Yuziuk J A, Nakagawa H, Hasegawa A, Kiso M, Li S C, Li Y T
Department of Biochemistry, Tulane University School of Medicine, New Orleans, LA 70112, USA.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):1041-8. doi: 10.1042/bj3151041.
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 (Ki approximately 0.007 mM); however, both NeuAc2en and KDN2en are moderate inhibitors of KS (K1 approximately 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-methyl-umbelliferyl-alpha-KDN (MU-KDN) 20 times faster than 4-methylumbelliferyl-alpha-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-GM3 (where GM3 is NeuAc alpha 2 --> 3Gal beta 1 --> 4Glc beta 1-1' Cer, and Cer is ceramide), KDN alpha 2 --> 3lactose, KDN alpha 2 --> 6lactose, KDN alpha 2 --> 6N-acetylgalactosaminitol, KDN alpha 2 --> 6 (KDN alpha 2 --> 3)N-acetylgalactosaminitol and KDN alpha 2 --> 6(GlcNAc beta 1 --> 3) N-acetylgalactosaminitol. However, under the same conditions, these KDN-containing glycoconjugates are refractory to RS. Conversely, GM3, NeuAc alpha 2 --> 3lactose and NeuAc alpha 2 --> 6lactose are effectively hydrolysed by RS but not by KS.
我们发现，在多棘海盘车的内脏中存在两种不同的唾液酸酶：一种是常规唾液酸酶（RS），可从唾液酸糖缀合物中裂解唾液酸；另一种是KDN-唾液酸酶（KS），它能从对RS有抗性的含KDN的糖缀合物中释放出唾液酸类似物KDN（2-酮-3-脱氧-D-甘油-D-半乳糖壬糖酸）。我们制备了纯化6700倍的KS和纯化1300倍的RS，以研究这两种唾液酸酶的性质。多棘海盘车的KS和RS除了糖基特异性外，在其他几个性质上也有所不同，包括分子量、等电点（pI）以及对竞争性和非竞争性抑制剂的敏感性。KS的分子量为31 kDa，pI为8.3，而RS的分子量为128 kDa，pI约为4.8。2,3-脱氢-2-脱氧-N-乙酰神经氨酸（NeuAc2en）是RS的有效竞争性抑制剂（Ki约为0.007 mM），但2,3-脱氢-2-脱氧-KDN（KDN2en）不是；然而，NeuAc2en和KDN2en都是KS的中度抑制剂（K1约为0.04 mM）。Hg2+是RS的有效非竞争性抑制剂，但不是KS的。我们检测了KS和RS水解含KDN和NeuAc的糖缀合物的能力。在最适pH 4.0时，KS水解4-甲基伞形酮基-α-KDN（MU-KDN）的速度比4-甲基伞形酮基-α-NeuAc（MU-NeuAc）快20倍，而RS水解MU-NeuAc的速度比MU-KDN快88倍。KS能有效水解KDN-GM3（其中GM3是NeuAcα2→3Galβ1→4Glcβ1-1'Cer，Cer是神经酰胺）、KDNα2→3乳糖、KDNα2→6乳糖、KDNα2→6N-乙酰半乳糖胺醇、KDNα2→6（KDNα2→3）N-乙酰半乳糖胺醇和KDNα2→6（GlcNAcβ1→3）N-乙酰半乳糖胺醇。然而，在相同条件下，这些含KDN的糖缀合物对RS具有抗性。相反，GM3、NeuAcα2→3乳糖和NeuAcα2→6乳糖能被RS有效水解，但不能被KS水解。
