Mediators of Galactose Sensitivity in UDP-Galactose 4′-Epimerase-impaired Mammalian Cells
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m414045200
ISSN1083-351X
AutoresJenny M. Schulz, Kerry L. Ross, Kerstin Malmström, Monty Krieger, Judith L. Fridovich‐Keil,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoUDP-galactose 4′-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia. UDP-galactose 4′-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia. UDP-galactose 4′-epimerase (GALE, 1The abbreviations used are: GALE, UDP-galactose 4′-epimerase; BrdUrd, bromodeoxyuridine; CHO, Chinese hamster ovary; eGALE, E. coli GALE; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GALT, galactose-1-phosphate uridylyltransferase; HA, hemagglutinin; hGALE, human GALE; HPLC, high performance liquid chromatography; LPDS, lipoprotein-deficient serum; mGALT, mouse GALT; PBS, phosphate-buffered saline. EC 5.1.3.2) catalyzes the third step in the highly conserved Leloir pathway of galactose metabolism in species ranging from bacteria to humans (1.Holden H.M. Rayment I. Thoden J.B. J. Biol. Chem. 2003; 278: 43885-43888Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). As illustrated in Fig. 1, GALE is a reversible enzyme, interconverting UDP-galactose (UDP-Gal) and UDP-glucose (UDP-Glc, reaction 1). Although Escherichia coli GALE catalyzes only the interconversion of UDP-Gal and UDP-Glc, mammalian GALE enzymes also interconvert UDP-N-acetylgalactosamine (UDP-GalNAc) and UDP-N-acetylglucosamine (UDP-GlcNAc, reaction 2) (2.Piller F. Hanlon M.H. Hill R.L. J. Biol. Chem. 1983; 258: 10774-10778Abstract Full Text PDF PubMed Google Scholar, 3.Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. J. Biol. Chem. 2001; 276: 15131-15136Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4.Thoden J. Henderson J. Fridovich-Keil J. Holden H. J. Biol. Chem. 2002; 277: 27528-27534Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5.Schulz J. Watson A. Sanders R. Ross K. Thoden J. Holden H. Fridovich-Keil J. J. Biol. Chem. 2004; 279: 32796-32803Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). UDP-GalNAc is the obligate first sugar donor for all mucin-type O-linked glycosylation reactions in mammalian cells (6.Hanisch F. Biol. Chem. 2001; 382: 143-149Crossref PubMed Scopus (272) Google Scholar), and all four UDP-sugars serve as activated substrate donors for both N- and O-linked glycosylation reactions. GALE therefore not only catalyzes an essential step in the Leloir pathway, but also serves as a gatekeeper overseeing the ratios of key substrate pools required for the synthesis of glycogen, glycosaminoglycans, proteoglycans, glycoproteins, and glycolipids in mammals. Defects in human GALE (hGALE) result in the inherited metabolic disorder epimerase-deficiency galactosemia (OMIM 230350) (7.Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2000: 1553-1587Google Scholar). The majority of patients with epimerase deficiency demonstrate an apparently benign form of the disorder in which enzyme impairment is restricted to the circulating red and white blood cells (7.Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2000: 1553-1587Google Scholar). These patients are said to have peripheral epimerase deficiency. A small number of patients have also been identified who demonstrate a more generalized enzyme impairment that extends to other tissues, resulting in potentially significant clinical disease (8.Holton J.B. Gillett M.G. MacFaul R. Young R. Arch. Dis. Child. 1981; 56: 885-887Crossref PubMed Scopus (84) Google Scholar, 9.Sardharwalla I.B. Wraith J.E. Bridge C. Fowler B. Roberts S.A. J. Inher. Metab. Dis. 1988; 11: 249-251Crossref PubMed Scopus (40) Google Scholar, 10.Walter J.H. Roberts R.E.P. Besley G.T.N. Wraith J.E. Cleary M.A. Holton J.B. MacFaul R. Arch. Dis. Child. 1999; 80: 374-376Crossref PubMed Scopus (66) Google Scholar). Finally, several reports suggest that intermediate forms of epimerase deficiency may also exist (11.Quimby B.B. Alano A. Almashanu S. DeSandro A.M. Cowan T.M. Fridovich-Keil J.L. Am. J. Hum. Gen. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 12.Alano A. Almashanu S. Chinsky J.M. Costeas P. Blitzer M.G. Wulfsberg E.A. Cowan T.M. J. Inher. Metab. Dis. 1998; 21: 341-350Crossref PubMed Scopus (48) Google Scholar, 13.Shin Y.S. Korenke G.C. Huddke P. Knerr I. Podskarbi T. J. Inher. Metab. Dis. 2000; 23: 383-386Crossref PubMed Scopus (11) Google Scholar). Although the basis for this biochemical and clinical spectrum of epimerase deficiency remains unknown, studies of patient GALE sequences have demonstrated significant variability (11.Quimby B.B. Alano A. Almashanu S. DeSandro A.M. Cowan T.M. Fridovich-Keil J.L. Am. J. Hum. Gen. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 14.Wohlers T.M. Christacos N.C. Harreman M.T. Fridovich-Keil J.L. Am. J. Hum. Gen. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15.Wohlers T. Fridovich-Keil J.L. J. Inher. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 16.Henderson J.M. Huguenin S.M. Cowan T.M. Fridovich-Keil J.L. Clin. Genet. 2001; 60: 350-355Crossref PubMed Scopus (10) Google Scholar, 17.Wasilenko J. Lucas M. Thoden J. Holden H. Fridovich-Keil J. Mol. Gen. Metab. 2004; 84: 32-38Crossref Scopus (25) Google Scholar), suggesting that allelic heterogeneity may be one contributing factor. Nonetheless, the precise role of human GALE in mediating both galactose metabolism and the pathophysiology of galactose sensitivity in epimerase deficiency remains unclear. As one approach toward addressing this question, both we and others have developed and applied yeast model systems in which GALE (GAL10) is either deleted or impaired (11.Quimby B.B. Alano A. Almashanu S. DeSandro A.M. Cowan T.M. Fridovich-Keil J.L. Am. J. Hum. Gen. 1997; 61: 590-598Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 14.Wohlers T.M. Christacos N.C. Harreman M.T. Fridovich-Keil J.L. Am. J. Hum. Gen. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15.Wohlers T. Fridovich-Keil J.L. J. Inher. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 18.Douglas H.C. Hawthorne D.C. Genetics. 1964; 49: 837-844Crossref PubMed Google Scholar, 19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). GALE-deficient yeast are not only unable to grow on media containing galactose as the sole carbon source; they also fail to grow on media containing alternate carbon sources, such as glycerol/ethanol or raffinose, if even trace amounts of galactose are added (e.g. 0.0025%) (19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). These results demonstrate that GALE-null yeast are not only Gal– or unable to metabolize galactose fully, they are also galactose-sensitive (15.Wohlers T. Fridovich-Keil J.L. J. Inher. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 18.Douglas H.C. Hawthorne D.C. Genetics. 1964; 49: 837-844Crossref PubMed Google Scholar, 19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). Prior and ongoing studies have helped to identify potential factors mediating galactose sensitivity in GALE-null yeast (15.Wohlers T. Fridovich-Keil J.L. J. Inher. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 18.Douglas H.C. Hawthorne D.C. Genetics. 1964; 49: 837-844Crossref PubMed Google Scholar, 19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar); nonetheless, how these results relate to galactose metabolism and sensitivity in GALE-deficient mammalian cells has remained unknown. With the experiments presented here we have begun to address the mechanism of galactose sensitivity in GALE-impaired mammalian cells utilizing a strain of Chinese hamster ovary cells called ldlD. ldlD cells were originally isolated more than 20 years ago on the basis of impaired low density lipoprotein receptor function (20.Krieger M. Brown M.S. Goldstein J.L. J. Mol. Biol. 1981; 150: 167-184Crossref PubMed Scopus (74) Google Scholar), a phenotype later revealed as secondary to a complete absence of GALE activity, which resulted in the aberrant processing of both N- and O-linked glycoproteins, including the low density lipoprotein receptor (21.Kingsley D. Kozarsky K.F. Hobbie L. Krieger M. Cell. 1986; 44: 749-759Abstract Full Text PDF PubMed Scopus (241) Google Scholar). It is important to note that although ldlD cells are viable in standard glucose-containing media despite their lack of GALE, no other truly GALE-null mammalian cells have been reported. In particular, cells derived from even the most severely affected epimerase-deficient patients are not fully GALE-null (14.Wohlers T.M. Christacos N.C. Harreman M.T. Fridovich-Keil J.L. Am. J. Hum. Gen. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15.Wohlers T. Fridovich-Keil J.L. J. Inher. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar, 22.Kingsley D.M. Krieger M. Holton J.B. N. Engl. J. Med. 1986; 314: 1257-1258PubMed Google Scholar). This observation stands in stark contrast to transferase deficiency, in which patients demonstrating no detectable activity, and even large genomic deletions, have been reported (23.Berry G.T. Leslie N. Reynolds R. Yager C.T. Segal S. Mol. Genet. Metab. 2001; 72: 316-321Crossref PubMed Scopus (37) Google Scholar). Although viable, ldlD cells clearly exist in a precarious balance. Because of their lack of GALE, they cannot synthesize endogenous galactose or GalNAc and therefore depend on exogenous sources of these sugars for normal glycosylation (21.Kingsley D. Kozarsky K.F. Hobbie L. Krieger M. Cell. 1986; 44: 749-759Abstract Full Text PDF PubMed Scopus (241) Google Scholar). These same cells, however, exhibit impaired growth when exposed to greater than 0.125 mm galactose (24.Krieger M. Reddy P. Kozarsky K. Kingsley D. Hobbie L. Penman M. Methods Cell Biol. 1989; 32: 57-84Crossref PubMed Scopus (62) Google Scholar). The mechanism behind this impaired growth phenotype has remained unclear and is addressed in part by the experiments presented here. In particular, we have asked three questions: 1) What are the metabolic changes associated with galactose sensitivity in ldlD cells? 2) How much hGALE activity is required to relieve the apparent galactose sensitivity of ldlD cells? 3) What is the relationship among GALE substrate specificity, metabolic imbalance, and galactose sensitivity in ldlD-derived cells? To address these questions, we first characterized both ldlD and wild-type CHO cells cultured in the presence or absence of 0.25 mm galactose. As expected, we observed a number of galactose-specific abnormalities in the ldlD cells, including impaired cell growth, elevated Gal-1-P and UDP-Gal, and depressed UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. To define the level of wild-type human GALE activity required to complement the ldlD cell growth impairment and metabolic abnormalities, we generated transfectants stably expressing either low (5–10%) or near normal levels of hGALE activity. The near normal hGALE-expressing cells (ldlD[hGALE-wt]) demonstrated full complementation of some abnormalities, including the growth, Gal-1-P, and UDP-Gal phenotypes and significant complementation of others, including the UDP-Glc, UDP-GlcNAc, and UDP-GalNAc phenotypes. In contrast, the 5–10% hGALE-expressing cells (ldlD[hGALE-low]) exhibited only marginal complementation of each of these abnormalities. To probe the role of GALE substrate specificity in galactose sensitivity, we generated ldlD transfectants expressing E. coli GALE, which completely lacks UDP-GalNAc activity. As expected, both galactose consumption and internal metabolite levels, including Gal-1-P, UDP-Gal, UDP-Glc, and UDP-GlcNAc, were complemented as well in these ldlD[eGALE] cells as in the ldlD[hGALE-wt] cells, although UDP-GalNAc remained undetectable. Nonetheless, the ldlD[eGALE] cells grew as well in the presence of 0.25 mm galactose as did their hGALE-expressing counterparts, demonstrating that the apparent growth impairment of GALE-null cells exposed to galactose was independent of UDP-GalNAc deficiency and therefore likely independent of O-linked glycosylation defects. Finally, as a first step toward addressing the issue of mechanism, we tested the impact of nucleoside supplementation and found that uridine alone was sufficient to relieve the galactose-specific growth impairment of ldlD cells. It is particularly interesting to note that although uridine supplementation did correct the UDP-Glc and, to a lesser extent, UDP-GlcNAc depletion of galactose-exposed ldlD cells, both the Gal-1-P and UDP-Gal levels in these cells remained abnormally high. These results suggest that it is not the dramatic accumulation of Gal-1-P or UDP-Gal, but rather the depletion of uridine that is cytostatic to ldlD cells exposed to galactose. Combined, these results provide a first step toward understanding the mechanism of galactose sensitivity in GALE-impaired mammalian cells and suggest a potential novel therapy for epimerase-deficiency galactosemia. Plasmids—HA-tagged forms of both the human GALE (hGALE, JF4169-4170) and E. coli GALE (eGALE, JF4387) enzymes were expressed in ldlD cells using a derivative of the pCDNA3 (Invitrogen) vector in which the cytomegalovirus promoter had been removed and replaced with the mouse galactose-1-phosphate uridylyltransferase (mGALT) promoter. The mGALT promoter sequence (25.Leslie N. Bai S. Mol. Genet. Metab. 2001; 72: 31-38Crossref PubMed Scopus (6) Google Scholar) was generated through PCR amplification of wild-type mouse genomic DNA (kind gift from Dr. David Weinshenker, Emory University) using the following primers, which contained the restriction sites MluI and HindIII for ease of subcloning: mGALTproMluIf1, 5′-CGCGACGCGTATCCGTGGCGGGACGAATGGACACAGCAAC-3′; and mGALTproHindIIIr1, 5′-CGCGAAGCTTATCGGCTCCGCTATGCGACGTGAGGCC-3′. The wild-type E. coli GALE sequence was obtained via PCR amplification from genomic E. coli DNA using the following primers, which engineered an HA tag onto the 3′-end of the open reading frame: eGALEf2, 5′-GCGCGAATTCCCAAGGTGCCATGAGAGTTCTGGTTACCGGTGG-3′; and eGALEHAr1, 5′-GCGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAATCGGGATATCCCTGTGGATG-3′. All amplified, subcloned alleles were confirmed by dideoxy sequencing. Cell Culture and Generation of Transfected Cell Lines—All mammalian cell lines used in these studies are listed in Table I. The control cell line, CHO-K1 (kind gift from Dr. Curt Hagedorn, Emory University), and mutant ldlD cell line were maintained in Ham's F-12 medium containing glutamine and 10 mm glucose (Cellgro) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% (v/v) fetal bovine serum (FBS) (Invitrogen). All cells were maintained at 37 °C in a humidified 5% CO2 incubator (NuAire) and harvested for splitting or analysis by standard protocols using trypsin/EDTA.Table IEnzyme activities in ldlD cells stably expressing human GALE (hGALE) or E. coli GALE (eGALE)Strain (comments)GALE activity (UDP-Gal)GALE activity (UDP-GalNAc)pmol / μg / minpmol / μg / minCHO-K1 (wild-type GALE)2.08 ± 0.212.27 ± 0.54ldlD (GALE-null, from CHO-K1)UndetectableUndetectableJFm342 (ldlD[hGALE-wt])1.64 ± 0.132.54 ± 0.49JFm348 (ldlD[hGALE-low])0.10 ± 0.050.28 ± 0.03JFm361 (ldlD[eGALE])0.82 ± 0.06Undetectable Open table in a new tab Cells were transfected in 6-well dishes using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. 24 h after transfection, cultures were trypsinized and replated at >1:10 dilution into Ham's F-12 medium supplemented with 10% FBS. G418 selection was initiated 24 h later by the addition of appropriate levels of drug (200 μg/ml G418, Cellgro). After ∼14 days of drug selection, individual clones were isolated by trypsinization within cloning rings and purified by further subculture in medium containing G418. Expression of GALE in each clone was confirmed by Western blot analysis against the HA tag and by GALE activity assays. Clonal lines of transfectants were maintained in Ham's F-12 medium containing glutamine and 10 mm glucose, supplemented with 10% FBS and 200 μg/ml G418. Generation of Lipoprotein-deficient Serum (LPDS)—Newborn calf LPDS was prepared according to methods described by Goldstein et al. (26.Goldstein J. Basu S. Brown M. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar) and modified by Krieger (27.Krieger M. Methods Enzymol. 1986; 129: 227-237Crossref PubMed Scopus (14) Google Scholar). Briefly, whole newborn calf serum (Invitrogen) was adjusted to a final density of 1.215 g/ml with solid KBr (Sigma). The serum was separated by ultracentrifugation at 59,000 rpm for 36 h at 4 °C in a Beckman 70 Ti rotor. The top, lipoprotein-rich fraction was removed through aspiration. The lipoprotein-deficient fraction was then subjected to five sequential rounds of dialysis against a total of 30 liters of 150 mm NaCl over 72 h at 4 °C. The thoroughly dialyzed LPDS was then sterilized by filtration (0.45 μ Millipore) and adjusted to a protein concentration of 60 mg/ml by dilution with sterile 150 mm NaCl. Cell Viability Assays—ldlD and wild-type CHO cells were plated in triplicate into Ham's F-12 medium containing glutamine, 10 mm glucose, and 2% LPDS. After cells reached 90% confluence (48 h), the medium was removed and replaced by fresh medium either with or without 0.25 mm galactose. After 72 h, cells were harvested and tested for viability by exclusion of the vital stain, trypan blue (Sigma), according to the manufacturer's instructions. Briefly, a 0.2-ml cell suspension in phosphate-buffered saline (PBS) was added to 0.5 ml of 0.4% trypan blue and 0.3 ml of PBS. The suspension was incubated for 5 min before microscopic examination, in which both stained and unstained cells were counted using a hemacytometer. A minimum of 100 cells were counted. Uridine Rescue—ldlD cells were plated into the wells of a 24-well plate at 10,000 cells/well in Ham's F-12 medium containing 10 mm glucose and supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, and 3% LPDS. After 1 day of culture the medium from each well was removed and replaced with fresh medium containing 0, 0.125, or 0.25 mm galactose with or without the addition of 100 μm uridine, thymidine, guanosine, adenosine, or cytidine. After an additional 5 days of culture, monolayers were fixed and stained with crystal violet, as described previously (28.Krieger M. Cell. 1983; 33: 413-422Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Metabolite studies on ldlD cells grown in the presence and absence of 100 μm uridine were performed as described below. Preparation of Protein Lysates and Enzyme Assays—Proteins were extracted by repeated freeze/thaw in 100 mm glycine buffer containing protease inhibitors (0.3 mm aprotinin, 0.63 μg/ml pepstatin, 2 mm antipain, 1 mm phosphoramidon, 0.2 μg/ml chymostatin, 8 mm E64, 1 mm phenylmethylsulfonyl fluoride, and 0.50 μg/ml leupeptin). Crude protein extracts were fractionated over P-30 Bio-Spin columns (Bio-Rad) to remove small metabolites prior to further analysis. Protein determinations were made using the Bio-Rad protein assay reagent, as recommended by the manufacturer, and quantified using a standard curve of bovine serum albumin. Individual Leloir pathway enzyme activities were measured using standard enzymatic assay procedures with quantification of reactants and products by HPLC. In brief, all enzymatic reactions were carried out at 37 °C for 30 min, essentially as described previously (19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar) with appropriate levels of protein included to remain within the linear range of the assay. Enzyme activity in each assay is defined as pmol of product formed per μg of protein/min. The following mobile phase buffers were used for separation of carbohydrate substrates and products: Buffer A, 15 mm NaOH; Buffer B, 50 mm NaOH, 1 m sodium acetate; and Buffer C, 15 mm NaOH, 1 mm Na2B4O7·10H2O. The following procedures were used for HPLC separation and quantification of enzymatic substrates and products. For galactokinase: 98% A and 2% B (–5 to 3 min), a linear increase of B to 25% (3 to 25 min), hold at 75% A and 25% B (25 to 27 min), and a linear decrease of B to 2% (27 to 30 min). For GALT: 85% A and 15% B (–10 to 5 min), a linear increase of B to 25% (5 to 10 min), a linear increase of B to 70% (10 to 15 min), hold at 30% A and 70% B (15 to 30 min), and a linear decrease of B to 15% (30 to 32 min). For GALE, UDP-Gal: 35% C and 65% B for 20 min. For GALE, UDP-GalNAc: 45% C and 55% B for 30 min. The flow rate was maintained at 1 ml/min for all separation procedures. BrdUrd Incorporation Assay—The colorimetric Cell Proliferation ELISA kit (Roche Applied Science), which monitors incorporation of BrdUrd into newly synthesized DNA, was used according to the manufacturer's recommendations to quantify the proliferation of cells grown in the presence versus absence of galactose. Briefly, cells were plated at 100 cells/200 μl of medium into each well of a 96-well plate, using medium containing 2% LPDS, and grown for 72 h. As described under "Results," some wells also contained small amounts of galactose or mannose. At the end of the 72-h growth period, cells were exposed to BrdUrd for 8 h and then fixed for 30 min with the provided solution. Next, cells were incubated for 1.5 h with a monoclonal peroxidase-labeled antibody directed against BrdUrd, provided by the manufacturer. After removal, cells were washed three times with 200 μl of 1× washing solution. Finally, the substrate was allowed to develop for 10 min before 1 m H2SO4 was added to stop the reaction. The plate was incubated for 30 s on low shake in an EL 808 Ultra Microplate Reader (Bio-tek Instruments) before absorbance was read at 450 nm (reference wavelength, 690 nm). In all cases, background was defined as BrdUrd signal detected in wells on the plate that did not contain cells. Metabolite Analysis—Cultures were split 1:6 from confluent 10-cm plates into normal growth medium. After reaching confluence, cells were collected, washed once with sterile PBS, and divided equally into 26 plates of medium containing 2% LPDS. After reaching >90% confluence (∼48 h), the growth medium was removed. Two plates of cells were harvested at this step and used to determine metabolites at a 0 time point. The remaining plates were fed with fresh medium supplemented with 2% LPDS ± 0.25 mm galactose. Duplicate plates were then harvested using trypsin/EDTA at the time points specified in each figure. A 250-μl sample of medium from each plate was also collected at each time point for analysis of external galactose consumption. Trypsinized cells were harvested by centrifugation at 3,500 rpm for 5 min, after which the supernatant was aspirated, and the cells were washed once with 5 ml of cold PBS before further manipulation. Analysis of Internal Metabolites—Metabolite samples from cells were prepared using a modified form of the procedure originally described by Smits et al. (29.Smits H.P. Cohen A. Buttler T. Nielsen J. Olsson L. Anal. Biochem. 1998; 261: 36-42Crossref PubMed Scopus (71) Google Scholar). In brief, each cell pellet was resuspended in 11 ml of cold PBS, of which 1 ml was used for protein determination via the Bio-Rad Dc protein assay, as recommended by the manufacturer. The remaining 10 ml of cell suspension was quenched in 20 ml of 60% MeOH (–20 °C), after which cells were collected by 20-min centrifugation at 2,000 rpm, 4 °C in an Eppendorf 5810R centrifuge. Intracellular metabolites were extracted by vigorous agitation for 45 min at 4 °C in a 4:2:1 mixture of CHCl3/MeOH/water, with a final volume of 875 μl. The aqueous layer was collected after high speed centrifugation for 10 min at 4 °C. The remaining organic phase was extracted a second time with 125 μl of MeOH and 125 μl of water. Aqueous layers were combined and dried under vacuum without heat (∼10 h). Finally, dried metabolites were rehydrated with sterile Milli-Q water to a concentration corresponding to 1.5–2.75 μg of protein/μl, using protein values determined in parallel samples as described above. Rehydrated samples were filtered through 0.2-μm nylon filters (Alltech) before HPLC fractionation. Analysis of External Galactose Consumption—Each 250-μl sample of culture medium was added directly into 500 μl of 60% MeOH (–20 °C). External metabolites were extracted as described above from a 375-μl fraction of the medium/MeOH mixture. After drying, metabolite pellets were rehydrated in 390 μl of sterile Milli-Q water and filtered before HPLC fractionation. Small Metabolite Analysis by HPLC—HPLC analysis was performed using a DX600 HPLC system (Dionex) consisting of a Dionex AS50 autosampler, a Dionex GP50 gradient pump, and a Dionex ED50 electrochemical detector, as described previously (19.Ross K.L. Davis C.N. Fridovich-Keil J.L. Mol. Gen. Metab. 2004; 83: 103-116Crossref PubMed Scopus (66) Google Scholar). In brief, carbohydrates were separated on a CarboPac PA10 column (250 × 4 mm) with an amino trap (50 × 4 mm) placed before the analysis column and a borate trap (50 × 4 mm) placed before the injector port to remove trace amounts of borate f
Referência(s)