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 species- and 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 CMP-Neu5Ac 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.

Figure 1

Nucleotide and deduced amino acid sequence of murine CMP-Neu5Ac synthetase. Two potential N-glycosylation sites are indicated by asterisks, and a polyadenylation signal is underlined. Three potential nuclear localization signals are shown as open boxes. Stretches highly homologous to cloned bacterial CMP-Neu5Ac synthetases are shown as shaded boxes.

Figure 2

(Left) Western blot analysis of PSA expressed by CHO wt C6, mutant LEC29, and the mutant LEC29.Lec32 before and after transfection with the plasmid pAM15, encoding the murine CMP-Neu5Ac synthetase. The signal in LEC29.Lec32 is caused by the presence of Lec32 revertants. To confirm the specificity of the signal, samples were treated with endoNE (+). (Right) A third aliquot of cell lysate was developed with digoxigenin-labeled lectin MAA specific for α-2,3-linked sialic acid.

Figure 3

Nuclear localization of CMP-Neu5Ac synthetase by Western blot analysis. LEC29.Lec32 cells were transfected transiently with pAM16Flag or with empty vector pFlagB1. After 48 hours, cytosolic (c) and nuclear (n) extracts were prepared and subjected to Western blot analysis with the anti-Flag mAb M5. A band at ≈50 kDa is prominent in the nuclear extract of cells transfected with pAM16Flag.

Figure 4

Nuclear localization of CMP-Neu5Ac synthetase by immunofluorescence. NIH 3T3 cells were transfected transiently with pAM16Flag (A–C), and control samples were transfected with empty vector pFlagB1 (D–F). Immunofluorescence was carried out with the anti-Flag mAb M5 and fluorescein (DTAF)-conjugated goat anti-mouse Ig (A and D). Nuclear staining was performed with Hoechst 32258 on the same microscopic samples (B and E). In C and F, a light microscopic image of the cells is shown (400×).

Figure 5

Functional expression of murine CMP-Neu5Ac synthetase in E. coli mutant EV5. E. coli EV5 cells were transformed with pQEAM16, encoding the murine CMP-Neu5Ac synthetase, or empty vector pQE30 or pUE17, containing the CMP-Neu5Ac synthetase from N. meningitidis. Expression of the transformed DNA was induced with 0.2 mM isopropyl-β-d-thiogalactoside, and the presence of PSA capsule was determined by Western blot analysis by using anti-PSA mAb 735. PSA was removed specifically if samples were treated with endoNE (+).

Figure 6

Northern blot analysis of total RNA isolated from CHO wt cells, mutant LEC29 and LEC29.Lec32, and the mouse pituitary tumor cell line AtT20 and from murine brain, muscle, heart, and liver samples. mRNA (15 μg per lane) was resolved on a 1% agarose formaldehyde gel and, after transfer to a nylon membrane, was developed with a digoxigenin-labeled RNA-probe, transcribed from the coding region of the CMP-Neu5Ac synthetase cDNA.