Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 μM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na+ and K+ (0.2 M) positively affect hyaluronidase activity; while Mg2+, Br2+, Ba2+, Cu2+, Zn2+, and Cd2+ reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/β) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms.

中文翻译：

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来自Botrops atrox秘鲁蛇毒的透明质酸酶的生化和分子表征。

蛇毒是酶的丰富来源，例如金属蛋白酶，丝氨酸蛋白酶，磷脂酶A2和肌毒素，这些酶在结构和功能上已得到很好的表征。但是，透明质酸酶（EC3.2.1.35）尚未得到广泛研究。在这项研究中，我们描述了从秘鲁蛇Bothrops atrox的毒液中分离出的透明质酸酶（Hyal-Ba）的生物化学和分子特征。通过结合离子交换色谱和凝胶过滤色谱法纯化Hyal-Ba。纯化的Hyal-Ba是一种69 kDa（SDS-PAGE）单体糖蛋白，其N端氨基酸序列与同源蛇毒透明质酸酶具有高度同一性。检测到的相关碳水化合物为己糖（16.38％），己糖胺（2.7％）和唾液酸（0.69％）。Hyal-Ba仅选择性水解透明质酸（HA;比活= 437。5 U / mg），但没有水解硫酸软骨素或肝素。最大活性的最佳pH和温度分别为6.0和40°C，Km为0.31μM。EDTA，碘乙酸盐，2-巯基乙醇，TLCK和地塞米松可抑制其活性。Na +和K +（0.2 M）对透明质酸酶活性有积极影响；而Mg2 +，Br2 +，Ba2 +，Cu2 +，Zn2 +和Cd2 +降低了催化活性。Hyal-Ba增强了整个毒液的出血和溶血活性，但减少了由l-氨基酸氧化酶（LAAO）引起的足底水肿。Hyal-Ba cDNA序列（2020 bp）编码449个氨基酸残基，包括催化位点残基（Glu135，Asp133，Tyr206，Tyr253和Trp328）和三个N-连接糖基化的功能性基序，与其他蛇透明质酸酶保守。Hyal-Ba的空间模型在C端部分显示TIM-Barrel（α/β）折叠和EGF样结构域。Hyal-Ba与其他同源Hyals的系统发育分析表明，该类化合物为蛇形。此外，Hyal-Ba研究可能会扩展我们对B. atrox毒素学的认识，并为改善治疗性抗蛇毒的中和策略提供见解。