Cloning, expression, and characterization of sialic acid synthases

https://doi.org/10.1016/j.bbrc.2005.10.113Get rights and content

Abstract

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.

Section snippets

Materials and methods

Materials. Human cDNA libraries were purchased from Origene Technologies (Rockville, MD). Genomic DNA of Neisseria meningitidis serogroup B was obtained from ATCC (Manassas, VA). Oligonucleotides were from the Midland Certified Reagents (Midland, TX). AmpliTaq DNA polymerase was from Applied Biosciences (Branchburg, NJ). The enzymes, NcoI, NdeI, XhoI, BamHI, and T4 DNA ligase, and DNA markers were purchased from New England Biolabs (Beverly, MA). QuikChange mutagenesis kit was purchased from

Cloning, expression, and purification of human NeuNAc-9-P synthase

An open reading frame encoding 359 amino acids of human NeuNAc-9-P synthase was amplified from human adult brain cDNA library. Authenticity of the gene was confirmed by DNA sequencing and the encoded protein sequence is identical to that of human NeuNAc-9-P synthase previously reported [22]. The NeuNAc-9-P synthase gene was subcloned in an expression plasmid for protein expression in E. coli. The overexpression of NeuNAc-9-P synthase in E. coli cells is very high representing approximately 30%

Discussion

In this paper, we report a detailed characterization of recombinant human NeuNAc-9-P synthase and NeuNAc synthase of N. meningitidis serogroup B, both expressed in E. coli.

One of the major problems with animal NeuNAc-9-P synthases is their stability. We developed prokaryotic expression system for large-scale purification of human enzyme and identified conditions to stabilize it suitable for structural and functional studies. The enzyme stored in the stabilizing buffer for extended period at

Acknowledgments

We acknowledge Mohammed Rafi and Ping Lin for technical assistance and Dr. Stephan Hinderlich for providing ManNAc-6-P substrate for enzyme kinetics experiments. This research was funded in part by the Development Fund from the Division of Nephrology, Vanderbilt University Medical Center and DK62524 (M.S.) from the National Institutes of Health.

References (33)

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Abbreviations: PEP, phosphoenolpyruvate; ManNAc, N-acetylmannosamine; ManNAc-6-P, N-acetylmannosamine-6-phosphate; NeuNAc, N-acetylneuraminic acid; NeuNAc-9-P, N-acetylneuraminic acid 9-phosphate; KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid. NCAM, Neuronal cell adhesion molecule; TBA, thiobarbituric acid assay; TIM, triosephosphate isomerase; AFP, antifreeze protein.

1

These authors contributed equally.

2

Present address: Harvard Medical School, Radiology, Goldenson B-142, 220 Longwood Ave., Boston, MA 02115, USA.

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