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 ␣-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) 1 and Golgi apparatus and therefore requires specific nucleotide sugar transport systems (1, 2). Nucleotide sugar transporters have been described for CMP-sialic acid, UDP-galactose, UDPGlcNAc, UDP-GalNAc, GDP-fucose, UDP-xylose, GDP-mannose, UDP-glucuronic acid, and UDP-glucose (2-4). 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 (2, 5). The high substrate specificity of the nucleotide sugar transporters, which has been demonstrated in biochemical and genetic analysis (2), 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 (6 -9). Since numerous sialyltransferases (for a review, see Ref. 10) but probably only a single CMP-sialic acid transporter (2) exist, the transporter may provide an effective target ...

Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.Nucleotide sugar transporters form a family of structurally related multimembrane-spanning proteins of the Golgi apparatus and the endoplasmic reticulum (ER).1 Their function resides in translocating activated sugars from the cytosol into the lumen of the ER and Golgi apparatus (1-3). Transporters therefore provide essential components of the glycosylation pathways in eukaryotic cells (for review, see Ref. 4). Recently, the first nucleotide sugar transporters have been identified at the molecular level. Complementation cloning in mutant cells lacking specific nucleotide sugar transport activities identified the mammalian transporters (Tr) for CMP-sialic acid (5, 6), UDP-galactose (UDP-Gal) (7,8), and UDP-N-acetylglucosamine (UDP-GlcNAc) (9), the yeast transporters for UDP-GlcNAc (10) and UDP-Gal (11), and the Leishmania GDP-mannose transporter (12, 13). Additional putative nucleotide sugar transporter sequences have been identified using sequence homologies (8,10,13). Surprisingly high sequence homology has been found between the mammalian transporters for CMPsialic acid, UDP-Gal, and UDP-GlcNAc (8, 9), whereas the conservation between transporters of identical specificity in different biological kingdoms can be low (9, 10).As mentioned above, cloning of transporters was achieved by complementation, and the cDNAs isolated were demonstrated to correct the mutant phenotype. Transport activity, however, has only been proven for the murine CMP-Sia-Tr, which could be functionally expressed in Saccharomyces cerevisiae (14). Because yeast cells lack sialic acids, this result clearly demonstrates that t...

5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K 198 RXR to be essential for nuclear transport and Arg 202 to be necessary to complete the transport process. Cytoplasmic forms of CMPNeu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg 199 and Arg 202 are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMPsialic acid synthetases reveals Arg 202 and Gln 203 as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.Sialic acids acids are a family of negatively charged, 9-carbon sugars that form terminal residues on cell surface glycoproteins and glycolipids and provide the bulk of the negative charge, which is characteristic for animal cell surfaces (for (8), as well as development (6, 9) and progression of malignancies (10 -12) are accompanied by alterations in the cellular sialylation pattern. In bacteria sialic acids are found as components of capsules and lipooligosaccharides and often are important virulence factors, mediating resistance to host defense mechanisms (reviewed in Refs. 13-15). For example, Neisseria meningitidis serogroup B (NmB), the major cause of meningitis outbreaks in the western hemisphere, expresses a capsular polysaccaride consisting of ␣2,8-linked sialic acid residues, exclusively. The polysialic acid (polySia) of the NmB capsule is identical to host expressed polySia, which represents a specific posttranslational modification of the neural cell adhesion molecule (for review, see Ref. 16). This classical example of antigenic mimicry explains the low serum response caused by NmB in infected individuals (17).A prerequisite for the incorporation of sialic acids into glycoconjugates is their activation as cytidine-monophosphate diester (CMP-Neu...

Poly-␣-2,8-sialic acid (polysialic acid) is a posttranslational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto-and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both autoand NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn 74 in ST8SiaIV and Asn 89 and Asn 219 in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylationnegative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.