Wang Wenshuang, Cai Xiaojuan, Han Naihan, Han Wenjun, Sugahara Kazuyuki, Li Fuchuan
National Glycoengineering Research Center, School of Life Science and Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, 27 South Shanda Rd, Jinan 250100, PR China.
Proteoglycan Signaling and Therapeutics Research Group, Faculty of Advanced Life Science, Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan.
Biochem J. 2017 Nov 9;474(22):3831-3848. doi: 10.1042/BCJ20170591.
Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris-HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications.
糖胺聚糖（GAGs）是一类化学性质各异的多糖家族，在生理和病理过程中发挥着重要作用。由于GAGs的结构复杂，其精细的化学结构和生物学功能尚未得到广泛研究。裂解GAGs的裂解酶是结构分析的重要工具。尽管已经鉴定出多种GAG裂解酶，但GAG测序迫切需要具有独特酶特性的外切裂解酶。在本研究中，一种来自海洋细菌的假定外切GAG裂解酶被重组表达并进行了详细表征。由于它对透明质酸（HA）、硫酸软骨素（CS）和硫酸皮肤素（DS）表现出外切裂解酶活性，因此被命名为HCDLase。这种新型外切酶在30°C的Tris-HCl缓冲液（pH 7.0）中表现出最高活性。特别是，它表现出从2-氨基苯甲酰胺（2-AB）标记的CS寡糖的还原端释放2-AB标记的二糖的比活性，这表明HCDLase不仅是一种新型外切裂解酶，能够从糖链的还原末端裂解二糖残基，也是CS链测序的有用工具。值得注意的是，HCDLase不能消化来自HA、DS或未硫酸化软骨素的2-AB标记的寡糖，这表明硫酸盐和键类型会影响HCDLase的催化活性。最后，这种酶与CSase ABC结合成功应用于几种具有复杂结构的CS六糖和八糖的测序。HCDLase的鉴定为CS相关的研究和应用提供了一种有用的工具。
