Mammalian cytidine 5′-monophosphate N -acetylneuraminic acid synthetase: A nuclear protein with evolutionarily conserved structural motifs

Abstract

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is speciesand tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMPNeu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.

Glycoproteins from Insect Cells: Sialylated or Not?

“…The CMP-NeuAc synthase, in contrast to other sugar-nucleotide synthases, is a nuclear resident as shown by biochemical studies (for a review see Kean, 1991) and, more recently, via expression of the cloned murine cDNA (Münster et al, 1998).…”

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

“…The formation of CMP-Kdo is catalyzed by the enzyme Kdo cytidyltransferase (CMP-Kdo synthetase), which is known as KdsB in Escherichia coli (4 -6). The enzyme is similar to CNS, which catalyzes the penultimate step in the addition of sialic acids to oligosaccharides in eukaryotes (7)(8)(9).…”

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

“…The membrane was washed twice in 2ϫ SSC, 0.1% SDS at room temperature for 15 min and twice in 0.1ϫ SSC, 0.1% SDS at 68°C for 20 min. Bound probe was detected by incubation with anti-digoxigenin Ig-alkaline phosphate conjugate (Roche Molecular Biochemicals) and chemiluminescence detection using disodium-3-(4-methoxyspiro{1,2-dioxetane-3,2Ј-(5Ј-chloro)tricyclo-[3.3.1.1 3,7 ]decan}-4-yl)phenylphosphate (Roche Molecular Biochemicals) as a substrate.…”

“…PSA can be detected with high sensitivity and has been shown in recent studies to be an excellent indicator for transformants in which defects in the sialylation pathway are complemented. Expression cloning in combination with PSA detection has been used to identify the polysialyltransferase ST8SiaIV (24,25), the CST from mouse (26) and hamster (27), and the CMP-Neu5Ac synthetase (3). In this study, PSA was a marker in analytical steps carried out to determine the functional consequences of lec8 mutations.…”