Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.
Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.
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DOI：
10.1093/glycob/cwu132
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发表时间：
2015-04
期刊：
影响因子：
4.3
通讯作者：
Rendic D
中科院分区：
文献类型：
--
作者：
Dragosits M;Yan S;Razzazi-Fazeli E;Wilson IB;Rendic D
融合裂片（FDL）己糖氨酸酶是在无脊椎动物中参与n -聚糖生物合成的最新遗传定义的糖苷酶，其狭窄的特异性对于昆虫中产生少糖苷n -聚糖至关重要。在这项研究中，我们探索了FDL己糖氨酸酶利用不同人工和天然底物的潜力，无论是作为纯化的天然化合物还是使用各种相关的糖基转移酶在体外生成。除了已知的黑腹果蝇（Drosophila melanogaster） FDL酶外，我们现在还鉴定和表征了蜜蜂（Apis mellifera） FDL同源物。将亲和纯化的昆虫FDL酶的可溶性形式（在酵母和昆虫细胞中均表达）与系统发育不同的重组秀丽隐杆线虫FDL样酶和n -乙酰半乳糖胺（GalNAc）特异性秀丽隐杆线虫己糖氨酸酶hexx -4的酶学性质进行了比较。在对一系列底物（包括天然n -聚糖）的测试中，我们发现无脊椎动物FDL（类）酶对附着在α1,3-甘露糖上的n -乙酰氨基葡萄糖胺具有高度特异性，但在极端条件下也能去除其他末端GalNAc和n -乙酰氨基葡萄糖胺残基。重组FDL也被证明可用于分析野生型和突变型隐杆线虫菌株中n -聚糖的复杂混合物，从而有助于确定该生物体内的少糖苷和杂交n -聚糖的异构体。此外，两个果蝇FDL位点突变体的活性和特异性存在差异，这与昆虫中几丁质分解酶和n -聚糖降解外己糖氨酸酶的高度结构相似性相一致。我们的研究表明GH20己糖氨酸酶家族在真核生物糖缀合物的分解代谢和生物合成中具有重要的结构和功能方面的多样性。
Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog. The enzymatic properties of the soluble forms of the affinity-purified insect FDL enzymes, expressed in both yeast and insect cells, were compared with those of the phylogenetically distinct recombinant Caenorhabditis elegans FDL-like enzymes and the N-acetylgalactosamine (GalNAc)-specific Caenorhabditis hexosaminidase HEX-4. In tests with a range of substrates, including natural N-glycans, we show that the invertebrate FDL(-like) enzymes are highly specific for N-acetylglucosamine attached to the α1,3-mannose, but under extreme conditions also remove other terminal GalNAc and N-acetylglucosamine residues. Recombinant FDL also proved useful in the analysis of complex mixtures of N-glycans originating from wild-type and mutant Caenorhabditis strains, thereby aiding isomeric definition of paucimannosidic and hybrid N-glycans in this organism. Furthermore, differences in activity and specificity were shown for two site-directed mutants of Drosophila FDL, compatible with the high structural similarity of chitinolytic and N-glycan degrading exohexosaminidases in insects. Our studies are another indication for the variety of structural and function aspects in the GH20 hexosaminidase family important for both catabolism and biosynthesis of glycoconjugates in eukaryotes.
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影响因子：
13.8
作者：
Bieniossek C;Imasaki T;Takagi Y;Berger I
通讯作者：
Berger I
DOI：
10.1042/bj20081630
发表时间：
2009-04-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Gutternigg M;Rendić D;Voglauer R;Iskratsch T;Wilson IB
通讯作者：
Wilson IB
影响因子：
7.2
作者：
Fitchette-Lainé, AC;Gomord, V;Faye, L
通讯作者：
Faye, L
影响因子：
3.9
作者：
Haslam, SM;Dell, A
通讯作者：
Dell, A
DOI：
10.1074/jbc.m704235200
发表时间：
2007-09-21
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Gutternigg M;Kretschmer-Lubich D;Paschinger K;Rendić D;Hader J;Geier P;Ranftl R;Jantsch V;Lochnit G;Wilson IB
通讯作者：
Wilson IB