Rashid Md Harun-Or, Sadik Golam, Alam Ahm Khurshid, Tanaka Toshihisa
Institute of Biological Science, University of Rajshahi, Rajshahi, 6205, Bangladesh.
Department of Pharmacy, University of Rajshahi, Rajshahi, 6205, Bangladesh.
BMC Biochem. 2017 May 25;18(1):9. doi: 10.1186/s12858-017-0085-1.
The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown.
Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity.
The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.
海洋无脊椎动物海星被发现含有一种新型α - N - 乙酰半乳糖胺酶，即α - GalNAcase II，它催化末端α - N - 乙酰半乳糖胺（α - GalNAc）的去除，此外还含有一种典型的α - N - 乙酰半乳糖胺酶，α - GalNAcase I，它催化末端α - N - 乙酰半乳糖胺（α - GalNAc）的去除，在较小程度上也催化半乳糖的去除。α - GalNAcase I和α - GalNAcase II之间的相互关系以及它们底物特异性差异的分子基础尚不清楚。
使用免疫染色、N端氨基酸测序和肽分析对α - GalNAcase I和II进行化学和结构比较，结果表明它们彼此之间以及与其他糖苷水解酶家族（GHF）27成员具有高度同源性。肽的氨基酸序列显示出与典型α - GalNAcase中活性位点相同的保守残基。在α - GalNAcase II中发现了一些保守氨基酸残基的取代，这些取代位于催化位点附近。其中，α - GalNAcase II中分别取代C171和W173的G171和A173被确定为导致α - GalNAcase II缺乏内在α - 半乳糖苷酶活性的原因。化学修饰支持丝氨酸、天冬氨酸和色氨酸作为活性位点残基的存在。两个色氨酸残基（W16和W173）参与α - 半乳糖苷酶活性，其中一个（W16）参与α - GalNAcase活性。
结果表明，α - GalNAcase I和II在一级结构和高级结构方面密切相关，它们的结构差异是底物特异性差异的原因。
