Ihara Hideyuki, Hanashima Shinya, Tsukamoto Hiroki, Yamaguchi Yoshiki, Taniguchi Naoyuki, Ikeda Yoshitaka
Department of Biomolecular Sciences, Saga University Faculty of Medicine, Saga, Japan.
Biochim Biophys Acta. 2013 Oct;1830(10):4482-90. doi: 10.1016/j.bbagen.2013.05.013. Epub 2013 May 18.
The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases.
The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated.
Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase.
The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases.
The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.
真核生物N - 聚糖的合成与根瘤菌的结瘤因子都涉及α1,6 - 岩藻糖基化。这些岩藻糖基化反应分别由真核生物的α1,6 - 岩藻糖基转移酶FUT8和根瘤菌酶NodZ催化。这两种酶具有相似的酶学性质和结构，但表现出不同的受体特异性：FUT8和NodZ分别偏好N - 聚糖和壳寡糖。本研究旨在检测FUT8和NodZ对壳寡糖的岩藻糖基化作用，并从其对糖苷酶水解的抗性方面对所得的二岩藻糖基化壳寡糖进行表征。
研究FUT8或NodZ是否能催化先由另一种酶进行单岩藻糖基化的壳寡糖的进一步岩藻糖基化。通过正相高效液相色谱、质谱和核磁共振对连续反应产生的寡糖产物进行分析。还研究了二岩藻糖基化对糖苷酶消化敏感性的影响。
FUT8和NodZ都能够对单岩藻糖基化的壳寡糖进行进一步的岩藻糖基化。对所得寡糖的结构分析表明，还原端的GlcNAc残基和非还原端的第三个GlcNAc残基通过α1,6 - 连接被岩藻糖基化。二岩藻糖基化保护寡糖不被己糖胺酶和溶菌酶广泛降解为GlcNAc，甚至也能防止被岩藻糖苷酶去岩藻糖基化。
FUT8和NodZ对共同底物的顺序作用有效地产生了位点特异性二岩藻糖基化的壳寡糖。这种修饰赋予寡糖对各种糖苷酶的抗性。
真核生物和细菌的α1,6 - 岩藻糖基转移酶组合作用于壳寡糖导致形成二岩藻糖基化产物，这有助于稳定壳寡糖抵抗糖苷酶的作用。
