Rodriguez-Boulan, 1993). Secretory protein transport through the Golgi apparatus has been proposed to be a bulk flow process (Pfeffer and Rothman, 1987); however, evidence for signal mediated exit of select proteins from the Golgi has been described recently (Musch et al., 1996). Our studies of LAMP-2 in MDCK cells suggest that mechanisms exist which regulate not only protein exit from the Golgi but also the rate of passage of proteins through the Golgi stack. Due to the retarded acquisition of endo H resistance and decreased efficiency of labeling, determination of Golgi transit time of LAMP-2 in nocodazole treated MDCK cells or in cells chased at 20°C is not feasible. Nevertheless, the equivalent increase in LAMP-2 size at 20°C in the presence of nocodazole indicates that the mechanisms regulating Golgi transit of LAMP-2 in MDCK cells function equally well in dispersed Golgi stacks as in a single unified Golgi apparatus. How the rate of protein transit through the Golgi apparatus is regulated and whether inefficient Golgi transit results in enhanced polylactosamine glycosylation in transformed and metastatic cells remain to be determined.

Materials and methods

Chemicals and glycosyltransferase substrates

UDP-6-[3H]-N-acetylglucosamine (26.8 Ci/mmol, NEN), and cytidine 5[prime]-monophosphate sialic acid [sialic-9-3H] (25.1 Ci/mmol, NEN) were diluted with the respective unlabeled sugar-nucleotides purchased from Sigma (St. Louis, MO). The GlcNAc-TV acceptor GlcNAc[beta]1-2Man[alpha]1-6Man[beta]-O(CH2)8COOCH3; the GlcNAc-TI acceptor, Man[alpha]1-3(Man[alpha]1-6)Man[beta]1-O-(CH2)8-COOCH3; and the GlcNAc-T(i) acceptor Gal[beta]4GlcNAc[beta]2Man[alpha]6Man[beta]-0- (CH2)8-COOCH3; were kindly provided by Dr. O. Hindsgaul, University of Alberta. Nocodazole was purchased from Sigma. ProMix containing 35S-methionine and cysteine was purchased from Amersham (Oakville, Ontario). Endo-[beta]-galactosidase was purchased from Boehringer-Mannheim (Laval, Quebec). Anti-LAMP-2 monoclonal antibody was used in the form of ascites fluid (AC17 hybridoma) as described previously (Nabi et al., 1991).

Cell culture

MDCK II were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, glutamine, and nonessential amino acids (Gibco Laboratories, Oakville, Ontario) in an air, 5% CO2 atmosphere at constant humidity. When grown on filters, 2 × 106 cells were seeded on 24 mm Transwell polycarbonate filters (0.4 µm pore size; CoStar Corp., Cambridge, MA) and cultured for 1 or 3 days with the medium of the 3 day cultures changed on the second day after plating (Nabi and Rodriguez-Boulan, 1993). Microtubule disruption was performed by incubating MDCK cells with 20 µM nocodazole at 4°C for 30 min followed by a 30 min incubation (minimum) with 20 µM nocodazole at 37°C.

Glycosyltransferase assays

Cells were washed in phosphate-buffered saline and lysed in 0.9% NaC1, 1% Triton X-100 at 0°C for all assays. The reactions contained 16 µl of cell lysate (8-12 mg/ml of protein), 0.5 µCi of [3H]sugar-nucleotide donor (~4400 c.p.m./nmol) in a total volume of 32 µl and were incubated for 1 or 2 h at 37°C, and then processed as indicated below for each substrate. For all transfer assays, endogenous activity measured in the absence of acceptor was subtracted from values determined in the presence of added acceptor. For assays using O(CH2)8COOCH3-coupled substrates, the reactions were diluted to 5 ml in H2O, applied to a C18 Sep-Pak (Millipore Waters) in H2O which was washed with 20 ml H2O. The products were then eluted with 5 ml of methanol and radioactivity counted in a [beta]-liquid scintillation counter.

[beta]1-2GlcNAc-TI (UDP-GlcNAc:[alpha]3Man[beta]2-N-acetylglucosaminyltransferase I): the reaction contained 50 mM MES pH 7.0, 1 mM UDP-GlcNAc, 0.5 µCi UDP-[3H]GlcNAc, 0.1 M GlcNAc, 25 mM MnCl2, and 1 mM of Man[alpha]1-3(Man[alpha]1-6)Man[beta]1- O(CH2)8COOCH3 as substrate. [beta]1-6GlcNAc-TV: the reactions contained 50 mM MES pH 7.0, 1 mM UDP-GlcNAc, 0.5 µCi UDP-[3H]GlcNAc, 0.1 M GlcNAc, 1 mM of GlcNAc[beta]1-2Man[alpha]1-6Man[beta]1-0(CH2)8COOCH3. [beta]1-3GlcNAc-T(i):the reactions contained 50 mM MES pH 7.0, 1 mM UDP-GlcNAc, 0.5 µCi UDP-[3H]GlcNAc, 0.1 M GlcNAc, 25 mM MnCl2, and 1 mM Gal[beta]1-4GlcNAc[beta]1-2Man[alpha]1-6Man[beta]-O-(CH2)8-COOCH3. [beta]1-4Gal-T: the reaction contained 20 mM MnCl2, 0.2 M MES, pH 6.7, 0.5% Triton X-100, 1 mM UDP-Gal, 0.5 µCi UDP-6-[3H]-galactose (20 Ci/mmol, Amersham), and 1 mM GlcNAc[beta]1-2Man[alpha]1-6Man[beta]1-O(CH2)8COOCH3 as acceptor. [alpha]2-3SA-T: the reaction mixture contained 0.8% Triton X-100, 50 mM Tris, pH 7.0, 5 mM AMP, 5 mM CMP-SA, 2 µCi cytidine 5[prime]-monophosphate sialic acid [sialic-9-3H] (NEN) and 0.1 mg of asialo-fetuin (Sigma). [beta]2-6SA-TII: the reaction mixture in total was the same as for [alpha]2-3SA-T with 0.1 mg asialo-[alpha]1-acid glycoproteins (Sigma) as acceptor. The product was precipitated and washed with TCA, and radioactivity measured in a [beta]-counter.

Immunofluorescence

MDCK cells grown on Transwell polycarbonate filters were rinsed three times with PBS/CM and fixed with 3% paraformaldehyde for 15 min. The filters were extensively rinsed with PBS/CM and then incubated in a blocking solution consisting of PBS/CM supplemented with 0.075% saponin and 0.2% BSA for 30 min. Filter sections were incubated with rabbit anti-[beta]-COP antibody (kindly provided by Dr. Jennifer Lippincott-Schwartz, NIHCD) and FITC anti-rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA) including appropriate washes in the blocking solution and nuclear labeling with propidium iodide (Nabi et al., 1993). Confocal microscopy was performed with the 60× Nikon Plan Apochromat objective of a dual channel Bio-Rad 600 laser scanning confocal microscope equipped with a krypton/argon laser and sections apical of the propidium iodide labeled nucleus were imaged.

Metabolic labeling, LAMP-2 immunoprecipitation, and glycosidase digestions

Metabolic labeling with 35S-methionine/cysteine, immunoprecipitation, endoglycosidase digestions, and surface immunoprecipitation of LAMP-2 from MDCK cells were essentially as described previously (Nabi et al., 1991; Nabi and Rodriguez-Boulan, 1993). To ensure sufficient labeling and complete processing of LAMP-2 in the nocodazole treated cultures, cells were labeled for 3 h (150 µCi/filter) and then chased for 16 h prior to cell lysis and LAMP-2 immunoprecipitation. The cells were starved for 30 min prior to labeling in pulse medium without methionine and cysteine. Nocodazole treated cells were incubated with 20 µM nocodazole at 4°C for 30 min prior to incubation with fresh nocodazole containing medium at 37°C for 30 or 120 min. Nocodazole was included in the starvation, pulse, and chase media. To identify the precursor form of LAMP-2, cells were pulsed for 30 min (250 µCi/filter) and not chased. For the 20°C block experiments, the cells were pulsed for 30 min at 37°C and then chased in DMEM minus bicarbonate with 25 mM Hepes, pH 7.3, at 20°C in an incubator without CO2 for the indicated times prior to lysis and LAMP-2 immunoprecipitation. To achieve the best separation of 1 day and 3 day LAMP-2, 7% or 8% polyacrylamide gels were run out until the 116 kDa marker was at the middle of the gel.

Acknowledgments

This study was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada.

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2To whom correspondence should be addressed at: Département de Pathologie et Biologie Cellulaire, Université de Montréal, C. P. 6128, Succursale A, Montréal, Québec, Canada H3C 3J7

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