A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization.
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization.
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DOI：
10.1371/journal.pntd.0002206
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发表时间：
2013
影响因子：
3.8
通讯作者：
Veiga SS
中科院分区：
文献类型：
--
作者：
Ferrer VP;de Mari TL;Gremski LH;Trevisan Silva D;da Silveira RB;Gremski W;Chaim OM;Senff-Ribeiro A;Nader HB;Veiga SS
Loxoscelism是指由Loxosceles蜘蛛叮咬引起的临床症状。临床表现包括皮肤坏死伴重力扩散和全身性紊乱。毒液含有几种酶毒素。在此，我们描述了一种新的棕色蜘蛛蛋白的克隆，表达，复性和生物学评价的特点是透明质酸酶。采用毒腺cDNA文库，我们克隆了透明质酸酶（1200 bp的cDNA），编码的信号肽和成熟蛋白。氨基酸序列比对显示与透明质酸酶家族成员，如蝎子和蛇物种的结构关系。重组透明质酸酶在包涵体中表达为N-末端His-标签融合蛋白（约45 kDa），并使用重折叠实现活性。免疫印迹分析表明，识别重组蛋白的抗体与来自全毒液的透明质酸酶交叉反应，以及抗蛇毒血清与重组蛋白反应。重组透明质酸酶能够降解纯化的透明质酸（HA）和硫酸软骨素（CS），而硫酸皮肤素（DS）和硫酸乙酰肝素（HS）不受影响。酶谱实验导致在透明质酸（HA）和硫酸软骨素（CS）底物中产生约45 kDa的溶解区。通过家兔皮肤坏死的体内实验，证明重组透明质酸酶可增强重组皮肤坏死毒素的皮肤坏死作用。intermedia venom（LiRecDT 1）。这些数据支持透明质酸酶是“扩散因子”的假设。重组透明质酸酶为生物技术目的提供了有用的工具。我们建议将这种酶命名为迪特里希的透明质酸酶，以荣誉卡尔·彼得·冯·迪特里希教授，他毕生致力于研究蛋白聚糖和糖胺聚糖。世界各地都有涉及棕色蜘蛛（Loxosceles属）的事故报道。巴西南部和东南部是这种蜘蛛的地方性地区。Loxosceles叮咬通常会引发局部症状，如肿胀，红斑，出血和标志性症状：重力扩散的皮肤坏死病变。全身性影响不太常见;然而，在更严重的病例中涉及。由于它们对细胞外组分的酶活性，在几种毒液中被称为“扩散因子”。这种活动促进了其他毒素通过受害者身体的渗透。事实上，先前的一项研究确定了L。中间毒液对糖胺聚糖的作用，糖胺聚糖是许多组织的细胞外基质中丰富的组分。为了进一步揭示透明质酸酶在这种毒液中的作用，我们研究了从L。中间毒液Dietrich透明质酸酶，正如它被命名的那样，是作为重组蛋白质产生的。通过使用Dietrich’s Hyp酸酶和皮肤坏死毒素进行兔皮肤坏死测定，我们表明Dietrich’s Hyp酸酶增加了由皮肤坏死毒素诱导的皮肤坏死面积。我们的结果证实透明质酸酶是L.中间毒液
Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans. Accidents involving brown spiders (Loxosceles genus) are reported throughout the world. South and Southeast of Brazil are endemic areas for this spider. Loxosceles bites commonly trigger local signs as swelling, erythema, hemorrhage and the hallmark symptom: a dermonecrotic lesion with gravitational spreading. Systemic effects are less common; however, are implicated in more severe cases. Hyaluronidases are referred in several venoms as “spreading factors” due to their enzymatic activity upon extracellular components. This activity facilitates the permeation of other toxins through the victim's body. In fact, a previous study identified the activity of L. intermedia venom upon glycosaminoglycans which are abundant components in the extracellular matrix of many tissues. Disclosing a little more about the role of hyaluronidases within this venom, we investigated the activities of a recombinant hyaluronidase from L. intermedia venom. Dietrich's hyaluronidase, as it was designated, was produced as a recombinant protein. By performing a rabbit skin dermonecrosis assay using Dietrich's Hyaluronidase and a dermonecrotic toxin, we showed that Dietrich's Hyaluronidase increased the dermonecrotic area induced by the dermonecrotic toxin. Our results confirm that hyaluronidases are a “spreading factor” of L. intermedia venom.
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影响因子：
3.1
作者：
FUTRELL, JM
通讯作者：
FUTRELL, JM
影响因子：
3.8
作者：
Baldo, Cristiani;Jamora, Colin;Moura-da-Silva, Ana M.
通讯作者：
Moura-da-Silva, Ana M.
DOI：
10.1016/0006-291x(83)91379-7
发表时间：
1983-01-01
影响因子：
3.1
作者：
DIETRICH, CP;NADER, HB;STRAUS, AH
通讯作者：
STRAUS, AH
影响因子：
1.1
作者：
Elston, DM;Eggers, JS;Fischer, JR
通讯作者：
Fischer, JR
影响因子：
2.8
作者：
Girish, KS;Kemparaju, K
通讯作者：
Kemparaju, K