scholarly journals Gene Cloning of Rat and Mouse Platelet Glycoprotein V: Identification of Megakaryocyte-Specific Promoters and Demonstration of Functional Thrombin Cleavage

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3253-3262 ◽  
Author(s):  
Catherine Ravanat ◽  
Martine Morales ◽  
David O. Azorsa ◽  
Sylvie Moog ◽  
Simone Schuhler ◽  
...  
Keyword(s):  
High Performance ◽  
Surface Protein ◽  
Platelet Glycoprotein ◽  
Recognition Sequence ◽  
Thrombin Cleavage ◽  
Human Thrombin ◽  
Recognition Motifs ◽  
Species Specific ◽  
Major Surface

Abstract Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V–IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (≈1,700 nucleotides) entirely contained in one exon and a single intron (≈900 nucleotides) in the 5′ untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8–amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2 -terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2 -terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2 -terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

IDENTIFICATION OF A NEUTROPHIL ELASTASE CLEAVAGE SITE ON THE Act -CHAIN OF PRIMATE FIBRINOGEN

1987 ◽  
Author(s):  
J Weitz ◽  
S Landman ◽  
S Birken
Keyword(s):  
Neutrophil Elastase ◽  
Cleavage Site ◽  
High Performance ◽  
Human Neutrophil Elastase ◽  
Amino Acid Sequence Analysis ◽  
Specific Radioimmunoassay ◽  
Human Thrombin ◽  
The Mean

Human neutrophil elastase (HNE) cleaves the Aα21-22 bond of fibrinogen thus releasing the fibrinopeptide A (FPA)-containing fragment Aαl-21. Plasma Aal-21 levels reflect in vivo HNE activity and peptide levels are increased in cigarette smokers and patients with chronic lung disease. To further explore the HNE-fibrinogen interaction, we set out to develop an animal model. The digestion of purified baboon and marmoset fibrinogen by human thrombin, HNE and extracts of baboon and marmoset neutrophils was monitored with a specific radioimmunoassay for human FPA. Thrcmbin produced quantitative release (2 mol/mol fibrinogen) of FPA. In contrast, HNE and the neutrophil extracts did not release FPA, but rather, produced quantitative release of a larger, FPA-containing fragment. Immunochemically, this fragment was clearly distinguishable from FPA in that in vitro thrombin treatment increased its immunoreactivity 1,000-fold (thrombin increasable FPA or TIFPA). TIFPA release by the neutrophil extracts was blocked by α1-proteinase inhibitor, a specific HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl) and an anti-HNE IgG, indicating that elastase was the responsible proteinase and that there was homology between the human and primate enzymes. The products of HNE and neutrophil extract proteolysis of the primate fibrinogens were then separated by high performance liquid chromatography and the TIFPA-containing fractions were subjected to amino acid sequence analysis. The FPA-containing fragments each consisted of 21 amino acids, had minor substitutions when compared with human A α] -21 [Baboon: Aα(3) Ser - Thr; Marmoset Aα(l) Ala - Thr, Aα(3) Ser - Thr, Aα(ll) Glu - Ala], and exhibited complete crossreactivity with the human peptide. Using the TIFPA assay, there was good recovery of primate or human Aαl-21 added to primate blood and the mean peptide level in 8 healthy marmosets was similar to that in man (0.5 nM and 0.4 nM, respectively). In conclusion, (1) the Aα;21 -22 bond of baboon and marmoset fibrinogen is a cleavage site for human and primate elastase, (2) baboon and marmoset Aal-21 can be measured with the assay for the human peptide, and (3) the primate serves as a useful model for the study of elastase-fibrinogen interactions.

scholarly journals Selective In Vivo Depletion of CD4+ T Lymphocytes with Anti-CD4 Monoclonal Antibody during Acute Infection of Calves with Anaplasma marginale

2002 ◽  
Vol 9 (2) ◽  
pp. 417-424 ◽  
Author(s):  
Reginald A. Valdez ◽  
Travis C. McGuire ◽  
Wendy C. Brown ◽  
William C. Davis ◽  
Jeffrey M. Jordan ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Lymphocytes ◽  
Acute Infection ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Treatment Groups ◽  
Peripheral Lymph ◽  
Sequential Experiments ◽  
Major Surface

ABSTRACT To investigate the in vivo role of CD4+ T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4+ T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginale in two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3+ CD4+ and CD4+ CD45R+ (naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginale antibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginale major surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4+-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4+ T lymphocytes would abrogate resistance to acute infection.

scholarly journals The Putative Proteinase Maturation Protein A ofStreptococcus pneumoniae Is a Conserved Surface Protein with Potential To Elicit Protective Immune Responses

2000 ◽  
Vol 68 (7) ◽  
pp. 4180-4188 ◽  
Author(s):  
K. Overweg ◽  
A. Kerr ◽  
M. Sluijter ◽  
M. H. Jackson ◽  
T. J. Mitchell ◽  
...  
Keyword(s):  
Protein Fraction ◽  
Pathogenic Bacteria ◽  
Protein A ◽  
Surface Protein ◽  
Cross Reactivity ◽  
Two Dimensional Gel Electrophoresis ◽  
Hyperimmune Serum ◽  
Maturation Protein ◽  
Species Specific

ABSTRACT Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.

scholarly journals pMGA Phenotypic Variation in Mycoplasma gallisepticumOccurs In Vivo and Is Mediated by Trinucleotide Repeat Length Variation

2000 ◽  
Vol 68 (10) ◽  
pp. 6027-6033 ◽  
Author(s):  
M. D. Glew ◽  
Glenn F. Browning ◽  
Philip F. Markham ◽  
Ian D. Walker
Keyword(s):  
Trinucleotide Repeat ◽  
Ex Vivo ◽  
Adaptive Immune Response ◽  
Surface Protein ◽  
Length Variation ◽  
Major Surface Protein ◽  
Adaptive Immune ◽  
Major Surface ◽  
Successful Colonization

ABSTRACT Chickens were infected with a pathogenic strain of Mycoplasma gallisepticum, and the expression of pMGA, the major surface protein, was inferred by examination of colonies from ex vivo cells. Within 2 days postinfection, 40% of cells had ceased the expression of the original pMGA surface protein (pMGA1.1), and by day 6, the majority of recovered cells were in this category. The switch in pMGA phenotype which had occurred in vivo was reversible, since most colonies produced from ex vivo progenitors exhibited frequent pMGA1.1+sectors. After prolonged in vivo habitation, increasing proportions of recovered cells gave rise to variant pMGA colonies which had switched from the expression of pMGA1.1 to another gene, pMGA1.2, concomitant with the acquisition of a (GAA)12 motif 5′ to its promoter. Collectively, the results suggest that changes in M. gallisepticum pMGA gene expression in vivo are normal, common, and possibly obligate events for successful colonization of the host. Surprisingly, the initial cessation of pMGA1.1 expression occurred in the absence of detectable pMGA antibodies and seemed to precede the adaptive immune response.

scholarly journals Expression of Anaplasma marginale Major Surface Protein 2 Variants during Persistent Cyclic Rickettsemia

1998 ◽  
Vol 66 (3) ◽  
pp. 1200-1207 ◽  
Author(s):  
Dorothy M. French ◽  
Terry F. McElwain ◽  
Travis C. McGuire ◽  
Guy H. Palmer
Keyword(s):  
Genetic Variants ◽  
Protective Immunity ◽  
Genetic Variant ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Competitive Pcr ◽  
Cloning And Sequencing ◽  
Major Surface Protein ◽  
Major Surface

ABSTRACT Anaplasma marginale is an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. Persistent A. marginale infection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence of A. marginale antigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles,msp-2 transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing ofmsp-2 cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverse A. marginale population. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistent A. marginale infection and suggest that emergent variants play an important role in persistence.

Engineering and functional evaluation of neutralizing antibody fragments against congenital toxoplasmosis

2021 ◽  
Author(s):  
Anne di Tommaso ◽  
Matthieu O Juste ◽  
Zineb Lakhrif ◽  
Marie-Noëlle Mévélec ◽  
Coraline Borowczyk ◽  
...  
Keyword(s):  
Specific Treatment ◽  
Congenital Toxoplasmosis ◽  
Surface Protein ◽  
Neutralizing Antibody ◽  
Antibody Fragments ◽  
Functional Evaluation ◽  
Maternal Fetal Transmission ◽  
Major Surface

Abstract Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current gold-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments – scFv, scFv-Fc – directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments’ biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having a better efficacy. These data provide a proof-of-concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

scholarly journals The Antibacterial Activity of LL-37 against Treponema denticola Is Dentilisin Protease Independent and Facilitated by the Major Outer Sheath Protein Virulence Factor

2011 ◽  
Vol 80 (3) ◽  
pp. 1107-1114 ◽  
Author(s):  
Graciela Rosen ◽  
Michael N. Sela ◽  
Gilad Bachrach
Keyword(s):  
Periodontal Disease ◽  
Host Defense ◽  
Surface Protein ◽  
Human Saliva ◽  
Treponema Denticola ◽  
Host Defense Peptides ◽  
Content Type ◽  
Outer Sheath ◽  
Major Surface

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease.Treponema denticolais an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. TheT. denticolamajor surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 andT. denticola. The threeT. denticolastrains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility ofT. denticolato LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of theT. denticolaMSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protectT. denticolafrom LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover,in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

scholarly journals The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion

2005 ◽  
Vol 201 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Alida Coppi ◽  
Consuelo Pinzon-Ortiz ◽  
Christina Hutter ◽  
Photini Sinnis
Keyword(s):  
Cell Invasion ◽  
Cysteine Protease ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Cysteine Protease Inhibitor ◽  
Target Cells ◽  
Malaria Vaccine Candidate ◽  
Major Surface

The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.

Role of heme modulation in inhibition of Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera hemotoxic venom activity

2018 ◽  
Vol 38 (2) ◽  
pp. 216-226 ◽  
Author(s):  
VG Nielsen ◽  
N Frank
Keyword(s):  
Potential Therapeutic Target ◽  
Venomous Snake ◽  
Human Thrombin ◽  
Specific Variation ◽  
Coagulation Kinetics ◽  
Species Specific ◽  
Kinetics Of

Venomous snake bite and subsequent coagulopathy is a significant source of morbidity and mortality worldwide. The gold standard to treat coagulopathy caused by these venoms is the administration of antivenom; however, despite this therapy, coagulopathy still occurs and recurs. Of interest, our laboratory has demonstrated in vitro and in vivo that coagulopathy-inducing venom exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the African genera Atheris, Atractaspis, Causus, Cerastes, Echis, and Macrovipera that have no or limited antivenoms available could be inhibited with CO or with the metheme-inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms were exposed in isolation to CO or PHA. Eight species were found to have procoagulant activity consistent with the generation of human thrombin, while one was likely fibrinogenolytic. All venoms were significantly inhibited by CO/PHA with species-specific variation noted. These data demonstrate indirectly that the heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, future investigation is warranted to determine if heme could serve as a potential therapeutic target to be modulated during treatment of envenomation by hemotoxic enzymes.

scholarly journals Snake Venom Proteomics of Samar Cobra (Naja samarensis) from the Southern Philippines: Short Alpha-Neurotoxins as the Dominant Lethal Component Weakly Cross-Neutralized by the Philippine Cobra Antivenom

2021 ◽  
Vol 12 ◽  
Author(s):  
Praneetha Palasuberniam ◽  
Yi Wei Chan ◽  
Kae Yi Tan ◽  
Choo Hock Tan
Keyword(s):  
Snake Venom ◽  
High Performance ◽  
Lethal Dose ◽  
Secretory Proteins ◽  
Venomous Snake ◽  
Neutralization Activity ◽  
Medical Importance ◽  
Species Specific ◽  
Venom Proteins

The Samar Cobra, Naja samarensis, is endemic to the southern Philippines and is a WHO-listed Category 1 venomous snake species of medical importance. Envenomation caused by N. samarensis results in neurotoxicity, while there is no species-specific antivenom available for its treatment. The composition and neutralization of N. samarensis venom remain largely unknown to date. This study thus aimed to investigate the venom proteome of N. samarensis for a comprehensive profiling of the venom composition, and to examine the immunorecognition as well as neutralization of its toxins by a hetero-specific antivenom. Applying C18 reverse-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (LC-MS/MS), three-finger toxins (3FTx) were shown to dominate the venom proteome by 90.48% of total venom proteins. Other proteins in the venom comprised snake venom metalloproteinases, phospholipases A2, cysteine-rich secretory proteins, venom nerve growth factors, L-amino acid oxidases and vespryn, which were present at much lower abundances. Among all, short-chain alpha-neurotoxins (SαNTX) were the most highly expressed toxin within 3FTx family, constituting 65.87% of the total venom proteins. The SαNTX is the sole neurotoxic component of the venom and has an intravenous median lethal dose (LD50) of 0.18 μg/g in mice. The high abundance and low LD50 support the potent lethal activity of N. samarensis venom. The hetero-specific antivenom, Philippine Cobra Antivenom (PCAV, raised against Naja philippinensis) were immunoreactive toward the venom and its protein fractions, including the principal SαNTX. In efficacy study, PCAV was able to cross-neutralize the lethality of SαNTX albeit the effect was weak with a low potency of 0.20 mg/ml (defined as the amount of toxin completely neutralized per milliliter of the antivenom). With a volume of 5 ml, each vial of PCAV may cross-neutralize approximately 1 mg of the toxin in vivo. The findings support the potential para-specific use of PCAV in treating envenomation caused by N. samarensis while underscoring the need to improve the potency of its neutralization activity, especially against the highly lethal alpha-neurotoxins.

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