Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.
Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.
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DOI：
10.1371/journal.pone.0143963
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kuhn-Nentwig L
中科院分区：
文献类型：
--
作者：
Biner O;Trachsel C;Moser A;Kopp L;Langenegger N;Kämpfer U;von Ballmoos C;Nentwig W;Schürch S;Schaller J;Kuhn-Nentwig L
Hybronidases是重要的毒液成分，作为有毒化合物的扩散因子。在几项研究中，这种扩散效应在脊椎动物组织上进行了测试。然而，关于蜘蛛对无脊椎动物（蜘蛛的主要猎物生物）的传播活动的数据却很缺乏。在这里，透明质酸酶样酶是从蜘蛛Cupiennius salei的毒液中分离出来的。通过毒腺转录组的cDNA分析确定酶的氨基酸序列，并通过蛋白质分析确认。通过串联质谱法鉴定了两种类似于蜜蜂透明质酸酶糖基化的复杂N-连接聚糖。使用InterPro在蜘蛛透明质酸酶中鉴定了C-末端EGF样结构域。蜘蛛透明质酸酶样酶在40-60°C之间的酸性pH和0.2M KCl下显示出最大活性。二价离子没有增强HA降解活性，表明它们不被募集用于催化。除了透明质酸，该酶降解硫酸软骨素A，而硫酸乙酰肝素和硫酸皮肤素不受影响。透明质酸降解的终产物是四聚体，而硫酸软骨素A主要降解为六聚体。通过鉴定寡聚物还原末端的末端N-乙酰葡糖胺或N-乙酰半乳糖胺，将该酶鉴定为内切-β-N-乙酰-D-氨基己糖苷酶水解酶。通过将透明质酸酶样酶与Cupiennius salei主要神经毒素CsTx-1共注射到果蝇体内，研究了透明质酸酶样酶对无脊椎动物组织的扩散作用。该酶显著增强CsTx-1的神经毒性活性。比较底物降解试验与透明质酸，硫酸软骨素A，硫酸皮肤素，硫酸乙酰肝素与毒液从39个蜘蛛物种从21个家庭确定了一些蜘蛛家庭（Atypidae，Eresidae，Araneidae和Nephilidae）没有活性的透明质酸酶样酶。这被解释为这种酶的损失，并符合目前的系统发育的想法，这些家庭的一个更孤立的位置，也许可以解释为专门的猎物捕捉技术。
Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40–60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.