Molecular characterization of a partial sequence encoding a novel Schistosoma mansoni serine protease

Parasitology ◽  
1997 ◽  
Vol 115 (4) ◽  
pp. 395-402 ◽  
Author(s):  
C. COCUDE ◽  
C. PIERROT ◽  
C. CETRE ◽  
C. GODIN ◽  
A. CAPRON ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Serine Proteases ◽  
Mrna Level ◽  
Cross Reactivity ◽  
Reading Frame ◽  
Consensus Sequences ◽  
Degenerate Oligonucleotide ◽  
Sequence Encoding

A PCR strategy using degenerate oligonucleotide primers based upon consensus sequences of the active site of serine proteases yielded a 467 bp fragment from genomic DNA from Schistosoma mansoni cercariae. The sequence presented a continuous open reading frame and the deduced amino acid sequence (156 aa) presented homologies with various serine proteases, in particular the highest percentage identity was observed with a mammalian plasma kallikrein. The expression of this serine protease was studied first at the mRNA level and it was only detected by RT-PCR in cercariae and in adult worms. At the protein level we were able to detect it by Western blotting and by using antigen extracts from metabolically radio-isotope labelled worms. The absence of any positive signal in Northern blot and the detection of the protein suggest that the mRNA has a very short half-life, however the protein may be accumulated in the parasite. The significance of identity with mammalian kallikrein was confirmed by cross-immunoreactivity with a native porcine pancreatic kallikrein. However, no cross-reactivity was observed with S. mansoni elastase, another serine protease. Thus, we suggest that the serine protease described in this paper is a kallikrein-like protease.

scholarly journals Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

1992 ◽  
Vol 40 (1) ◽  
pp. 83-92 ◽  
Author(s):  
T Berg ◽  
I Wassdal ◽  
K Sletten
Keyword(s):  
Submandibular Gland ◽  
Serine Proteases ◽  
Cross Reactivity ◽  
Immunohistochemical Localization ◽  
Staining Reaction ◽  
Anatomic Distribution ◽  
Family Gene ◽  
Terminal Amino ◽  
Rat Submandibular Gland

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.

scholarly journals Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity from Bothrops barnetti Snake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Magaly Alejandra Brousett-Minaya ◽  
Paulo Aparecido Baldasso ◽  
Salomón Huancahuire-Vega ◽  
Sérgio Marangoni
Keyword(s):  
Serine Protease ◽  
Snake Venom ◽  
Serine Proteases ◽  
Kinetic Studies ◽  
Size Exclusion ◽  
Reverse Phase Hplc ◽  
Pharmacological Characterization ◽  
Coagulant Activity ◽  
Exclusion Chromatography

A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of Km and Vmax of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.

scholarly journals Characterization of an Immunogenic Outer Membrane Autotransporter Protein, Arp, of Bartonella henselae

2007 ◽  
Vol 75 (11) ◽  
pp. 5255-5263 ◽  
Author(s):  
Christine M. Litwin ◽  
Mindy L. Rawlins ◽  
Erica M. Swenson
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Outer Membrane ◽  
Dna Sequences ◽  
Bartonella Henselae ◽  
Cross Reactivity ◽  
Passenger Domain ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT Bartonella henselae is a recently recognized pathogenic bacterium associated with cat scratch disease, bacillary angiomatosis, and bacillary peliosis. This study describes the cloning, sequencing, and characterization of an antigenic autotransporter gene from B. henselae. A cloned 6.0-kb BclI-EcoRI DNA fragment expresses a 120-kDa B. henselae protein immunoreactive with 21.2% of sera from patients positive for B. henselae immunoglobulin G antibodies by indirect immunofluorescence, with 97.3% specificity and no cross-reactivity with antibodies against various other organisms. DNA sequencing of the clone revealed one open reading frame of 4,320 bp with a deduced amino acid sequence that shows homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane and consist of a passenger region, the α-domain, and an outer membrane transporter region, the β-domain. The passenger domain shows homology to a family of pertactin-like adhesion proteins and contains seven, nearly identical 48-amino-acid repeats not found in any other bacterial or Bartonella DNA sequences. The passenger α-domain has a calculated molecular mass of 117 kDa, and the transporter β-domain has a calculated molecular mass of 36 kDa. The clone expresses a 120-kDa protein and a protein that migrates at approximately 38 kDa exclusively in the outer membrane protein fraction, suggesting that the 120-kDa passenger protein remains associated with the outer membrane after cleavage from the 36-kDa transporter.

scholarly journals Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1614-1623 ◽  
Author(s):  
JW Heusel ◽  
EM Scarpati ◽  
NA Jenkins ◽  
DJ Gilbert ◽  
NG Copeland ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Catalytic Triad ◽  
Small Population ◽  
Cathepsin G ◽  
Protease Gene ◽  
Chromosome 14 ◽  
Gene Encoding ◽  
Specific Expression ◽  
Reading Frame

We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1–4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.

Identification of a developmentally regulated Schistosoma mansoni serine protease homologous to mouse plasma kallikrein and human factor I

Parasitology ◽  
1999 ◽  
Vol 118 (4) ◽  
pp. 389-396 ◽  
Author(s):  
C. COCUDE ◽  
C. PIERROT ◽  
C. CÊTRE ◽  
J. FONTAINE ◽  
C. GODIN ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Light Chain ◽  
Serine Proteases ◽  
Human Factor ◽  
Plasma Kallikrein ◽  
Parasite Life Cycle ◽  
Developmentally Regulated ◽  
Mouse Plasma ◽  
Factor I

The isolation of 2 genomic clones has allowed us to further characterize a Schistosoma mansoni serine protease designated SmSP1. The deduced amino acid sequence (248aa) considered as a ‘light chain’ encoding the active site, presents significant homologies with mouse plasma kallikrein and human factor I light chain. The secondary structure of SmSP1 ‘light chain’ is correctly predicted and may be sufficient by itself to constitute an active enzyme. The biological function of SmSP1 is unknown, however, the homology with 2 serine proteases suggests that SmSP1 may play a role in the evasion of the host immune response. This is supported by the presence of the native protein corresponding to SmSP1 particularly in schistosomula released products (SRP) and in male dorsal spines. The expression of this enzyme is differentially regulated throughout the parasite life-cycle. However, infected animals with S. mansoni did not produce specific antibodies to recombinant SmSP1. The lack of such response could be advantageous to the parasite by protecting itself from host effector mechanisms.

Molecular characterization of serine protease inhibitor isoform 3, SmSPI, from Schistosoma mansoni

2016 ◽  
Vol 115 (8) ◽  
pp. 2981-2994 ◽  
Author(s):  
Pattarakul Pakchotanon ◽  
Patamaporn Molee ◽  
Supaporn Nuamtanong ◽  
Yanin Limpanont ◽  
Phiraphol Chusongsang ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Molecular Characterization ◽  
Serine Protease Inhibitor

The investigation of nematocidal activity in Stenotrophomonas maltophilia G2 and characterization of a novel virulence serine protease

2009 ◽  
Vol 55 (8) ◽  
pp. 934-942 ◽  
Author(s):  
Xiaowei Huang ◽  
Junwei Liu ◽  
Junmei Ding ◽  
Qiusheng He ◽  
Rui Xiong ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Stenotrophomonas Maltophilia ◽  
Bursaphelenchus Xylophilus ◽  
Panagrellus Redivivus ◽  
Bacterial Culture Supernatant ◽  
Free Living Nematode ◽  
Nematocidal Activity ◽  
Nematode Cuticle

The Gram-negative bacterium Stenotrophomonas maltophilia G2 was isolated from a soil sample and was found to have high nematotoxic activity against a free-living nematode, Panagrellus redivivus, and a plant-parasitic nematode, Bursaphelenchus xylophilus . The analysis of virulence factors revealed that although the small molecular metabolites participated in nematode killing, the crude extracellular protein extract from the bacterial culture supernatant contributed significantly to its nematocidal activity. An extracellular protease was purified by chromatography, and its effects on degrading purified nematode cuticle and killing living nematodes were confirmed experimentally. Characterization of this purified protease revealed that the application of phenylmethylsulphonyl fluoride, an inhibitor of serine proteases, could completely abolish its proteolytic activity. The results from N-terminal amino acid sequencing showed no similarity with any known serine protease in S. maltophilia, suggesting a novel virulence serine protease was obtained. Our study is the first to show the nematocidal activity of S. maltophilia, and we identified a novel serine protease as an important pathogenicity factor.

scholarly journals Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1614-1623 ◽  
Author(s):  
JW Heusel ◽  
EM Scarpati ◽  
NA Jenkins ◽  
DJ Gilbert ◽  
NG Copeland ◽  
...  
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Catalytic Triad ◽  
Small Population ◽  
Cathepsin G ◽  
Protease Gene ◽  
Chromosome 14 ◽  
Gene Encoding ◽  
Specific Expression ◽  
Reading Frame

Abstract We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1–4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.

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