Refolding of β-Stranded Class I Chitinases of Hippophae rhamnoides Enhances the Antifreeze Activity during Cold Acclimation

Class I chitinases hydrolyse the β-1,4-linkage of chitin and also acquire antifreeze activity in some of the overwintering plants during cold stress. Two chitinases, HrCHT1a of 31 kDa and HrCHT1b of 34 kDa, were purified from cold acclimated and non-acclimated seabuckthorn seedlings using chitin affinity chromatography. 2-D gels of HrCHT1a and HrCHT1b showed single spots with pIs 7.0 and 4.6 respectively. N-terminal sequence of HrCHT1b matched with the class I chitinase of rice and antifreeze proteins while HrCHT1a could not be sequenced as it was N-terminally blocked. Unlike previous reports, where antifreeze activity of chitinase was cold inducible, our results showed that antifreeze activity is constitutive property of class I chitinase as both HrCHT1a and HrCHT1b isolated even from non-acclimated seedlings, exhibited antifreeze activity. Interestingly, HrCHT1a and HrCHT1b purified from cold acclimated seedlings, exhibited 4 and 2 times higher antifreeze activities than those purified from non-acclimated seedlings, suggesting that antifreeze activity increased during cold acclimation. HrCHT1b exhibited 23–33% higher hydrolytic activity and 2–4 times lower antifreeze activity than HrCHT1a did. HrCHT1b was found to be a glycoprotein; however, its antifreeze activity was independent of glycosylation as even deglycosylated HrCHT1b exhibited antifreeze activity. Circular dichroism (CD) analysis showed that both these chitinases were rich in unusual β-stranded conformation (36–43%) and the content of β-strand increased (∼11%) during cold acclimation. Surprisingly, calcium decreased both the activities of HrCHT1b while in case of HrCHT1a, a decrease in the hydrolytic activity and enhancement in its antifreeze activity was observed. CD results showed that addition of calcium also increased the β-stranded conformation of HrCHT1a and HrCHT1b. This is the first report, which shows that antifreeze activity is constitutive property of class I chitinase and cold acclimation and calcium regulate these activities of chitinases by changing the secondary structure.I类几丁质酶（Class I chitinases）可水解几丁质的β-1,4糖苷键，部分越冬植物在冷胁迫条件下还会赋予其抗冻活性。研究人员从经冷驯化与未冷驯化的沙棘幼苗中，通过几丁质亲和层析纯化得到两种几丁质酶：分子量31 kDa的HrCHT1a与34 kDa的HrCHT1b。HrCHT1a与HrCHT1b的二维凝胶电泳（2-D gels）结果显示，二者分别仅存在等电点（isoelectric point, pI）为7.0和4.6的单一蛋白斑点。HrCHT1b的N端序列与水稻I类几丁质酶及抗冻蛋白相符，而HrCHT1a因N端封闭无法完成测序。与既往报道中几丁质酶的抗冻活性为冷诱导型不同，本研究结果表明，抗冻活性是I类几丁质酶的组成型特性：即便是从未经冷驯化的幼苗中分离得到的HrCHT1a与HrCHT1b，均表现出抗冻活性。值得注意的是，从冷驯化幼苗中纯化得到的HrCHT1a与HrCHT1b，其抗冻活性分别比未冷驯化幼苗中纯化的对应酶高4倍与2倍，提示冷驯化过程中几丁质酶的抗冻活性会增强。HrCHT1b的水解活性较HrCHT1a高23%~33%，但其抗冻活性则比HrCHT1a低2~4倍。HrCHT1b属于糖蛋白，但其抗冻活性并不依赖糖基化修饰：即便是去糖基化后的HrCHT1b，仍可检测到抗冻活性。圆二色性（Circular Dichroism, CD）分析结果显示，两种几丁质酶均富含罕见的β折叠构象（占比36%~43%），且冷驯化过程中β折叠的占比提升了约11%。令人意外的是，钙离子会同时降低HrCHT1b的两种酶活性；而对于HrCHT1a，其水解活性下降，但抗冻活性却得到增强。圆二色性（CD）结果还显示，添加钙离子可提升HrCHT1a与HrCHT1b的β折叠构象占比。本研究首次报道，抗冻活性是I类几丁质酶的组成型特性，且冷驯化与钙离子可通过改变几丁质酶的二级结构，调控其两类酶活性。