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Mutants of the CMP-sialic Acid Transporter Causing the Lec2 Phenotype

1998; Elsevier BV; Volume: 273; Issue: 32 Linguagem: Inglês

10.1074/jbc.273.32.20189

1083-351X

Matthias Eckhardt, Birgit Gotza, Rita Gerardy‐Schahn,

Viral Infectious Diseases and Gene Expression in Insects

Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker α-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter. Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker α-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter. Carbohydrates added to cell surface proteins and lipids provide major contact and communication elements for animal cells. The biosynthesis of the carbohydrate structures occurs mainly in the luminal parts of the endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumCHOChinese hamster ovaryCMP-Sia-TrCMP-sialic acid transporterHAhemagglutininPCRpolymerase chain reactionRT-PCRreverse transcription PCRUDP-Gal-TrUDP-galactose transporterMOPS4-morpholinepropanesulfonic acidmAbmonoclonal antibodyPBSphosphate-buffered salinePSApolysialic acid.1The abbreviations used are: ERendoplasmic reticulumCHOChinese hamster ovaryCMP-Sia-TrCMP-sialic acid transporterHAhemagglutininPCRpolymerase chain reactionRT-PCRreverse transcription PCRUDP-Gal-TrUDP-galactose transporterMOPS4-morpholinepropanesulfonic acidmAbmonoclonal antibodyPBSphosphate-buffered salinePSApolysialic acid. and Golgi apparatus and therefore requires specific nucleotide sugar transport systems (1Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar). Nucleotide sugar transporters have been described for CMP-sialic acid, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, UDP-xylose, GDP-mannose, UDP-glucuronic acid, and UDP-glucose (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar, 3Hirschberg C.B. Snider M.D. Annu. Rev. Biochem. 1987; 56: 63-88Crossref PubMed Scopus (439) Google Scholar, 4Verbert A. Cacan R. Cecchelli R. Biochimie. 1987; 69: 91-99Crossref PubMed Scopus (15) Google Scholar). These proteins function as antiporters in an ATP- and ion-independent manner by exchanging the nucleotide sugar with the corresponding nucleoside monophosphate generated in the organellar lumen through the action of glycosyltransferases and nucleoside diphosphatases (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar, 5Abeijon C. Mandon E.C. Hirschberg C.B. Trends Biochem. Sci. 1997; 22: 203-207Abstract Full Text PDF PubMed Scopus (100) Google Scholar). The high substrate specificity of the nucleotide sugar transporters, which has been demonstrated in biochemical and genetic analysis (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar), makes these molecules ideal targets for the selective inhibition of glycoconjugate maturation. Increased sialylation has been described for tumor cell surfaces and has been shown to correlate positively with malignant potential (6Santer U.V. DeSantis R. Hard K.J. van Kuik J.A. Vliegenthart J.F. Won B. Glick M.C. Eur. J. Biochem. 1989; 181: 249-260Crossref PubMed Scopus (54) Google Scholar, 7Saitoh O. Wang W.C. Lotan R. Fukuda M. J. Biol. Chem. 1992; 267: 5700-5711Abstract Full Text PDF PubMed Google Scholar, 8Bresalier R.S. Ho S.B. Schoeppner H.L. Kim Y.S. Sleisenger M.H. Brodt P. Byrd J.C. Gastroenterology. 1996; 110: 1354-1367Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Gorelik E. Xu F. Henion T. Anaraki F. Galili U. Cancer Res. 1997; 57: 332-336PubMed Google Scholar). Since numerous sialyltransferases (for a review, see Ref. 10Tsuji S. J. Biochem. ( Tokyo ). 1996; 120: 1-13Crossref PubMed Scopus (217) Google Scholar) but probably only a single CMP-sialic acid transporter (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar) exist, the transporter may provide an effective target to inhibit cell surface sialylation. Accordingly, the inhibition of the UDP-galactose transporter and CMP-sialic acid transporter by somatic mutations and synthetic inhibitors resulted in strong reduction of the metastatic potential in the murine MDAY-D2 tumor cell line and in human colorectal cancer lines in nude mice (11Harvey B.E. Toth C.A. Wagner H.E. Steele G.D. Thomas P. Cancer Res. 1992; 52: 1775-1779PubMed Google Scholar, 12Harvey B.E. Thomas P. Biochem. Biophys. Res. Commun. 1993; 190: 571-575Crossref PubMed Scopus (33) Google Scholar, 13Takano R. Muchmore E. Dennis J.W. Glycobiology. 1994; 4: 665-674Crossref PubMed Scopus (74) Google Scholar).Considerable progress in studying the transport of nucleotide sugars into the Golgi lumen has been made by the molecular cloning of nucleotide sugar transporter genes. CMP-Sia-Tr and UDP-Gal-Tr cDNAs were cloned from mammalian species (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar, 15Miura N. Ishida N. Hoshino M. Yamauchi M. Hara T. Ayusawa D. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 236-241Crossref PubMed Scopus (119) Google Scholar, 16Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 1074-1078Crossref PubMed Scopus (91) Google Scholar, 17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), and the GDP-mannose transporter from Leishmania donovani (18Descoteaux A. Luo Y. Turco S.J. Beverly S.M. Science. 1995; 269: 1869-1872Crossref PubMed Scopus (143) Google Scholar, 19Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and the UDP-GlcNAc- and UDP-Gal-transporters were cloned from yeast (20Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (110) Google Scholar, 21Tabuchi M. Tanaka N. Iwahara S. Takegawa K. Biochem. Biophys. Res. Commun. 1997; 232: 121-125Crossref PubMed Scopus (77) Google Scholar). Related cDNAs from human, Saccharomyces cerevisiae, andCaenorhabditis elegans were also identified by homology searches in the gene data bases (16Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 1074-1078Crossref PubMed Scopus (91) Google Scholar, 19Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 20Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (110) Google Scholar). Heterologous expression of the murine CMP-Sia-Tr on the zero background of S. cerevisiae was used to confirm the biological function of the protein (22Berninsone P. Eckhardt M. Gerardy-Schahn R. Hirschberg C.B. J. Biol. Chem. 1997; 272: 12616-12619Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The transfected yeast cells acquired the ability to translocate CMP-sialic acid.All nucleotide-sugar transporters cloned to date have been identified by complementation cloning in glycosylation mutants. The murine and hamster CMP-Sia-Tr were isolated by expression cloning in a clone of the Lec2 complementation group. Although the Lec2 mutation is known to inhibit translocation of CMP-sialic acid into the Golgi lumen (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (196) Google Scholar), the molecular basis responsible for the asialo phenotype is still unknown. This study was undertaken to determine the molecular basis of the Lec2 phenotype. Thereby, we took advantage of the fact that several independent Lec2 mutants have been isolated in the laboratory (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar). The results summarized in this study demonstrate that Lec2 cells carry defects in the CMP-Sia-Tr gene leading to aberrations in the transporter protein. The analysis of these mutants provides a useful system to gain insight into structure-function relationships of the transporter protein.DISCUSSIONCells of the Lec2 complementation group are defective in the transport of CMP-NeuAc into the lumen of the Golgi apparatus (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Clones exhibiting the lec2 defect have been isolated by lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar, 31Briles E.B. Li E. Kornfeld S. J. Biol. Chem. 1977; 252: 1107-1116Abstract Full Text PDF PubMed Google Scholar) or immunoselection (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar). The lec2mutation causes the expression of asialo cell surfaces. Among the carbohydrate epitopes missing is PSA. Reexpression of PSA was therefore used to isolate cDNAs encoding the murine and hamster CMP-Sia-Tr cDNAs via complementation cloning (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar, 17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar). The present study was carried out to determine the lec2 mutation at the molecular level. Four independent mutants (1E3, 6B2, 8G8, and 9D3), which arose from chemical mutation experiments (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar), together with the clone Lec2 isolated via lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar), were analyzed by Northern blotting and RT-PCR. Deletions, insertions, and point mutations in the CMP-Sia-Tr coding region were found that demonstrated that the gene defective in these cells encodes the CMP-Sia-Tr. The high frequency by which these mutants occur after chemical mutagenesis (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 33Stanley P. Annu. Rev. Genet. 1984; 18: 525-552Crossref PubMed Scopus (167) Google Scholar) makes this approach an attractive way to identify functionally important primary sequence elements and to investigate structure-function relationships of nucleotide-sugar transporters.Deletions observed in clones 6B2, 8G8, and Lec2, are likely to result from mutations in splice acceptor or donor sites. Support for this assumption comes from the observation that the deleted sequence sections share common boundaries. The extended sequence changes associated with the mutations lec2, 6b2, and8g8, cause mistargeting of the translation products. While co-localization of the wild-type CMP-Sia-Tr with α-mannosidase II indicated transport to the Golgi apparatus (Ref. 14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar and Figs. 4 and 5), all internally deleted or C-terminally truncated transporter mutants were not in the α-mannosidase II compartment but produced a diffuse staining throughout the cytoplasma. The staining pattern observed for the mutants might be explained by retention of the proteins in the ER. The extended primary sequence changes caused by the deletions or truncations most probably lead to misfolded proteins, which do not escape the process of "ER quality control" (34Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar). Consistent with this, reduced expression levels were observed for the mutant proteins.Analysis of the CMP-Sia-Tr mRNA from clone 9D3 revealed a single missense mutation, resulting in exchange of glycine at position 189 for glutamic acid. Like the other mutants, the steady-state level of mRNA expression in this mutant was comparable with that of wild-type cells (Fig. 1), and Western blot analysis of cells transiently transfected with the epitope-tagged G189E cDNA indicated that this protein was expressed at the same level as the epitope-tagged wild-type protein. In contrast to the mutants described above, this protein co-localizes with α-mannosidase II, indicating correct targeting to the Golgi apparatus. Therefore, the G189E mutation seems to directly affect the transport activity of the CMP-Sia-Tr. Changing glycine 189 to alanine did not influence the activity of CMP-Sia-Tr, measured as polysialic acid expression in clones of the Lec2 complementation group. In contrast, the activities of G189Q and G189I mutants were drastically decreased and resembled that of the G189E mutant. These results suggest that not the charge repulsion between glutamine and CMP-sialic acid, but rather the size of the amino acid at position 189, is a critical factor for the transport activity. Large amino acids at this position may lead to steric hindrance of a hydrophilic "channel" required to translocate CMP-sialic acid through the membrane. This hypothesis is in good agreement with the transporter model proposed recently (17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), where Gly189 is part of a transmembrane helix, closely located to the cytosolic face of the membrane. Another explanation would be that the mutation destroys a potential site involved in the binding of cofactors or in protein dimerization. So far, however, there are no experimental data supporting the idea.The G189E mutation identified a functionally important region of CMP-Sia-Tr. In the mutant 9D3, sialic acid is undetectable by either Western blotting or immunocytochemistry. Overexpression of the mutant G189E in 9D3 cells, however, restored transport activity at a very low level. Due to the high sensitivity of the anti-PSA mAb 735, a very faint PSA signal was visible in immunocytochemistry and Western blot after transient transfection of cells. The signal intensity is however too low to be reproduced in Fig. 6, and sialic acid reexpression was not detectable with Maackia amurensis lectin. Thus, the mutant transporter from 9D3 cells is not completely inactive, but the endogenous expression of the mutated protein in 9D3 cells is insufficient to translocate CMP-sialic acid at a rate necessary for detectable amounts of (poly)sialic acid at the cell surface. Transient overexpression of the deletion mutants in CHO cells of the Lec2 cells did not lead to a detectable complementation. Together with the above results, this strongly suggests that these mutants are completely inactive.The change Gly189 to Glu occurs in a region that is conserved among CMP-Sia-Tr and UDP-Gal-Tr isolated from mammals and the yeast Schizosacchaccharomyces pombe. Furthermore, this sequence is found in a putative nucleotide-sugar transporter ofC. elegans. All transporter sequences containing this motif are listed in Fig. 7. The high conservation strongly suggests that this amino acid stretch is essential for a functionally active transporter. On the other hand, the appearance of this domain in transporters of different specificity argues against a direct involvement in nucleotide-sugar binding or recognition. Additional studies are required to define the functional role of this sequence motif.An important aspect of this study consists in the fact that a CMP-Sia-Tr mutant exhibiting residual transport activity could be isolated after chemical mutation of CHO cells. Isolation and functional analysis of such mutants provides a powerful way to gain further insight into structure-function relationships for this structurally ambitious group of molecules. Carbohydrates added to cell surface proteins and lipids provide major contact and communication elements for animal cells. The biosynthesis of the carbohydrate structures occurs mainly in the luminal parts of the endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumCHOChinese hamster ovaryCMP-Sia-TrCMP-sialic acid transporterHAhemagglutininPCRpolymerase chain reactionRT-PCRreverse transcription PCRUDP-Gal-TrUDP-galactose transporterMOPS4-morpholinepropanesulfonic acidmAbmonoclonal antibodyPBSphosphate-buffered salinePSApolysialic acid.1The abbreviations used are: ERendoplasmic reticulumCHOChinese hamster ovaryCMP-Sia-TrCMP-sialic acid transporterHAhemagglutininPCRpolymerase chain reactionRT-PCRreverse transcription PCRUDP-Gal-TrUDP-galactose transporterMOPS4-morpholinepropanesulfonic acidmAbmonoclonal antibodyPBSphosphate-buffered salinePSApolysialic acid. and Golgi apparatus and therefore requires specific nucleotide sugar transport systems (1Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar). Nucleotide sugar transporters have been described for CMP-sialic acid, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, UDP-xylose, GDP-mannose, UDP-glucuronic acid, and UDP-glucose (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar, 3Hirschberg C.B. Snider M.D. Annu. Rev. Biochem. 1987; 56: 63-88Crossref PubMed Scopus (439) Google Scholar, 4Verbert A. Cacan R. Cecchelli R. Biochimie. 1987; 69: 91-99Crossref PubMed Scopus (15) Google Scholar). These proteins function as antiporters in an ATP- and ion-independent manner by exchanging the nucleotide sugar with the corresponding nucleoside monophosphate generated in the organellar lumen through the action of glycosyltransferases and nucleoside diphosphatases (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar, 5Abeijon C. Mandon E.C. Hirschberg C.B. Trends Biochem. Sci. 1997; 22: 203-207Abstract Full Text PDF PubMed Scopus (100) Google Scholar). The high substrate specificity of the nucleotide sugar transporters, which has been demonstrated in biochemical and genetic analysis (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar), makes these molecules ideal targets for the selective inhibition of glycoconjugate maturation. Increased sialylation has been described for tumor cell surfaces and has been shown to correlate positively with malignant potential (6Santer U.V. DeSantis R. Hard K.J. van Kuik J.A. Vliegenthart J.F. Won B. Glick M.C. Eur. J. Biochem. 1989; 181: 249-260Crossref PubMed Scopus (54) Google Scholar, 7Saitoh O. Wang W.C. Lotan R. Fukuda M. J. Biol. Chem. 1992; 267: 5700-5711Abstract Full Text PDF PubMed Google Scholar, 8Bresalier R.S. Ho S.B. Schoeppner H.L. Kim Y.S. Sleisenger M.H. Brodt P. Byrd J.C. Gastroenterology. 1996; 110: 1354-1367Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Gorelik E. Xu F. Henion T. Anaraki F. Galili U. Cancer Res. 1997; 57: 332-336PubMed Google Scholar). Since numerous sialyltransferases (for a review, see Ref. 10Tsuji S. J. Biochem. ( Tokyo ). 1996; 120: 1-13Crossref PubMed Scopus (217) Google Scholar) but probably only a single CMP-sialic acid transporter (2Hirschberg C.B. Clapham D.E. Ehrlich B.E. Organellar Ion Channels and Transporters. Rockefeller University Press, New York1996: 105-120Google Scholar) exist, the transporter may provide an effective target to inhibit cell surface sialylation. Accordingly, the inhibition of the UDP-galactose transporter and CMP-sialic acid transporter by somatic mutations and synthetic inhibitors resulted in strong reduction of the metastatic potential in the murine MDAY-D2 tumor cell line and in human colorectal cancer lines in nude mice (11Harvey B.E. Toth C.A. Wagner H.E. Steele G.D. Thomas P. Cancer Res. 1992; 52: 1775-1779PubMed Google Scholar, 12Harvey B.E. Thomas P. Biochem. Biophys. Res. Commun. 1993; 190: 571-575Crossref PubMed Scopus (33) Google Scholar, 13Takano R. Muchmore E. Dennis J.W. Glycobiology. 1994; 4: 665-674Crossref PubMed Scopus (74) Google Scholar). endoplasmic reticulum Chinese hamster ovary CMP-sialic acid transporter hemagglutinin polymerase chain reaction reverse transcription PCR UDP-galactose transporter 4-morpholinepropanesulfonic acid monoclonal antibody phosphate-buffered saline polysialic acid. endoplasmic reticulum Chinese hamster ovary CMP-sialic acid transporter hemagglutinin polymerase chain reaction reverse transcription PCR UDP-galactose transporter 4-morpholinepropanesulfonic acid monoclonal antibody phosphate-buffered saline polysialic acid. Considerable progress in studying the transport of nucleotide sugars into the Golgi lumen has been made by the molecular cloning of nucleotide sugar transporter genes. CMP-Sia-Tr and UDP-Gal-Tr cDNAs were cloned from mammalian species (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar, 15Miura N. Ishida N. Hoshino M. Yamauchi M. Hara T. Ayusawa D. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 236-241Crossref PubMed Scopus (119) Google Scholar, 16Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 1074-1078Crossref PubMed Scopus (91) Google Scholar, 17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), and the GDP-mannose transporter from Leishmania donovani (18Descoteaux A. Luo Y. Turco S.J. Beverly S.M. Science. 1995; 269: 1869-1872Crossref PubMed Scopus (143) Google Scholar, 19Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and the UDP-GlcNAc- and UDP-Gal-transporters were cloned from yeast (20Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (110) Google Scholar, 21Tabuchi M. Tanaka N. Iwahara S. Takegawa K. Biochem. Biophys. Res. Commun. 1997; 232: 121-125Crossref PubMed Scopus (77) Google Scholar). Related cDNAs from human, Saccharomyces cerevisiae, andCaenorhabditis elegans were also identified by homology searches in the gene data bases (16Ishida N. Miura N. Yoshioka S. Kawakita M. J. Biochem. ( Tokyo ). 1996; 120: 1074-1078Crossref PubMed Scopus (91) Google Scholar, 19Ma D. Russell D.G. Beverly S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 20Abeijon C. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5963-5968Crossref PubMed Scopus (110) Google Scholar). Heterologous expression of the murine CMP-Sia-Tr on the zero background of S. cerevisiae was used to confirm the biological function of the protein (22Berninsone P. Eckhardt M. Gerardy-Schahn R. Hirschberg C.B. J. Biol. Chem. 1997; 272: 12616-12619Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The transfected yeast cells acquired the ability to translocate CMP-sialic acid. All nucleotide-sugar transporters cloned to date have been identified by complementation cloning in glycosylation mutants. The murine and hamster CMP-Sia-Tr were isolated by expression cloning in a clone of the Lec2 complementation group. Although the Lec2 mutation is known to inhibit translocation of CMP-sialic acid into the Golgi lumen (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (196) Google Scholar), the molecular basis responsible for the asialo phenotype is still unknown. This study was undertaken to determine the molecular basis of the Lec2 phenotype. Thereby, we took advantage of the fact that several independent Lec2 mutants have been isolated in the laboratory (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar). The results summarized in this study demonstrate that Lec2 cells carry defects in the CMP-Sia-Tr gene leading to aberrations in the transporter protein. The analysis of these mutants provides a useful system to gain insight into structure-function relationships of the transporter protein. DISCUSSIONCells of the Lec2 complementation group are defective in the transport of CMP-NeuAc into the lumen of the Golgi apparatus (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Clones exhibiting the lec2 defect have been isolated by lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar, 31Briles E.B. Li E. Kornfeld S. J. Biol. Chem. 1977; 252: 1107-1116Abstract Full Text PDF PubMed Google Scholar) or immunoselection (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar). The lec2mutation causes the expression of asialo cell surfaces. Among the carbohydrate epitopes missing is PSA. Reexpression of PSA was therefore used to isolate cDNAs encoding the murine and hamster CMP-Sia-Tr cDNAs via complementation cloning (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar, 17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar). The present study was carried out to determine the lec2 mutation at the molecular level. Four independent mutants (1E3, 6B2, 8G8, and 9D3), which arose from chemical mutation experiments (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar), together with the clone Lec2 isolated via lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar), were analyzed by Northern blotting and RT-PCR. Deletions, insertions, and point mutations in the CMP-Sia-Tr coding region were found that demonstrated that the gene defective in these cells encodes the CMP-Sia-Tr. The high frequency by which these mutants occur after chemical mutagenesis (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 33Stanley P. Annu. Rev. Genet. 1984; 18: 525-552Crossref PubMed Scopus (167) Google Scholar) makes this approach an attractive way to identify functionally important primary sequence elements and to investigate structure-function relationships of nucleotide-sugar transporters.Deletions observed in clones 6B2, 8G8, and Lec2, are likely to result from mutations in splice acceptor or donor sites. Support for this assumption comes from the observation that the deleted sequence sections share common boundaries. The extended sequence changes associated with the mutations lec2, 6b2, and8g8, cause mistargeting of the translation products. While co-localization of the wild-type CMP-Sia-Tr with α-mannosidase II indicated transport to the Golgi apparatus (Ref. 14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar and Figs. 4 and 5), all internally deleted or C-terminally truncated transporter mutants were not in the α-mannosidase II compartment but produced a diffuse staining throughout the cytoplasma. The staining pattern observed for the mutants might be explained by retention of the proteins in the ER. The extended primary sequence changes caused by the deletions or truncations most probably lead to misfolded proteins, which do not escape the process of "ER quality control" (34Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar). Consistent with this, reduced expression levels were observed for the mutant proteins.Analysis of the CMP-Sia-Tr mRNA from clone 9D3 revealed a single missense mutation, resulting in exchange of glycine at position 189 for glutamic acid. Like the other mutants, the steady-state level of mRNA expression in this mutant was comparable with that of wild-type cells (Fig. 1), and Western blot analysis of cells transiently transfected with the epitope-tagged G189E cDNA indicated that this protein was expressed at the same level as the epitope-tagged wild-type protein. In contrast to the mutants described above, this protein co-localizes with α-mannosidase II, indicating correct targeting to the Golgi apparatus. Therefore, the G189E mutation seems to directly affect the transport activity of the CMP-Sia-Tr. Changing glycine 189 to alanine did not influence the activity of CMP-Sia-Tr, measured as polysialic acid expression in clones of the Lec2 complementation group. In contrast, the activities of G189Q and G189I mutants were drastically decreased and resembled that of the G189E mutant. These results suggest that not the charge repulsion between glutamine and CMP-sialic acid, but rather the size of the amino acid at position 189, is a critical factor for the transport activity. Large amino acids at this position may lead to steric hindrance of a hydrophilic "channel" required to translocate CMP-sialic acid through the membrane. This hypothesis is in good agreement with the transporter model proposed recently (17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), where Gly189 is part of a transmembrane helix, closely located to the cytosolic face of the membrane. Another explanation would be that the mutation destroys a potential site involved in the binding of cofactors or in protein dimerization. So far, however, there are no experimental data supporting the idea.The G189E mutation identified a functionally important region of CMP-Sia-Tr. In the mutant 9D3, sialic acid is undetectable by either Western blotting or immunocytochemistry. Overexpression of the mutant G189E in 9D3 cells, however, restored transport activity at a very low level. Due to the high sensitivity of the anti-PSA mAb 735, a very faint PSA signal was visible in immunocytochemistry and Western blot after transient transfection of cells. The signal intensity is however too low to be reproduced in Fig. 6, and sialic acid reexpression was not detectable with Maackia amurensis lectin. Thus, the mutant transporter from 9D3 cells is not completely inactive, but the endogenous expression of the mutated protein in 9D3 cells is insufficient to translocate CMP-sialic acid at a rate necessary for detectable amounts of (poly)sialic acid at the cell surface. Transient overexpression of the deletion mutants in CHO cells of the Lec2 cells did not lead to a detectable complementation. Together with the above results, this strongly suggests that these mutants are completely inactive.The change Gly189 to Glu occurs in a region that is conserved among CMP-Sia-Tr and UDP-Gal-Tr isolated from mammals and the yeast Schizosacchaccharomyces pombe. Furthermore, this sequence is found in a putative nucleotide-sugar transporter ofC. elegans. All transporter sequences containing this motif are listed in Fig. 7. The high conservation strongly suggests that this amino acid stretch is essential for a functionally active transporter. On the other hand, the appearance of this domain in transporters of different specificity argues against a direct involvement in nucleotide-sugar binding or recognition. Additional studies are required to define the functional role of this sequence motif.An important aspect of this study consists in the fact that a CMP-Sia-Tr mutant exhibiting residual transport activity could be isolated after chemical mutation of CHO cells. Isolation and functional analysis of such mutants provides a powerful way to gain further insight into structure-function relationships for this structurally ambitious group of molecules. Cells of the Lec2 complementation group are defective in the transport of CMP-NeuAc into the lumen of the Golgi apparatus (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I.B. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Clones exhibiting the lec2 defect have been isolated by lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar, 31Briles E.B. Li E. Kornfeld S. J. Biol. Chem. 1977; 252: 1107-1116Abstract Full Text PDF PubMed Google Scholar) or immunoselection (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar). The lec2mutation causes the expression of asialo cell surfaces. Among the carbohydrate epitopes missing is PSA. Reexpression of PSA was therefore used to isolate cDNAs encoding the murine and hamster CMP-Sia-Tr cDNAs via complementation cloning (14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar, 17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar). The present study was carried out to determine the lec2 mutation at the molecular level. Four independent mutants (1E3, 6B2, 8G8, and 9D3), which arose from chemical mutation experiments (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar), together with the clone Lec2 isolated via lectin resistance (24Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar), were analyzed by Northern blotting and RT-PCR. Deletions, insertions, and point mutations in the CMP-Sia-Tr coding region were found that demonstrated that the gene defective in these cells encodes the CMP-Sia-Tr. The high frequency by which these mutants occur after chemical mutagenesis (32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 33Stanley P. Annu. Rev. Genet. 1984; 18: 525-552Crossref PubMed Scopus (167) Google Scholar) makes this approach an attractive way to identify functionally important primary sequence elements and to investigate structure-function relationships of nucleotide-sugar transporters. Deletions observed in clones 6B2, 8G8, and Lec2, are likely to result from mutations in splice acceptor or donor sites. Support for this assumption comes from the observation that the deleted sequence sections share common boundaries. The extended sequence changes associated with the mutations lec2, 6b2, and8g8, cause mistargeting of the translation products. While co-localization of the wild-type CMP-Sia-Tr with α-mannosidase II indicated transport to the Golgi apparatus (Ref. 14Eckhardt M. Mühlenhoff M. Bethe A. Gerardy-Schahn R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7572-7576Crossref PubMed Scopus (148) Google Scholar and Figs. 4 and 5), all internally deleted or C-terminally truncated transporter mutants were not in the α-mannosidase II compartment but produced a diffuse staining throughout the cytoplasma. The staining pattern observed for the mutants might be explained by retention of the proteins in the ER. The extended primary sequence changes caused by the deletions or truncations most probably lead to misfolded proteins, which do not escape the process of "ER quality control" (34Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar). Consistent with this, reduced expression levels were observed for the mutant proteins. Analysis of the CMP-Sia-Tr mRNA from clone 9D3 revealed a single missense mutation, resulting in exchange of glycine at position 189 for glutamic acid. Like the other mutants, the steady-state level of mRNA expression in this mutant was comparable with that of wild-type cells (Fig. 1), and Western blot analysis of cells transiently transfected with the epitope-tagged G189E cDNA indicated that this protein was expressed at the same level as the epitope-tagged wild-type protein. In contrast to the mutants described above, this protein co-localizes with α-mannosidase II, indicating correct targeting to the Golgi apparatus. Therefore, the G189E mutation seems to directly affect the transport activity of the CMP-Sia-Tr. Changing glycine 189 to alanine did not influence the activity of CMP-Sia-Tr, measured as polysialic acid expression in clones of the Lec2 complementation group. In contrast, the activities of G189Q and G189I mutants were drastically decreased and resembled that of the G189E mutant. These results suggest that not the charge repulsion between glutamine and CMP-sialic acid, but rather the size of the amino acid at position 189, is a critical factor for the transport activity. Large amino acids at this position may lead to steric hindrance of a hydrophilic "channel" required to translocate CMP-sialic acid through the membrane. This hypothesis is in good agreement with the transporter model proposed recently (17Eckhardt M. Gerardy-Schahn R. Eur. J. Biochem. 1997; 248: 187-192Crossref PubMed Scopus (40) Google Scholar), where Gly189 is part of a transmembrane helix, closely located to the cytosolic face of the membrane. Another explanation would be that the mutation destroys a potential site involved in the binding of cofactors or in protein dimerization. So far, however, there are no experimental data supporting the idea. The G189E mutation identified a functionally important region of CMP-Sia-Tr. In the mutant 9D3, sialic acid is undetectable by either Western blotting or immunocytochemistry. Overexpression of the mutant G189E in 9D3 cells, however, restored transport activity at a very low level. Due to the high sensitivity of the anti-PSA mAb 735, a very faint PSA signal was visible in immunocytochemistry and Western blot after transient transfection of cells. The signal intensity is however too low to be reproduced in Fig. 6, and sialic acid reexpression was not detectable with Maackia amurensis lectin. Thus, the mutant transporter from 9D3 cells is not completely inactive, but the endogenous expression of the mutated protein in 9D3 cells is insufficient to translocate CMP-sialic acid at a rate necessary for detectable amounts of (poly)sialic acid at the cell surface. Transient overexpression of the deletion mutants in CHO cells of the Lec2 cells did not lead to a detectable complementation. Together with the above results, this strongly suggests that these mutants are completely inactive. The change Gly189 to Glu occurs in a region that is conserved among CMP-Sia-Tr and UDP-Gal-Tr isolated from mammals and the yeast Schizosacchaccharomyces pombe. Furthermore, this sequence is found in a putative nucleotide-sugar transporter ofC. elegans. All transporter sequences containing this motif are listed in Fig. 7. The high conservation strongly suggests that this amino acid stretch is essential for a functionally active transporter. On the other hand, the appearance of this domain in transporters of different specificity argues against a direct involvement in nucleotide-sugar binding or recognition. Additional studies are required to define the functional role of this sequence motif. An important aspect of this study consists in the fact that a CMP-Sia-Tr mutant exhibiting residual transport activity could be isolated after chemical mutation of CHO cells. Isolation and functional analysis of such mutants provides a powerful way to gain further insight into structure-function relationships for this structurally ambitious group of molecules. We thank Michael Cahill for critical remarks on the manuscript, K. Moremen for kindly providing the mannosidase II antiserum, and D. Bitter-Suermann for continuous support.