title: A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases.
abstract: Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, 30624 Hannover, Germany.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Keys
authors.forename: Friedrich
authors.initials: F
authors.lastname: Freiberger
authors.forename: Jörg
authors.initials: J
authors.lastname: Ehrit
authors.forename: Jonas
authors.initials: J
authors.lastname: Krueger
authors.forename: Katinka
authors.initials: K
authors.lastname: Eggers
authors.forename: Falk F R
authors.initials: FF
authors.lastname: Buettner
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives*; D038541:Neisseria meningitidis, Serogroup B / Q000201:enzymology*; D010655:Phenylenediamines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D012799:Sialyltransferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D002457:Cell Extracts; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010655:Phenylenediamines; D011994:Recombinant Proteins; C058886:1,2-diamino-4,5-methylenedioxybenzene; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid