Abstract
KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.
Highlights
KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature
Despite the structural similarity between KDN and Neu5Ac, ␣-ketosides of KDN are refractory to conventional sialidases
We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase
Summary
C. virginica, collected from Barataria Bay in southern Louisiana, were purchased from P&J Oyster Company, New Orleans, LA. Sephacryl S-200 Gel Filtration—The crude enzyme preparation was applied onto a Sephacryl S-200 column (5 ϫ 100 cm) equilibrated with 50 mM sodium acetate buffer, pH 4.2, containing 0.15 M NaCl. The column was eluted with the same buffer at 1 ml/min, and 20-ml fractions were collected. Purification of KDN-sialidase—The enzyme preparation obtained after octyl Sepharose chromatography (Fig. 1B, fractions 20 to 29) was applied onto a Sephacryl S-100 gel filtration column (1.5 ϫ 90 cm) equilibrated with 40 mM sodium phosphate buffer, pH 6.7. The column was eluted with the same buffer at 0.3 ml/min, and 1-ml fractions were collected The fractions containing both MU-KDN- and MU-Neu5Accleaving activities were pooled and concentrated to 0.2 ml using a Centricon-10 microconcentrator.
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