scholarly journals Serine Protease Variants Encoded byEchis ocellatusVenom Gland cDNA: Cloning and Sequencing Analysis

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
S. S. Hasson ◽  
R. A. Mothana ◽  
T. A. Sallam ◽  
M. S. Al-balushi ◽  
M. T. Rahman ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Indian Subcontinent ◽  
Sequence Similarity ◽  
Sequence Conservation ◽  
Sequencing Analysis ◽  
Cdna Sequences ◽  
Venom Glands ◽  
High Degree

Envenoming byEchissaw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands ofEchis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All theEchis ocellatus EoSPgroups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the newEchis ocellatus EoSPson the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by theEchis ocellatus EoSPsand analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

scholarly journals Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma

1993 ◽  
Vol 294 (2) ◽  
pp. 387-390 ◽  
Author(s):  
L C Au ◽  
S B Lin ◽  
J S Chou ◽  
G W Teh ◽  
K J Chang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Serine Proteases ◽  
Sequence Similarity ◽  
Active Form ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Binding Reaction ◽  
Venom Glands ◽  
High Degree

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.

scholarly journals Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands

2020 ◽  
Vol 8 (11) ◽  
pp. 1755
Author(s):  
Evert Drijver ◽  
Joep Stohr ◽  
Jaco Verweij ◽  
Carlo Verhulst ◽  
Francisca Velkers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Sequencing Analysis ◽  
Nucleotide Polymorphisms ◽  
Pan Genome ◽  
Next Generation Sequencing Analysis ◽  
Long Read ◽  
Short Read Sequence ◽  
High Degree

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

scholarly journals Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease fromBothrops pirajaiSnake Venom

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Kayena D. Zaqueo ◽  
Anderson M. Kayano ◽  
Rodrigo Simões-Silva ◽  
Leandro S. Moreira-Dill ◽  
Carla F. C. Fernandes ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Size Exclusion ◽  
Coagulation Cascade ◽  
Activate Factor ◽  
High Sequence Similarity ◽  
Fibrin Networks ◽  
Bothrops Pirajai

This paper presents a novel serine protease (SP) isolated fromBothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated fromBothrops pirajaisnake venom.

scholarly journals Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin.

1989 ◽  
Vol 109 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
T J Byers ◽  
A Husain-Chishti ◽  
R R Dubreuil ◽  
D Branton ◽  
L S Goldstein
Keyword(s):  
Sequence Similarity ◽  
Expression Library ◽  
Cdna Sequences ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Conserved Domain ◽  
Alpha Spectrin ◽  
Amino Terminal Domain ◽  
Related Proteins ◽  
High Degree

We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gt11 expression library. Analysis of 800 residues of deduced amino acid sequence at the amino-terminal end revealed a strikingly conserved domain of integral of 230 residues that shows a high degree of sequence similarity to the amino-terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrin-related proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino-terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.

scholarly journals Modules of Structure and Function in the Evolution of Prothrombin, Plasminogen and Factors X and IX

1977 ◽  
Author(s):  
S. Magnusson ◽  
L. Sottrup-Jensen ◽  
T.E. Petersen
Keyword(s):  
Serine Protease ◽  
Heavy Chain ◽  
Light Chain ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Amino Acid Sequences ◽  
Factor Ix ◽  
Evolutionary Origin ◽  
Factor X

Comparison of the amino acid sequences of prothrombin (Magnusson et al.), factor X1 (Enfield et al.: light chain; Titani et al.: heavy chain), plasminogen (Wiman: PAP? Sottrup-Jensen et al.: heavy chain region, Wallen & Wiman, Sottrup-Jensen et al.: light chain region) and what is known about factor IX (Fujikawa et al.) indicates that these zymogens contain three major structure modules of separate evolutionary origin: 1) A C-terminal serine protease module is common to all four zymogens and to other serine proteases. 2) The “middle” module is either a kringle · structure (duplicated in prothrombin, quintiplicated in plasminogen; all seven kringle structures mutually homologous), or a “pseudo-kringle” structure (one in factor X, residues appr. 45-142 of the light chain; probably one in factor IX, both mutually homologous). 3) The “N-terminal” module is either the vitamin-K dependent region (residues 1-41/42) of prothrombin, factor X and factor IX (all mutually homologous), or the PAP-region of plasminogen. Apparently then different variants of type 2) and 3) modules and also different numbers of type 2) modules can be “fused” to a C-terminal serine protease module during evolution to produce large serine protease zymogens with strictly defined binding affinities and activation characteristics. The “three-module” evolutionary origin of these zymogens leads to the assumption that each module in the zymogen has its own tertiary structure, largely independent of the rest of the molecule. This view is strengthened by the fact that large (1 mm x 0.5 mm x 0.5 mm) high-resolution (2.8 Å) crystals of native A-fragment (residues 1-156) from bovine prothrombin have been obtained (Olsson, Lindkvist, Sottrup-Jensen, Petersen & Magnusson).

Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1518-1523 ◽  
Author(s):  
R Devraj-Kizuk ◽  
DH Chui ◽  
EV Prochownik ◽  
CJ Carter ◽  
FA Ofosu ◽  
...  
Keyword(s):  
Serine Protease ◽  
Point Mutation ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Antithrombin Iii ◽  
Control Value ◽  
Reactive Center ◽  
Length Polymorphisms ◽  
History Of ◽  
Thrombin Antithrombin Iii Complex

Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.

scholarly journals Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1518-1523 ◽  
Author(s):  
R Devraj-Kizuk ◽  
DH Chui ◽  
EV Prochownik ◽  
CJ Carter ◽  
FA Ofosu ◽  
...  
Keyword(s):  
Serine Protease ◽  
Point Mutation ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Antithrombin Iii ◽  
Control Value ◽  
Reactive Center ◽  
Length Polymorphisms ◽  
History Of ◽  
Thrombin Antithrombin Iii Complex

Abstract Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.

scholarly journals Crystal structure of the M14R mutant of oryctin in complex with trypsin

2014 ◽  
Vol 70 (a1) ◽  
pp. C465-C465
Author(s):  
Desheng Liu ◽  
Tatsuya Suzuki ◽  
Shoichiro Horita ◽  
Takeshi Kawai ◽  
Jun Ishibashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Serine Protease ◽  
Serine Proteases ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Structural Similarity ◽  
Leukocyte Elastase ◽  
Subtilisin Carlsberg ◽  
Domain 3 ◽  
Α Helix

Oryctin is a 66-amino-acid protein purified from the larval haemolymph of the coconut rhinoceros beetle Oryctes rhinoceros, which shows no sequence similarity to any other protein known. We determined the solution NMR structure of oryctin, and found that oryctin had a similar backbone fold to the turkey ovomucoid domain 3, OMTKY3, a Kazal-type serine protease inhibitor [1]. Based on the structural similarity, we tested the serine protease inhibitory activity of oryctin, and found that oryctin does inhibit some serine proteases, such as α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase [1]. However, oryctin cannot inhibit trypsin at all. In this study, we have introduced point mutations to the putative inhibition loop of oryctin to obtain oryctin mutants that can inhibit trypsin. Then, we have solved the crystal structure of such an oryctin mutant, M14R-oryctin with a Ki value of 3.410.8 nM, in complex with trypsin to reveal how it binds to and inhibits trypsin. As predicted, the putative inhibition loop lay on the substrate binding cleft of trypsin. Particularly, the side chain of R14 fit into the S1 pocket of trypsin by forming hydrogen/ionic bonds with D191, S192 and G216 at the bottom of the S1 pocket and G195, D196, S197 and S212 at its entrance. In addition, R65 located in the C-terminal α-helix of M14R-oryctin formed hydrogen bonds with S40 and F44 of trypsin. The latter interaction, which is unique to oryctin, enhances its binding affinity to trypsin.

Elastic Network Normal Mode Analysis for Microtubule Mechanics

2009 ◽  
Author(s):  
Marco A. Deriu ◽  
Monica Soncini ◽  
Mario Orsi ◽  
Mishal Patel ◽  
Jonathan W. Essex ◽  
...  
Keyword(s):  
Building Block ◽  
Tertiary Structure ◽  
Sequence Similarity ◽  
Normal Mode Analysis ◽  
Mode Analysis ◽  
Outer Diameter ◽  
Elastic Network ◽  
Level 1 ◽  
High Degree ◽  
Β Tubulin

The cellular microtubules MTs are hollow cylinder-shaped biopolymers with inner and outer diameter of about 17 and 25 nm and length ranging from 1 to 10 μm. They are constituted by αβ-tubulins arranged in protofilaments with a head-to-tail motif [1]. The protofilaments bind together laterally along the MT’s long axis with a slight shift generating a spiral with a pitch of 2, 3 or 4 monomers’ length (Fig.1a). The building-block of the MT, the αβ-tubulin, is a hetero-dimer made of two globular monomers, α- and β-tubulin, each of them consisting of about 450 residues with high degree of sequence similarity from the primary to the tertiary structure level [1].

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