Structures and functions of venom metalloproteinases and C-type lectin-like (CTLL) glycoproteins families from Russell’s viper

• Main Authors: Hong-Sen Chen, 陳鴻森
• Other Authors: Inn-Ho Tsai
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/61939077172166354466

博士 === 國立臺灣大學 === 生化科學研究所 === 96 === Envenomings by Russell’s vipers (Daboia russelii and Daboia siamensis) are one of the major health hazards in many countries of South and Southeast Asia. Systemic bleeding and acute renal failure due to consumptive coagulopathy and thrombocytopenia are rather common clinical manifestations in the snakebite patients. Great geographic variations of their symptoms have also been noted. To better understand the venom proteins contributing to these symptoms, two venom families, metalloproteinase and C-type lectin-like (CTLL) have been purified and characterized in the present study. In addition, cDNA encoding these venom proteins were cloned and sequenced. Their evolutionary relationships and possible functional roles (sites) were also studied based on bioinformatic and phylogenetic analyses. Part I. To characterize the highly hemorrhagic symptoms elicited by Myanmar and eastern India Daboia envenoming, two similar P-III metalloproteinases were purified from Daboia venom of both regions, and designated as daborhagin-M and daborhagin-K, respectively. They induced dermal hemorrhage in mice with a minimum hemorrhagic dose of 0.8-0.9 mg. Aa-chain of fibrinogen, fibronectin, and type IV collagen were specifically hydrolyzed by the P-III in vitro. Analyses of its cleavage sites on insulin chain B and the kinetic parameters of five representative P-IIIs toward oligopeptides suggested that daborhagin and other hemorrhagins prefers hydrophobic residues at the P1, P1'', and P2'' positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65 kDa. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 sequences of P-IIIs revealed the co-evolution of two major classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs. Part II. RVV-X, the factor X activator from Russell’s viper venom, is a potent pro-coagulating and lethal toxin. As a heterotrimeric glycoprotein, the detailed structures of RVV-X and possible physiological significance of its N-glycans remain to be elucidated. We cloned and sequenced all its three subunits from the cDNAs of Indonesia D. siamensis venom glands. It is found the deduced heavy chain sequence contains a C-terminal extension of four residues more than that previously published. Both light chains showed 77-81% identities to those of a homologus factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X strongly binds protein S besides factor X and IX. If this binding inactivates protein S, it may potentiate the disseminated intravascular coagulation syndrome observed. The N-glycans released from each subunit and the glycopeptides were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans showed a heterogeneous pattern with a combination of variable terminal fucosylation and sialylation on multiantennary complex type of sugars. Among the notable features are the presences of terminal Lewis and sialyl-Lewis epitopes, as confirmed by Western blotting analyses. These glyco-epitopes possibly contributed to rapid homing of RVV-X to vascular system, as supported by the observation that slower and fewer fibrinogen-degradation products were released by desialylated RVV-X as compared with the native RVV-X. Part III. To understand the evolution of CTLLs and their roles in the clinical manifestations of Daboia envenoming, we designed a pair of degenerative primers to clone and study the CTLL isoforms. Three set of cDNAs prepared from venom glands of Daboia subspecies from India, Myanmar and Indonesia, respectively, were used. Totally, eight distinct cDNAs encoding the subunits of four paralogous CTLLs—designated as RVV-X LC, dabocetin, p31, and p68—were cloned and sequenced. Among them, p31 and p68 were first identified in the present study. By biochemical analyses of the purified proteins, and cladogram analyses of sequences of both A/B subunits, the venom CTLLs are categorized into three distinct subtypes. We noticed that these CTLL subunits are differentially expressed in the geographic samples, and both subunits of each CTLL appear to be coherently expressed. Remarkably, subunits of orthologous CTLLs of different Daboia subspecies exhibited almost identical sequences, except the LC2 subunits of RVV-X show substantial variations. In the phylogenetic trees, p31 subunits are closely related to those of EMS16 from an Echis venom. In contrast, p68 seemed to be a new subtype, with a dimeric heterodimer (a2b2) structure. Our study showed that p68 does not inhibit the platelet aggregations induced by ADP and collagen, nor agglutinate the platelets in human platelet-rich plasma.