Induction, Localization, and Purification of a Novel Sialidase, Deaminoneuraminidase (KDNase), from Sphingobacterium multivorum

Abstract

Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDN␣233Gal␤133GalNAc␣133[KDN␣23 (8KDN␣23) n 36]GalNAcol, as a sole carbon source induced KDNase SM activity 15-40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid.The natural occurrence of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), 1 deaminated neuraminic acid, was first discovered in 1986 by Nadano et al. (1). Subsequently, an increasing number of other KDN-glycoconjugates have been reported (2-14). Substitution of the aminoacyl group at C-5 on Neu5Ac or Neu5Gc by a hydroxyl group blocks almost completely the action of bacterial exosialidases that are commonly used for the identification and structure-function studies of sialoglycoconjugates (1,8,15).Recently, we isolated and identified a Gram-negative soil bacterium, Sphingobacterium multivorum, that produced a new sialidase that selectively catalyzed the hydrolysis of different types of KDN-ketosidic linkages (16). The partial purification of this enzyme, designated KDNase SM, and its substrate specificity were also described previously (16). The most important feature of KDNase SM was the complete absence of sialidase activity that could release Neu5Ac and Neu5Gc residues from a variety of naturally occurring Neu5Ac-and Neu5Gc-containing glycoconjugates. This unique specificity was in distinct contrast with other eukaryotic KDN-sialidases that hydrolyzed both KDN and N-acylneuraminic acid ␣-ketosidic linkages (17, 18). Since the first report of KDN residues in fish egg polysialoglycoproteins (1), it has been presumed that KDN residues would not likely be restricted in occurrence to only lower vertebrates but rather would also be expressed on mammalian cells. With the recent findings that KDN-glycoconjugates are implicated in fertilization in fish egg polysialoglycoproteins (4,7,8), it became important to determine the extent of the natural occurrence of KDN-glycoconjugates in other eukaryotic organisms. While anti-KDN antibodies have been used to show that K...

Catalysis by a New Sialidase, Deaminoneuraminic Acid Residue-cleaving Enzyme (KDNase Sm), Initially Forms a Less Stable α-Anomer of 3-Deoxy-D-glycero-D-galacto-nonulosonic Acid and Is Strongly Inhibited by the Transition State Analogue, 2-Deoxy-2,3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic Acid, but Not by 2-Deoxy-2,3-didehydro-N-acetylneuraminic Acid

“…KDNase Sm is an inducible enzyme in the periplasm of the bacteria (15). The purified enzyme consists of a single polypeptide chain with molecular mass of 47.5 kDa (15). Significantly, KDNase Sm completely lacks any N-acylneuraminidase activity that releases Neu5Ac and Neu5Gc from a variety of Neu5Ac-and Neu5Gc-containing glycoconjugates (14).…”

“…KDN␣233Gal, KDN␣23 6GalNAc, and KDN␣238KDN, in a diverse range of oligosaccharides, glycoproteins, and glycolipids as well as the synthetic substrate, 4-methylumbelliferyl KDN (KDN␣2MeUmb) (14). KDNase Sm is an inducible enzyme in the periplasm of the bacteria (15). The purified enzyme consists of a single polypeptide chain with molecular mass of 47.5 kDa (15).…”

“…KDNase Sm was isolated and purified from S. multivorum as described previously (15). One unit of enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of 1 nmol of KDN␣2MeUmb per min at 25°C.…”

The Aspergillus fumigatus Sialidase Is a 3-Deoxy-d-glycero-d-galacto-2-nonulosonic Acid Hydrolase (KDNase)

“…A sialidase specific for KDN ketosidic linkages was first discovered in the bacterium Sphingobacterium multivorum, and this so-called KDNase released KDN from naturally occurring substrates, including KDN␣2-3Gal, KDN␣2-6GalNAc, and KDN␣2-8KDN linkages, but was not inhibited by Neu5Ac2en (28,29). The KDNase was, however, inhibited by 2,3-didehydro-2,3-dideoxy-D-glycero-D-galacto-nonulosonic acid (KDN2en, 4 in Fig.…”

Diversity in sialic and polysialic acid residues and related enzymes

“…Enzyme catalyzing the hydrolytic removal of KDN residues from their a-ketosidic linkage was anticipated to be an important reagent to complement immunochemical probes in identi®cation of KDN-glycoconjugates and studying their function. We found a KDNase-inducing microorganism, Sphingobacterium multivorum [43,44]. This bacterium induced KDNase that hydrolyzes speci®cally KDN-ketosidic linkage but not N-acylneuraminyl linkages (Table 3).…”