Inhibition of CMP-Sialic Acid Transport into Golgi Vesicles by Nucleoside Monophosphates

Chiaramonte, Molly; Koviach, Jennifer L.; Moore, Chris; Iyer, Vidhya V.; Wagner, Carston R.; Halcomb, Randall L.; Miller, Wayne H.; Melançon, Paul; Kuchta, Robert D.

doi:10.1021/bi011262w

Cited by 18 publications

(26 citation statements)

References 42 publications

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“…It should be noted here that intracellular nucleotide sugar concentrations are estimated to be 60 -200 M for UDPGal and 70 -500 M for CMP-Sia (40,41). In view of these values, the K m value of chimera G1 for UDP-Gal may not be too high for the transporter to work with reasonable efficiency in cells.…”

Section: Discussionmentioning

confidence: 78%

“…hUGT1 and hCST may not strictly discriminate between UMP and CMP, although the affinity of hCST for UMP may not be very high because direct measurement of transport did not detect accumulation of UMP in CST-expressing membrane vesicles. Chiaramonte et al (40) recently examined the substrate specificity of CST in detail and reported that CST can transport UMP, although less efficiently than CMP.…”

Section: Discussionmentioning

confidence: 99%

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Substrate Recognition by Nucleotide Sugar Transporters

Aoki

Ishida

Kawakita

2003

Journal of Biological Chemistry

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Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.Nucleotide sugar transporters are membrane proteins localized in the endoplasmic reticulum and Golgi apparatus. They play an indispensable role in constructing the sugar chains of glycoconjugates. The transporters carry nucleotide sugars into the endoplasmic reticulum and Golgi apparatus, in which they are used by specific transferases as precursors of sugar chains (for recent reviews, see Refs. 1-5). More than simply functioning as a passive entrance route of nucleotide sugars into the organelles, the transporters may regulate the amounts of nucleotide sugars available in the lumen of the endoplasmic reticulum or Golgi apparatus and consequently may affect the sugar chain composition of a cell (6). To better understand the roles of transporters in regulating sugar chains and, in turn, various aspects of cellular functions, it is indispensable to define which substrate(s) each transporter recognizes and to understand how and with what stringency the transporter discriminates between its own substrate(s) and irrelevant nucleotide sugars.The functions of nucleotide sugar transporters have long been studied by analyzing the properties of transporter-deficient mutant cells and by in vitro characterization of the transport activity using microsomal fractions from natural sources. In recent years, the genes encoding several kinds of nucleotide sugar transporters have been cloned from several species (Refs. 7-12 and references therein) and have provided us with useful tools for studying the molecular and functional features of the transporters. For instance, functionally important subregions of the transporter molecules were defi...

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Substrate Recognition by Nucleotide Sugar Transporters

Aoki

Ishida

Kawakita

2003

Journal of Biological Chemistry

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“…P-3F ax -Neu5Ac presumably enters the intracellular sialic acid synthesis pathway via a salvage pathway and is converted to CMP-P-3F ax -Neu5Ac (23). Only CMP-activated sialic acids can be recognized by the CMP-sialic acid transporter and undergo active transportation into the Golgi lumen where they serve as substrate for sialyltransferases (24,25). Rillahan and colleagues proposed that P-3F ax -Neu5Ac blocks sialylation globally via 2 mechanisms.…”

Section: Introductionmentioning

confidence: 99%

“…Initially, CMP-activated P-3F ax -Neu5Ac inhibits sialyltransferases directly and thereby prevents incorporation of natural sialic acids into assembling glycans. As a consequence, CMP sialic acids accumulate in the cell and disable UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE/MNK), a key enzyme upstream in the de novo synthesis pathway of sialic acids (25,26). In a 2-step reaction, GNE/MNK catalyzes intracellular sialic acid precursors (UDP-N-Acetylglucosamine) to NAcetylmannosamine (ManNAc) and ManNAc-6-phosphate (ManNAc-6P) which is finally converted into CMP-Neu5Ac (27).…”

Section: Introductionmentioning

confidence: 99%

Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth

Büll

Boltje

Wassink

et al. 2013

Molecular Cancer Therapeutics

151

133

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Cancer cells decorate their surface with a dense layer of sialylated glycans by upregulating the expression of sialyltransferases and other glycogenes. Although sialic acids play a vital role in many biologic processes, hypersialylation in particular has been shown to contribute to cancer cell progression and metastasis. Accordingly, selective strategies to interfere with sialic acid synthesis might offer a powerful approach in cancer therapy. In the present study, we assessed the potential of a recently developed fluorinated sialic acid analogue (P-3F ax -Neu5Ac) to block the synthesis of sialoglycans in murine melanoma cells and the consequences on cell adhesion, migration, and in vivo growth. The results showed that P-3F ax -Neu5Ac readily caused depletion of a2,3-/a2,6-linked sialic acids in B16F10 cells for several days. Long-term inhibition of sialylation for 28 days was feasible without affecting cell viability or proliferation. Moreover, P-3F ax -Neu5Ac proved to be a highly potent inhibitor of sialylation even at high concentrations of competing sialyltransferase substrates. P-3F ax -Neu5Ac-treated cancer cells exhibited impaired binding to poly-L-lysine, type I collagen, and fibronectin and diminished migratory capacity. Finally, blocking sialylation of B16F10 tumor cells with this novel sialic acid analogue reduced their growth in vivo. These results indicate that P-3F ax -Neu5Ac is a powerful glycomimetic capable of inhibiting aberrant sialylation that can potentially be used for anticancer therapy. Mol Cancer Ther; 12(10); 1935-46. Ó2013 AACR.

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“…Since CMP is an inhibitor of sialyltransferases, its rapid removal from the Golgi complex is important to optimize sialyltransferase activity (33,34). The mammalian CST has been cloned by phenotypic correction of the Lec2 CHO cell CMPsialic acid transport mutant (31), and its identity has been further confirmed by functional expression in organisms that lack an endogenous CST, such as Saccharomyces cerevisiae and Escherichia coli (32,35).…”

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confidence: 99%

The CMP-sialic Acid Transporter Is Localized in the Medial-Trans Golgi and Possesses Two Specific Endoplasmic Reticulum Export Motifs in Its Carboxyl-terminal Cytoplasmic Tail

Zhao

Chen

Vertel

et al. 2006

Journal of Biological Chemistry

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The addition of sialic acid to glycoproteins and glycolipids requires Golgi sialyltransferases to have access to their glycoconjugate substrates and nucleotide sugar donor, CMP-sialic acid. CMP-sialic acid is transported into the lumen of the Golgi complex through the CMP-sialic acid transporter, an antiporter that also functions to transport CMP into the cytosol. We localized the transporter using immunofluorescence and deconvolution microscopy to test the prediction that it is broadly distributed across the Golgi stack to serve the many sialyltransferases involved in glycoconjugate sialylation. The transporter co-localized with ST6GalI in the medial and trans Golgi, showed partial overlap with a medial Golgi marker and little overlap with early Golgi or trans Golgi network markers. Endoplasmic reticulumretained forms of sialyltransferases did not redistribute the transporter from the Golgi to the endoplasmic reticulum, suggesting that transporter-sialyltransferase complexes are not involved in transporter localization. Next we evaluated the role of the transporter's N-and C-terminal cytoplasmic tails in its trafficking and localization. The N-tail was not required for either endoplasmic reticulum export or Golgi localization. The C-tail was required for endoplasmic reticulum export and contained di-Ile and terminal Val motifs at its very C terminus that function as independent endoplasmic reticulum export signals. Deletion of the last four amino acids of the C-tail (IIGV) eliminated these export signals and prevented endoplasmic reticulum export of the transporter. This form of the transporter supplied limited amounts of CMP-sialic acid to Golgi sialyltransferases but was unable to completely rescue the transporter defect of Lec2 Chinese hamster ovary cells.

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Inhibition of CMP-Sialic Acid Transport into Golgi Vesicles by Nucleoside Monophosphates

Cited by 18 publications

References 42 publications

Substrate Recognition by Nucleotide Sugar Transporters

Substrate Recognition by Nucleotide Sugar Transporters

Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth

The CMP-sialic Acid Transporter Is Localized in the Medial-Trans Golgi and Possesses Two Specific Endoplasmic Reticulum Export Motifs in Its Carboxyl-terminal Cytoplasmic Tail

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