Dolichol metabolism in Chinese hamster ovary cells ADINAKAIDENAND SHARONS.
KRAG'
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Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205, U.S.A. Received October 17, 1991
KAIDEN,A., and KRAG,S. S. 1992. Dolichol metabolism in Chinese hamster ovary cells. Biochem. Cell Biol. 70: 385-389. The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar K, values for this lipid. Key words: dolichol, dolichyl phosphate, metabolism, Chinese hamster ovary cells. KAIDEN,A., et KRAG,S. S. 1992. Dolichol metabolism in Chinese hamster ovary cells. Biochem. Cell Biol. 70 : 385-389. L'addition d'oligosaccharides aux rtsidus asparagine des prottines solubles et des proteines assocites aux membranes dans les cellules eucaryotes requiert la presence d'un transporteur de lipides polyisoprtno'ides, le dolichol. Dans les cellules ovariennes du hamster chinois, le principal isomtre de ce polyisoprtnol renferme 19 unitts isoprtniques. I'unitt terminale Ctant saturte. Dans notre laboratoire, nous avons dtveloppe une technique pour analyser les quantitts et la nature des dkrivts dolichyl des cellules. Les cellules ovariennes du hamster chinois renferment surtout des dtrivCs anioniques activts du dolichol comme I'oligosaccharyl pyrophosphoryldolichol, les phosphoryldolichols monoglyosylts et le dolichyl phosphate. Nos ttudes dtmontrent que dans les cellules en croissance, il y a synthtse continuelle du dolichol total. De plus, les donntes prtliminaires suggtrent I'absence de catabolisme ou de stcrttion de ce lipide. Nos ttudes montrent que le taux du dolichyl phosphate ne change pas de facon importante dans diverses conditions oh les taux des activitts enzymatiques utilisant le dolichyl phosphate changent. D'aprts nos rtsultats, ces enzymes auraient accts au mCme pool de dolichyl phosphate et auraient les mCmes valeurs de K, pour ce lipide. Mots clks : dolichol, dolichyl phosphate, mttabolisme, cellules ovariennes du hamster chinois. [Traduit par la rtdaction] Introduction Glycosylation of asparagine residues in eukaryotic cells involves a lipid carrier, dolichyl phosphate (for reviews, see Hubbard and Ivatt 1981; Kornfeld and Kornfeld 1985; Krag 1985; Waechter 1989). The biosynthesis of the myriad of oligosaccharide moieties found attached to asparagine residues has many common steps that involve dolichyl phosphate. All cells synthesize dolichyl phosphate (for a review, see Kaiden and Krag 1991), which functions as a carrier lipid in the assembly of an oligosaccharide of the structure Glc3Man9GlcNAc2. This oligosaccharide is transferred from dolichyl pyrophosphate to an asparagine residue in the Asn-X-Ser(Thr) consensus sequence of a nascent protein. Modification of the transferred oligosaccharide by trimming reactions and further monosaccharide additions give rise to diverse mature structures. In mammalian cells, dolichol, a polyisoprenoid lipid, is found as a family of isomers containing 17-21 isoprenyl groups. Distinctive of dolichol is that its alpha-isoprenyl group is saturated. The first few steps in the biosynthesis of dolichol (up to and including the synthesis of farnesyl pyrophosphate) are identical to those for cholesterol. cisPolyprenyl transferase (also known as dehydrodolichyl ABBREVIATIONS: CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; HMG-CoA, hydromethylglutaryl-coenzyme A; MPD, mannosylphosphoryldolichol; GPD. glucosylphosphoryldolichol; TLC, thin-layer chromatogravhy. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
diphosphate synthase) catalyzes the synthesis of polyprenyl pyrophosphate (dehydrodolichyl diphosphate) by sequential head-to-tail cis additions of multiple molecules of the activated isoprenyl unit, isopentenyl pyrophosphate, t o farnesyl pyrophosphate. The final steps in the synthesis of dolichyl phosphate are reduction of the alpha isoprenyl group and dephosphorylation. Our laboratory has been studying dolichol metabolism in a mammalian cell line, CHO cells. This article reviews our work during the past 4 years. We have concentrated thus far on CHO cells, since this fibroblastic cell line is easy to culture in vitro, is genetically stable, and is functionally haploid at many loci, which facilitates isolation of mutants. Labelling and detecting dolichol metabolites in CHO cells We have developed techniques for labelling and detecting total dolichol and the various forms or metabolites of dolichol in CHO cells using [3~]mevalonateas a metabolic precursor. In wild-type cells, total dolichol accounts for only 2-5% of the labelled lipid in a steady-state labelling experiment (Stoll et al. 1988; Rosenwald and Krag 1990). When analyzing total dolichol, cells are saponified and labelled, and nonsaponifiable lipids are extracted and separated into neutral and anionic pools by DEAE-cellulose chromatography (Rosenwald and Krag 1990). Neutral lipids are then separated by gel filtration chromatography and the radioactivity coeluting with dolichol is subjected to HPLC using
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DOLICHYL ESTER
1
DOLICHYL PHOSPHATE
DOLICHOL
FIG. 1. The dolichol cycle for the assembly of oligosaccharide-lipid involved in N-linked glycosylation. This figure illustrates the various interconversions of the various dolichol derivatives or forms. TABLE1. Outline of procedure for obtaining level of incorporation into various dolichol forms" Step
Procedure
1 2 3
Incubation of cells with [3~]mevalonate for 72 h Methanol extraction of cells Partitioning into chloroform-methanol-water mixtures Chloroform-methanol (2:l)fraction (a) DEAE-cellulose chromatography (b) Neutral lipid is analyzed by fractogel chromatography 4-13-AA'S (c) Anionic lipid is separated by borate paper chromatography using chloroform - methanol ammonium hydroxide - water (60:25:2:2) Chloroform-methanol-water fraction Oligosaccharide lipid was fractionated on TLC in chloroform-methanol-water (10:10:3)
4
5
These methods are detailed in Stoll el al. (1988) and Rosenwald el al. (1990).
either normal or reverse-phase systems. Anionic lipids are first treated with acid phosphatase and then gel filtration chromatography, followed by HPLC. The major isomer of dolichol in CHO cells was found to have 19 isoprenyl groups (Stoll et al. 1988; Rosenwald and Krag 1990). Despite the small amount of dolichol in cells, we are able through a variety of procedures (Table 1) to determine the level of each form or metabolite of dolichol (Fig. 1) under control and altered conditions. In this case, labelled cells are not saponified, but rather are first extracted with chloroform-methanol-water mixtures to yield different lipid forms and then the various lipids are separated from each other by various chromatographic procedures (Table 1).
Using this technique of labelling dolichol with [ 3 ~ ] mevalonate, we were able to address questions which were hitherto not feasible using sugar labels, as each mono- or oligo-saccharide requires a different metabolic precursor and some derivatives of dolichol (e.g., dolichyl phosphate) are not labelled with sugar precursors. Previously, steady-state labelling of dolichol metabolites had been described by incubating insect cells (Drosophila Kc cells) with [3~]mevalonate (Sagami and Lennarz 1987). The identity of the various dolichol metabolites had also been determined by incubating soybean embryo microsomes with [I-I4c]dolichol in the presence of unlabelled sugar nucleotides (Rip et al. 1990). Our work with CHO cells is the first report of an analysis of individual dolichol metabolites in mammalian cells. We perform the labelling under conditions in which endogenous mevalonate biosynthesis is inhibited by mevinolin, an inhibitor of HMG-CoA reductase. Since mevinolin is toxic to CHO cells, sufficient mevalonate was added to the medium to insure cell viability. Therefore, in our labelling procedure, the specific activity of the intracellular mevalonate is determined strictly by the added [ 3 ~ ] mevalonate. This procedure circumvents problems that may arise owing to potential changes in the pool size of mevalonate under different experimental conditions or in different mutants. We found using our labelling procedure that the steadystate level of total dolichol was 7.5 pmol/106 CHO cells (Rosenwald et al. 1990). This amount of total dolichol in CHO cells agrees with the value found by Kabakoff et al. (1990), who determined the mass quantities of dolichol in CHO cells by HPLC analyses. The level of incorporation of mevalonate into total dolichol in CHO cells parallels cell growth (A. Kaiden and
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LLJ
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 2. The distribution of dolichol forms in wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990). The data are presented as the percentage of the radioactivity ([3~]mevalonate)incorporated at steady state into each of the following dolichol derivatives: DOL + DE, dolichol and dolichyl ester; DOL-P, dolichyl phosphate; MPD, mannosylphosphoryldolichol; GPD, glucosylphosphoryldolichol;OSL, oligosaccharidelipids. The average of three different experiments using different wild-type clones were obtained and the SE was determined.
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 3. The distribution of dolichol forms in wild-type cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the final 12 h of incubation with the [3~]mevalonate.The open bars represent the distribution of label in wild-type cells incubated without tunicamycin and the shaded bars represent the distribution in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
S.S. Krag, unpublished data). Preliminary studies indicated that total dolichol was not catabolized or secreted from CHO cells during a pulse-chase experiment. No loss of label in total dolichol was seen after 51 h of chase (over two cell doublings, A. Kaiden and S.S. Krag, in preparation). It appears that in growing cells dolichol is synthesized at a rate which is sufficient to maintain a constant amount of total dolichol per cell with no dolichol catabolism. The level of cholesterol decreased 30% during the 51 h of chase in the pulse-chase experiment (A. Kaiden and S.S. Krag, in preparation). Alterations in the levels of dolichol metabolites When we examined the steady-state level of the various dolichol forms in CHO cells, we found that about 90% of the labelled dolichol was phosphorylated and (or) glycosylated, rather than free or esterified to fatty acid (Stoll et al. 1988). Moreover, the predominant form was oligosaccharidelipid (approximately 40% of the total). The other labelled species included dolichol and dolichyl ester, dolichyl phosphate, MPD, and GPD (each about 20% of the total) (Fig. 2; Rosenwald et al. 1990). We have not detected N-acetylglucosaminyl-P-P-dolichol, chitobiosyl-P-Pdolichol, or dolichyl pyrophosphate. Addition of tunicamycin (an inhibitor of N-acetylglucosaminyl-phosphatetransferase, the enzyme which synthesizes N-acetylglucosaminylpyrophosphoryldolichol; Elbein 1987) to the medium of CHO cells resulted as expected in a sevenfold reduction in the level of oligosaccharide-lipid (Fig. 3). A comparable increase in free dolichyl phosphate was not observed. Rather, the level of several activated forms of dolichol increased in tunicamycin-
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 4. The distribution of dolichol forms in 3Ell cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in 3Ell cells. The SEs in this experiment did not exceed 10%.
treated cells relative to untreated cells. In particular, the level of dolichyl phosphate increased 150070, the level of MPD increased 150070, and the level of GPD increased 200% (Rosenwald et al. 1990).
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DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 5. The distribution of dolichol forms in 3Ell cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the fmal 12 h of incubation with [3~mevalonate. The open bars represent the distribution of label in 3Ell cells in the absence of tunicamycin, while the shaded bars give the distribution of label in 3Ell cells in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 6. The distribution of dolichol forms in B4-2-1 cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in B4-2-1 cells. The SEs in this experiment did not exceed 10%.
We next examined the steady-state level of dolichol intermediates in two glycosylation mutants. The first mutant, 3El1, has 15-fold more N-acetylglucosaminylphosphate transferase activity as does wild-type cells
1992
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 7. The distribution of dolichol forms in B4-2-1 cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the final 12 h of incubation with [3~]mevalonate.The open bars represent the distribution of label in B4-2-1 cells in the absence of tunicamycin, while the shaded bars give the distribution of label in B4-2-1 cells in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
(Waldman et al. 1987). Not unexpectedly, this increased activity gave rise to a higher steady-state level of oligosaccharide-lipid in these cells, at the expense of dolichyl phosphate, MPD, and GPD levels (Fig. 4; Rosenwald et al. 1990). When 3Ell cells were treated with tunicamycin, similar changes occurred in the levels of the dolichol forms as had been seen with wild-type cells, but the changes were more pronounced. As shown in Fig. 5, the level of oligosaccharide-lipid was substantially reduced and the level of dolichyl phosphate increased 50%. Most striking, the levels of both MPD and GPD rose to about 40% of the total dolichol form (Rosenwald et al. 1990). The second glycosylation mutant we examined was B4-2-1 cells (Stoll et al. 1982). These cells lack MPD synthase activity and as shown in Fig. 6 have reduced levels of MPD. The only other dolichol form which has an altered level in B4-2-1 cells is oligosaccharide-lipid, the level of which is increased 40%. Again, the addition of tunicamycin to B4-2-1 cells resulted in a reduced level of oligosaccharide-lipid, a slight increase in dolichyl phosphate and MPD levels, and a significant increase in the level of GPD (Fig. 7; Rosenwald et al. 1990). In all of these experiments (Figs. 3-7) the total amount of dolichol did not change significantly, although there appeared to be a slight increase in dolichol levels upon addition of tunicamycin. More importantly, the level of dolichyl phosphate remained well buffered, while the levels of the other activated dolichols were altered relative to wild-type cells. Thus, there appears to be a common pool of dolichyl phosphate for the three enzymes which utilize it (Fig. I). Flux among the various dolichol forms may be dependent upon the relative activity of the various enzymes utilizing
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the mannosyl- and glucosyl-transferases that utilize MPD and GPD as substrates appear to be reduced in vivo by the alteration in the lipid. Further, the high level of free polyprenol may be explained by the specificity of the kinase, intracellular location of the free lipid, and (or) by catabolism of the anionic forms of polyprenol. Acknowledgements The work cited in this paper was supported by grants R01-CA20421 and R01-GM36570 to S.S.K.
FIG. 8. The distribution of dolichol forms in Lec9 cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald and Krag (1990). The data are presented as the percentage of the radioactivity ([3~]mevalonate)incorporated at steady state into each of the following dolichol derivatives: DOL + DE, dolichol and dolichyl ester; DOL-P, dolichyl phosphate; MPD, mannosylphosphoryldolichol; GPD, glucosylphosphoryldolichol; OSL, oligosaccharide-lipids. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in Lec9 cells. The SEs in this experiment did not exceed 10%.
dolichyl phosphate and their corresponding K,,, values for dolichyl phosphate, as well as on utilization of the various dolichol forms in the assembly of the oligosaccharide-lipid. In the wild-type and mutant CHO cells mentioned above and in the presence of tunicamycin, the level of free dolichol and dolichyl ester remained constant, between 10 and 20%. This was not the case for two glycosylation mutants that we have studied, which appear to be defective in the reduction of the alpha-isoprene unit of polyprenol to form dolichol (Stoll and Krag 1988; Stoll et al. 1988; Rosenwald et al. 1989; Rosenwald and Krag 1990). These cells contain the same amount of total polyisoprenyl lipid, but it is fully unsaturated polyprenol rather than dolichol. In these mutants, the level of free alcohol and ester is 60% of the total polyprenol pool (Fig. 8). In addition, these cells have significantly reduced levels of oligosaccharide-lipid, resulting in reduced glycosylation of proteins. The levels of mannosylphosphoryl lipid are increased twofold, the level of glucosylphosphoryl lipid remains the same, and the level of lipid phosphate is reduced in the polyprenol reductase mutants relative to wild-type cells. We are currently trying to understand what factors lead to the glycosylation defects in these mutants which lack polyprenol reductase. We hypothesize that in vivo N-acetylglucosaminyl-phosphate transferase is less active than in wild-type cells, since it is less likely to utilize polyprenyl phosphate than dolichyl phosphate for reasons of substrate specificity or accessibility. MPD synthase and GPD synthase seem to be less affected in vivo by the unsaturation of the alpha isoprenyl unit of the lipid substrate. The activities of
Elbein, A.D. 1987. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56: 497-534. Hubbard, S.C., and Ivatt, R.J. 1981. Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50: 555-583. Kabakoff, B.D., Doyle, J.W., and Kandutsch, A.A. 1990. Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth. Arch. Biochem. Biophys. 276: 382-389. Kaiden, A., and Krag, S.S. 1991. Regulation of glycosylation of asparagine-linked glycoproteins. Trends Glycosci. Glycotechnol. 3: 275-286. Kornfeld, R., and Kornfeld, S. 1985. Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54: 631-664. Krag, S.S. 1985. Mechanisms and functional role of glycosylation in membrane protein synthesis. Curr. Top. Membrs. Transp. 24: 181-249. Rip, J.W., Williams, J.A., Crick, D.C., and Carroll, K.K. 1990. Regulation of synthesis of oligosaccharyl pyrophosphoryl dolichol during seed germination. Biochem. Cell Biol. 68: 680-684. Rosenwald, A.G., and Krag, S.S. 1990. Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol. J. Lipid Res. 31: 523-533. Rosenwald, A.G., Stanley, P., and Krag, S.S. 1989. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis. Mol. Cell. Biol. 9: 914-924. Rosenwald, A.G., Stoll, J., and Krag, S.S. 1990. Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo. J. Biol. Chem. 265: 14 544 - 14 553. Sagami, H., and Lennarz, W.J. 1987. Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides. J. Biol. Chem. 262: 15 610 - 15 617. Stoll, J., and Krag, S.S. 1988. A mutant of Chinese hamster ovary cells with a reduction in levels of dolichyl phosphate available for glycosylation. J. Biol. Chem. 263: 10 766 - 10 773. Stoll, J., Robbins, A.R., and Krag, S.S. 1982. Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol. Proc. Natl. Acad. Sci. U.S.A. 79: 2296-2300. Stoll, J., Rosenwald, A.G., and Krag, S.S. 1988. A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions. J. Biol. Chem. 263: 10 774 - 10 782. Waechter, C.J. 1989. Biosynthesis of glycoproteins. In Neurobiology of glycoconjugates. Edited by R.U. Margolis and R.E. Margolis. Plenum Press, New York. pp. 127-149. Waldman, B.C., Oliver, C., and Krag, S.S. 1987. A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphatetransferase activity has altered asparagine-linked glycosylation. J. Cell. Physiol. 131: 302-3 17.
KRAG'
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Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205, U.S.A. Received October 17, 1991
KAIDEN,A., and KRAG,S. S. 1992. Dolichol metabolism in Chinese hamster ovary cells. Biochem. Cell Biol. 70: 385-389. The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar K, values for this lipid. Key words: dolichol, dolichyl phosphate, metabolism, Chinese hamster ovary cells. KAIDEN,A., et KRAG,S. S. 1992. Dolichol metabolism in Chinese hamster ovary cells. Biochem. Cell Biol. 70 : 385-389. L'addition d'oligosaccharides aux rtsidus asparagine des prottines solubles et des proteines assocites aux membranes dans les cellules eucaryotes requiert la presence d'un transporteur de lipides polyisoprtno'ides, le dolichol. Dans les cellules ovariennes du hamster chinois, le principal isomtre de ce polyisoprtnol renferme 19 unitts isoprtniques. I'unitt terminale Ctant saturte. Dans notre laboratoire, nous avons dtveloppe une technique pour analyser les quantitts et la nature des dkrivts dolichyl des cellules. Les cellules ovariennes du hamster chinois renferment surtout des dtrivCs anioniques activts du dolichol comme I'oligosaccharyl pyrophosphoryldolichol, les phosphoryldolichols monoglyosylts et le dolichyl phosphate. Nos ttudes dtmontrent que dans les cellules en croissance, il y a synthtse continuelle du dolichol total. De plus, les donntes prtliminaires suggtrent I'absence de catabolisme ou de stcrttion de ce lipide. Nos ttudes montrent que le taux du dolichyl phosphate ne change pas de facon importante dans diverses conditions oh les taux des activitts enzymatiques utilisant le dolichyl phosphate changent. D'aprts nos rtsultats, ces enzymes auraient accts au mCme pool de dolichyl phosphate et auraient les mCmes valeurs de K, pour ce lipide. Mots clks : dolichol, dolichyl phosphate, mttabolisme, cellules ovariennes du hamster chinois. [Traduit par la rtdaction] Introduction Glycosylation of asparagine residues in eukaryotic cells involves a lipid carrier, dolichyl phosphate (for reviews, see Hubbard and Ivatt 1981; Kornfeld and Kornfeld 1985; Krag 1985; Waechter 1989). The biosynthesis of the myriad of oligosaccharide moieties found attached to asparagine residues has many common steps that involve dolichyl phosphate. All cells synthesize dolichyl phosphate (for a review, see Kaiden and Krag 1991), which functions as a carrier lipid in the assembly of an oligosaccharide of the structure Glc3Man9GlcNAc2. This oligosaccharide is transferred from dolichyl pyrophosphate to an asparagine residue in the Asn-X-Ser(Thr) consensus sequence of a nascent protein. Modification of the transferred oligosaccharide by trimming reactions and further monosaccharide additions give rise to diverse mature structures. In mammalian cells, dolichol, a polyisoprenoid lipid, is found as a family of isomers containing 17-21 isoprenyl groups. Distinctive of dolichol is that its alpha-isoprenyl group is saturated. The first few steps in the biosynthesis of dolichol (up to and including the synthesis of farnesyl pyrophosphate) are identical to those for cholesterol. cisPolyprenyl transferase (also known as dehydrodolichyl ABBREVIATIONS: CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; HMG-CoA, hydromethylglutaryl-coenzyme A; MPD, mannosylphosphoryldolichol; GPD. glucosylphosphoryldolichol; TLC, thin-layer chromatogravhy. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
diphosphate synthase) catalyzes the synthesis of polyprenyl pyrophosphate (dehydrodolichyl diphosphate) by sequential head-to-tail cis additions of multiple molecules of the activated isoprenyl unit, isopentenyl pyrophosphate, t o farnesyl pyrophosphate. The final steps in the synthesis of dolichyl phosphate are reduction of the alpha isoprenyl group and dephosphorylation. Our laboratory has been studying dolichol metabolism in a mammalian cell line, CHO cells. This article reviews our work during the past 4 years. We have concentrated thus far on CHO cells, since this fibroblastic cell line is easy to culture in vitro, is genetically stable, and is functionally haploid at many loci, which facilitates isolation of mutants. Labelling and detecting dolichol metabolites in CHO cells We have developed techniques for labelling and detecting total dolichol and the various forms or metabolites of dolichol in CHO cells using [3~]mevalonateas a metabolic precursor. In wild-type cells, total dolichol accounts for only 2-5% of the labelled lipid in a steady-state labelling experiment (Stoll et al. 1988; Rosenwald and Krag 1990). When analyzing total dolichol, cells are saponified and labelled, and nonsaponifiable lipids are extracted and separated into neutral and anionic pools by DEAE-cellulose chromatography (Rosenwald and Krag 1990). Neutral lipids are then separated by gel filtration chromatography and the radioactivity coeluting with dolichol is subjected to HPLC using
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DOLICHYL ESTER
1
DOLICHYL PHOSPHATE
DOLICHOL
FIG. 1. The dolichol cycle for the assembly of oligosaccharide-lipid involved in N-linked glycosylation. This figure illustrates the various interconversions of the various dolichol derivatives or forms. TABLE1. Outline of procedure for obtaining level of incorporation into various dolichol forms" Step
Procedure
1 2 3
Incubation of cells with [3~]mevalonate for 72 h Methanol extraction of cells Partitioning into chloroform-methanol-water mixtures Chloroform-methanol (2:l)fraction (a) DEAE-cellulose chromatography (b) Neutral lipid is analyzed by fractogel chromatography 4-13-AA'S (c) Anionic lipid is separated by borate paper chromatography using chloroform - methanol ammonium hydroxide - water (60:25:2:2) Chloroform-methanol-water fraction Oligosaccharide lipid was fractionated on TLC in chloroform-methanol-water (10:10:3)
4
5
These methods are detailed in Stoll el al. (1988) and Rosenwald el al. (1990).
either normal or reverse-phase systems. Anionic lipids are first treated with acid phosphatase and then gel filtration chromatography, followed by HPLC. The major isomer of dolichol in CHO cells was found to have 19 isoprenyl groups (Stoll et al. 1988; Rosenwald and Krag 1990). Despite the small amount of dolichol in cells, we are able through a variety of procedures (Table 1) to determine the level of each form or metabolite of dolichol (Fig. 1) under control and altered conditions. In this case, labelled cells are not saponified, but rather are first extracted with chloroform-methanol-water mixtures to yield different lipid forms and then the various lipids are separated from each other by various chromatographic procedures (Table 1).
Using this technique of labelling dolichol with [ 3 ~ ] mevalonate, we were able to address questions which were hitherto not feasible using sugar labels, as each mono- or oligo-saccharide requires a different metabolic precursor and some derivatives of dolichol (e.g., dolichyl phosphate) are not labelled with sugar precursors. Previously, steady-state labelling of dolichol metabolites had been described by incubating insect cells (Drosophila Kc cells) with [3~]mevalonate (Sagami and Lennarz 1987). The identity of the various dolichol metabolites had also been determined by incubating soybean embryo microsomes with [I-I4c]dolichol in the presence of unlabelled sugar nucleotides (Rip et al. 1990). Our work with CHO cells is the first report of an analysis of individual dolichol metabolites in mammalian cells. We perform the labelling under conditions in which endogenous mevalonate biosynthesis is inhibited by mevinolin, an inhibitor of HMG-CoA reductase. Since mevinolin is toxic to CHO cells, sufficient mevalonate was added to the medium to insure cell viability. Therefore, in our labelling procedure, the specific activity of the intracellular mevalonate is determined strictly by the added [ 3 ~ ] mevalonate. This procedure circumvents problems that may arise owing to potential changes in the pool size of mevalonate under different experimental conditions or in different mutants. We found using our labelling procedure that the steadystate level of total dolichol was 7.5 pmol/106 CHO cells (Rosenwald et al. 1990). This amount of total dolichol in CHO cells agrees with the value found by Kabakoff et al. (1990), who determined the mass quantities of dolichol in CHO cells by HPLC analyses. The level of incorporation of mevalonate into total dolichol in CHO cells parallels cell growth (A. Kaiden and
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LLJ
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 2. The distribution of dolichol forms in wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990). The data are presented as the percentage of the radioactivity ([3~]mevalonate)incorporated at steady state into each of the following dolichol derivatives: DOL + DE, dolichol and dolichyl ester; DOL-P, dolichyl phosphate; MPD, mannosylphosphoryldolichol; GPD, glucosylphosphoryldolichol;OSL, oligosaccharidelipids. The average of three different experiments using different wild-type clones were obtained and the SE was determined.
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 3. The distribution of dolichol forms in wild-type cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the final 12 h of incubation with the [3~]mevalonate.The open bars represent the distribution of label in wild-type cells incubated without tunicamycin and the shaded bars represent the distribution in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
S.S. Krag, unpublished data). Preliminary studies indicated that total dolichol was not catabolized or secreted from CHO cells during a pulse-chase experiment. No loss of label in total dolichol was seen after 51 h of chase (over two cell doublings, A. Kaiden and S.S. Krag, in preparation). It appears that in growing cells dolichol is synthesized at a rate which is sufficient to maintain a constant amount of total dolichol per cell with no dolichol catabolism. The level of cholesterol decreased 30% during the 51 h of chase in the pulse-chase experiment (A. Kaiden and S.S. Krag, in preparation). Alterations in the levels of dolichol metabolites When we examined the steady-state level of the various dolichol forms in CHO cells, we found that about 90% of the labelled dolichol was phosphorylated and (or) glycosylated, rather than free or esterified to fatty acid (Stoll et al. 1988). Moreover, the predominant form was oligosaccharidelipid (approximately 40% of the total). The other labelled species included dolichol and dolichyl ester, dolichyl phosphate, MPD, and GPD (each about 20% of the total) (Fig. 2; Rosenwald et al. 1990). We have not detected N-acetylglucosaminyl-P-P-dolichol, chitobiosyl-P-Pdolichol, or dolichyl pyrophosphate. Addition of tunicamycin (an inhibitor of N-acetylglucosaminyl-phosphatetransferase, the enzyme which synthesizes N-acetylglucosaminylpyrophosphoryldolichol; Elbein 1987) to the medium of CHO cells resulted as expected in a sevenfold reduction in the level of oligosaccharide-lipid (Fig. 3). A comparable increase in free dolichyl phosphate was not observed. Rather, the level of several activated forms of dolichol increased in tunicamycin-
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 4. The distribution of dolichol forms in 3Ell cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in 3Ell cells. The SEs in this experiment did not exceed 10%.
treated cells relative to untreated cells. In particular, the level of dolichyl phosphate increased 150070, the level of MPD increased 150070, and the level of GPD increased 200% (Rosenwald et al. 1990).
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BIOCHEM. CELL BIOL. VOL. 70,
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 5. The distribution of dolichol forms in 3Ell cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the fmal 12 h of incubation with [3~mevalonate. The open bars represent the distribution of label in 3Ell cells in the absence of tunicamycin, while the shaded bars give the distribution of label in 3Ell cells in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 6. The distribution of dolichol forms in B4-2-1 cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in B4-2-1 cells. The SEs in this experiment did not exceed 10%.
We next examined the steady-state level of dolichol intermediates in two glycosylation mutants. The first mutant, 3El1, has 15-fold more N-acetylglucosaminylphosphate transferase activity as does wild-type cells
1992
DOL+ DOL-P DE
MPD
GPD
OSL
FIG. 7. The distribution of dolichol forms in B4-2-1 cells in the absence and presence of tunicamycin. The original data for this figure were presented by Rosenwald et al. (1990) and the various dolichol pools are defined in the legend to Fig. 2. Tunicamycin was added during the final 12 h of incubation with [3~]mevalonate.The open bars represent the distribution of label in B4-2-1 cells in the absence of tunicamycin, while the shaded bars give the distribution of label in B4-2-1 cells in the presence of tunicamycin. The SEs in this experiment did not exceed 10%.
(Waldman et al. 1987). Not unexpectedly, this increased activity gave rise to a higher steady-state level of oligosaccharide-lipid in these cells, at the expense of dolichyl phosphate, MPD, and GPD levels (Fig. 4; Rosenwald et al. 1990). When 3Ell cells were treated with tunicamycin, similar changes occurred in the levels of the dolichol forms as had been seen with wild-type cells, but the changes were more pronounced. As shown in Fig. 5, the level of oligosaccharide-lipid was substantially reduced and the level of dolichyl phosphate increased 50%. Most striking, the levels of both MPD and GPD rose to about 40% of the total dolichol form (Rosenwald et al. 1990). The second glycosylation mutant we examined was B4-2-1 cells (Stoll et al. 1982). These cells lack MPD synthase activity and as shown in Fig. 6 have reduced levels of MPD. The only other dolichol form which has an altered level in B4-2-1 cells is oligosaccharide-lipid, the level of which is increased 40%. Again, the addition of tunicamycin to B4-2-1 cells resulted in a reduced level of oligosaccharide-lipid, a slight increase in dolichyl phosphate and MPD levels, and a significant increase in the level of GPD (Fig. 7; Rosenwald et al. 1990). In all of these experiments (Figs. 3-7) the total amount of dolichol did not change significantly, although there appeared to be a slight increase in dolichol levels upon addition of tunicamycin. More importantly, the level of dolichyl phosphate remained well buffered, while the levels of the other activated dolichols were altered relative to wild-type cells. Thus, there appears to be a common pool of dolichyl phosphate for the three enzymes which utilize it (Fig. I). Flux among the various dolichol forms may be dependent upon the relative activity of the various enzymes utilizing
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the mannosyl- and glucosyl-transferases that utilize MPD and GPD as substrates appear to be reduced in vivo by the alteration in the lipid. Further, the high level of free polyprenol may be explained by the specificity of the kinase, intracellular location of the free lipid, and (or) by catabolism of the anionic forms of polyprenol. Acknowledgements The work cited in this paper was supported by grants R01-CA20421 and R01-GM36570 to S.S.K.
FIG. 8. The distribution of dolichol forms in Lec9 cells as compared with wild-type cells. The original data for this figure were presented by Rosenwald and Krag (1990). The data are presented as the percentage of the radioactivity ([3~]mevalonate)incorporated at steady state into each of the following dolichol derivatives: DOL + DE, dolichol and dolichyl ester; DOL-P, dolichyl phosphate; MPD, mannosylphosphoryldolichol; GPD, glucosylphosphoryldolichol; OSL, oligosaccharide-lipids. The open bars represent the distribution of label in wild-type cells, while the shaded bars represent the distribution of label in Lec9 cells. The SEs in this experiment did not exceed 10%.
dolichyl phosphate and their corresponding K,,, values for dolichyl phosphate, as well as on utilization of the various dolichol forms in the assembly of the oligosaccharide-lipid. In the wild-type and mutant CHO cells mentioned above and in the presence of tunicamycin, the level of free dolichol and dolichyl ester remained constant, between 10 and 20%. This was not the case for two glycosylation mutants that we have studied, which appear to be defective in the reduction of the alpha-isoprene unit of polyprenol to form dolichol (Stoll and Krag 1988; Stoll et al. 1988; Rosenwald et al. 1989; Rosenwald and Krag 1990). These cells contain the same amount of total polyisoprenyl lipid, but it is fully unsaturated polyprenol rather than dolichol. In these mutants, the level of free alcohol and ester is 60% of the total polyprenol pool (Fig. 8). In addition, these cells have significantly reduced levels of oligosaccharide-lipid, resulting in reduced glycosylation of proteins. The levels of mannosylphosphoryl lipid are increased twofold, the level of glucosylphosphoryl lipid remains the same, and the level of lipid phosphate is reduced in the polyprenol reductase mutants relative to wild-type cells. We are currently trying to understand what factors lead to the glycosylation defects in these mutants which lack polyprenol reductase. We hypothesize that in vivo N-acetylglucosaminyl-phosphate transferase is less active than in wild-type cells, since it is less likely to utilize polyprenyl phosphate than dolichyl phosphate for reasons of substrate specificity or accessibility. MPD synthase and GPD synthase seem to be less affected in vivo by the unsaturation of the alpha isoprenyl unit of the lipid substrate. The activities of
Elbein, A.D. 1987. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56: 497-534. Hubbard, S.C., and Ivatt, R.J. 1981. Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50: 555-583. Kabakoff, B.D., Doyle, J.W., and Kandutsch, A.A. 1990. Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth. Arch. Biochem. Biophys. 276: 382-389. Kaiden, A., and Krag, S.S. 1991. Regulation of glycosylation of asparagine-linked glycoproteins. Trends Glycosci. Glycotechnol. 3: 275-286. Kornfeld, R., and Kornfeld, S. 1985. Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54: 631-664. Krag, S.S. 1985. Mechanisms and functional role of glycosylation in membrane protein synthesis. Curr. Top. Membrs. Transp. 24: 181-249. Rip, J.W., Williams, J.A., Crick, D.C., and Carroll, K.K. 1990. Regulation of synthesis of oligosaccharyl pyrophosphoryl dolichol during seed germination. Biochem. Cell Biol. 68: 680-684. Rosenwald, A.G., and Krag, S.S. 1990. Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol. J. Lipid Res. 31: 523-533. Rosenwald, A.G., Stanley, P., and Krag, S.S. 1989. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis. Mol. Cell. Biol. 9: 914-924. Rosenwald, A.G., Stoll, J., and Krag, S.S. 1990. Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo. J. Biol. Chem. 265: 14 544 - 14 553. Sagami, H., and Lennarz, W.J. 1987. Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides. J. Biol. Chem. 262: 15 610 - 15 617. Stoll, J., and Krag, S.S. 1988. A mutant of Chinese hamster ovary cells with a reduction in levels of dolichyl phosphate available for glycosylation. J. Biol. Chem. 263: 10 766 - 10 773. Stoll, J., Robbins, A.R., and Krag, S.S. 1982. Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol. Proc. Natl. Acad. Sci. U.S.A. 79: 2296-2300. Stoll, J., Rosenwald, A.G., and Krag, S.S. 1988. A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions. J. Biol. Chem. 263: 10 774 - 10 782. Waechter, C.J. 1989. Biosynthesis of glycoproteins. In Neurobiology of glycoconjugates. Edited by R.U. Margolis and R.E. Margolis. Plenum Press, New York. pp. 127-149. Waldman, B.C., Oliver, C., and Krag, S.S. 1987. A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphatetransferase activity has altered asparagine-linked glycosylation. J. Cell. Physiol. 131: 302-3 17.
