Snake Venom Hemotoxic Enzymes: Biochemical Comparison between Crotalus Species from Central Mexico

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A2 (PLA2), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA2 activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA2 activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms.

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Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model

Toxins ◽

10.3390/toxins12070455 ◽

2020 ◽

Vol 12 (7) ◽

pp. 455 ◽

Cited By ~ 1

Author(s):

Edgar Neri-Castro ◽

Melisa Bénard-Valle ◽

Dayanira Paniagua ◽

Leslie V. Boyer ◽

Lourival D. Possani ◽

...

Keyword(s):

Animal Model ◽

Snake Venom ◽

Serine Proteases ◽

Large Animal Model ◽

Large Animal ◽

Clinical Effects ◽

Differential Absorption ◽

Protein Families ◽

Ovine Model ◽

Viper Venoms

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

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Fine structural and biochemical characterization of phloem proteins

Canadian Journal of Botany ◽

10.1139/b80-102 ◽

1980 ◽

Vol 58 (7) ◽

pp. 802-806 ◽

Cited By ~ 16

Author(s):

Rainer Kollmann

Keyword(s):

Molecular Weight ◽

Conformational Changes ◽

Biochemical Characterization ◽

Protein Pattern ◽

Molecular Weights ◽

P Protein ◽

Sieve Tubes ◽

Species Specific

The heterogeneous group of proteins occurring in the exudate of cut phloem tissue will be termed phloem proteins. Their molecular weights range from less than 30 000 to over 100 000. The protein pattern is a species-specific constant. Depending on the plant species the bulk of phloem proteins is either basic or acidic. In Cucurbita maxima up to 40% of the total phloem proteins are basic proteins with a molecular weight of 116 000; it shows G–F transition properties in vitro and represents the so-called P-protein in the sieve tubes of angiosperms. Depending on the stage of cell differentiation the P-protein shows various conformational changes in situ: amorphous, filamentous tubular, and paracrystalline structures are described. P-protein differs from actin- or tubulinlike proteins with regard to their molecular weight and most biochemical characteristics. The main discrepancy is the failure to bind either heavy meromyosin or any nucleotides and colchicine. No structural evidence exists for cytoplasmic streaming in mature sieve tubes based on an actin–myosin system. The function of P-protein is still questionable; sieve pore plugging after wounding, sites of enzymatic activity, and surface interaction are discussed.

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Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

Biochimie ◽

10.1016/j.biochi.2012.07.007 ◽

2012 ◽

Vol 94 (12) ◽

pp. 2545-2558 ◽

Cited By ~ 31

Author(s):

Danilo Luccas Menaldo ◽

Carolina Petri Bernardes ◽

Norival Alves Santos-Filho ◽

Laura de Andrade Moura ◽

André Lopes Fuly ◽

...

Keyword(s):

Comparative Analysis ◽

Snake Venom ◽

Serine Proteases ◽

Biochemical Characterization ◽

Bothrops Pirajai

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Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Biochemical Journal ◽

10.1042/bj20102131 ◽

2011 ◽

Vol 435 (3) ◽

pp. 733-742 ◽

Cited By ~ 20

Author(s):

Pitter F. Huesgen ◽

Helder Miranda ◽

XuanTam Lam ◽

Manuela Perthold ◽

Holger Schuhmann ◽

...

Keyword(s):

Serine Proteases ◽

Protein Quality ◽

Biochemical Characterization ◽

Pdz Domain ◽

Control Mechanisms ◽

Ph Optimum ◽

Periplasmic Proteins ◽

Synechocystis Sp ◽

Pcc 6803

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

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Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2006.08.023 ◽

2006 ◽

Vol 1760 (12) ◽

pp. 1762-1771 ◽

Cited By ~ 18

Author(s):

Eladio F. Sanchez ◽

Liza F. Felicori ◽

Carlos Chavez-Olortegui ◽

Henrique B.P. Magalhaes ◽

Ana L. Hermogenes ◽

...

Keyword(s):

Molecular Cloning ◽

Plasminogen Activator ◽

Snake Venom ◽

Biochemical Characterization

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Structure of plasma and tissue kallikreins

Thrombosis and Haemostasis ◽

10.1160/th12-11-0840 ◽

2013 ◽

Vol 110 (09) ◽

pp. 423-433 ◽

Cited By ~ 40

Author(s):

Monika Pathak ◽

Szu Shen Wong ◽

Ingrid Dreveny ◽

Jonas Emsley

Keyword(s):

Molecular Weight ◽

Serine Proteases ◽

Molecular Mechanisms ◽

Substrate Recognition ◽

Coagulation Factor ◽

Structural Data ◽

Pressure Control ◽

Factor Xii ◽

Zymogen Activation ◽

Kinin System

SummaryThe kallikrein kinin system (KKS) consists of serine proteases involved in the production of peptides called kinins, principally bradykinin and Lys-bradykinin (kallidin). The KKS contributes to a variety of physiological processes including inflammation, blood pressure control and coagulation. Here we review the protein structural data available for these serine proteases and examine the molecular mechanisms of zymogen activation and substrate recognition focusing on plasma kallikrein (PK) and tissue kallikrein (KLK1) cleavage of kininogens. PK circulates as a zymogen bound to high-molecular-weight kininogen (HK). PK is activated by coagulation factor XIIa and then cleaves HK to generate bradykinin and factor XII to generate further XIIa. A structure has been described for the activated PK protease domain in complex with the inhibitor benzamidine. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and the central nervous system. They cleave a wide variety of substrates including low-molecular-weight kininogen (LK) and matrix proteins. Crystal structures are available for KLK1, 3, 4, 5, 6 and 7 activated protease domains typically in complex with S1 pocket inhibitors. A substrate mimetic complex is described for KLK3 which provides insight into substrate recognition. A zymogen crystal structure determined for KLK6 reveals a closed S1 pocket and a novel mechanism of zymogen activation. Overall these structures have proved highly informative in understanding the molecular mechanisms of the KKS and provide templates to design inhibitors for treatment of a variety of diseases.

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Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

International Journal of Molecular Sciences ◽

10.3390/ijms19082405 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2405 ◽

Cited By ~ 1

Author(s):

Caroline Costa ◽

Mariana Belchor ◽

Caroline Rodrigues ◽

Daniela Toyama ◽

Marcos de Oliveira ◽

...

Keyword(s):

Serine Protease ◽

Snake Venom ◽

Serine Proteases ◽

Three Dimensional ◽

Negative Control ◽

Protease Activated Receptors ◽

Crotalus Durissus Terrificus ◽

Three Dimensional Modeling ◽

Cox 2 ◽

Crotalus Durissus

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.

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