Two different sialidases, KDN-sialidase and regular sialidase in the starfish Asterina pectinifera

Abstract

We have found the coexistence of two different sialidases in the entrails of the starfish Asterina pectinifera: a regular sialidase (RS), which cleaves sialic acid from sialoglycoconjugates, and a KDN-sialidase (KS) which releases the sialic acid analogue KDN (2-keto-3-deoxy-D-glycero-d-galacto-nononic acid) from KDN-containing glycoconjugates that are resistant to RS. The 6700-fold purified KS and 1300-fold purified RS were prepared to study the properties of these two sialidases. KS and RS from Asterina starfish differ in several properties other than glycon specificity, including molecular mass, isoelectric point (pI) and susceptibility to competitive and non-competitive inhibitors. KS has a molecular mass of 31 kDa and a pI of 8.3 while RS has a molecular mass of 128 kDa and a pI of about 4.8. 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (NeuAc2en), but not 2,3-dehydro-2-deoxy-KDN (KDN2en) is a potent competitive inhibitor of RS (K(i)~0.007 mM); however, both NeuAc2en and KDN2en are moderate inhibitors of RS (K(i)~0.04 mM). Hg2+ is a potent non-competitive inhibitor of RS but not of KS. KS and RS were examined for their ability to hydrolyse KDN- and NeuAc-containing glycoconjugates. KS hydrolyses 4-methylumbelliferyl-α-KDN (MU-KDN) 20 times faster than 4-methylumbelliferyl-α-NeuAc (MU-NeuAc), while RS hydrolyses MU-NeuAc 88 times faster than MU-KDN at the pH optimum of 4.0. KS effectively hydrolyses KDN-G(M3) (where G(M3) is NeuAcα2→3Galβ1→4Glcβ1-1'Cer, and Cer is ceramide), KDNα2→3lactose, KDNαa2→6lactose, KDNα2→6N-acetylgalactosaminitol, KDNαu2→6(KDNα2→3)N-acetylgalactosaminitol and KDNα2→6(GlcNAcβ1→3)N-acetylgalactosaminitol. However, under the same conditions, these KDN-containing glycoconjugates are refractory to RS. Conversely, G(M3), NeuAcα2→3lactose and NeuAcα2→6lactose are effectively hydrolysed by RS but not by KS.