Discovery of a new type of sialidase, "KDNase," which specifically hydrolyzes deaminoneuraminyl (3-deoxy-D-glycero-D-galacto-2-nonulosonic acid) but not N-acylneuraminyl linkages.
The release of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN, deaminoneuraminic acid) residues from their alpha-ketosidic linkage is required to determine the structural and functional role of KDN-glycoconjugates in sources as disparate as trout egg polysialoglycoproteins and human cancers. We report for the first time the isolation and characterization of a novel type of sialidase (KDNase), which specifically hydrolyzes KDN ketosidic but not N-acylneuraminyl linkages. KDNase activity was assayed using 4-methylumbelliferyl KDN (4-MU-KDN). A KDNase-producing microorganism was identified as Sphingobacterium multivorum. The affinity-purified enzyme was designated KDNase SM to denote its origin and that it was free of N-acylneuraminidase, proteolytic, and other glycosidase activities. KDNase SM activity toward 4-MU-KDN was not inhibited by the N-acylneuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. KDNase SM released free KDN from naturally occurring substrates, including (KDN)GM3, KDN-glycoprotein, which bears a number of O-linked chains of KDN alpha 2-->3Gal beta 1-->3GalNAc alpha 1-->3 (KDN alpha 2-->(-->8KDN alpha 2-->)n-->6)GalNAc alpha 1-->, and the biantennary complex-type of N-glycan, KDN alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(KDN alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. KDNase SM thus exhibited a broad linkage specificity and was able to hydrolyze the KDN residues ketosidically linked alpha 2-->3, alpha 2-->6, and alpha 2-->8. The enzyme did not release Neu5Ac or Neu5Gc from 4-MU-Neu5Ac, N-acetyl-neuraminyllactose, colominic acid, or other Sia(Neu5Ac or Neu5Gc)-containing glycoconjugates.
Kitajima
K
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
Kuroyanagi
H
H
Inoue
S
S
Ye
J
J
Troy
F A
FA
Inoue
Y
Y
D001419:Bacteria / Q000201:enzymology; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D006001:Glycoconjugates / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism*; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism*
D002413:Cations, Divalent; D006001:Glycoconjugates; D006027:Glycosides; D012794:Sialic Acids; D013400:Sugar Acids; C050008:3-deoxyglycero-galacto-nonulosonic acid; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid