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, de aminoneuraminic acid) residues from their alpha-ketosidic linkage is r equired to determine the structural and functional role of KDN-glycoco njugates in sources as disparate as trout egg polysialoglycoproteins a nd human cancers. We report for the first time the isolation and chara cterization of a novel type of sialidase (KDNase), which specifically hydrolyzes KDN ketosidic but not N-acylneuraminyl linkages. KDNase act ivity 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 o rigin and that it was free of N-acylneuraminidase, proteolytic, and ot her 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 occ urring substrates, including (KDN)GM(3), KDN-glycoprotein, which bears a number of O-linked chains of KDN alpha 2-->3Gal beta 1-->3GalNAc al pha 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 bet a 1-->, 4GlcNAc beta 1-->2Man alpha 1--6(KDN alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->2 Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNA c. KDNase SM thus exhibited a broad linkage specificity and was able t o hydrolyze the KDN residues ketosidically linked alpha 2-->3, alpha 2 -->6, and alpha 2-->8. The enzyme did not release Neu5Ac or Neu5Gc fro m 4-MU-Neu5Ac, N-acetylneuraminyllactose, colominic acid, or other Sia (Neu5Ac or Neu5Gc)-containing glycoconjugates.