Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in FEBS Journal 273 (2006): 2779-2788, doi:10.1111/j.1742-4658.2006.05355_1.x.The cone snail is the only invertebrate system in which the vitamin K dependent carboxylase (or γ-carboxylase) and its product γ-carboxyglutamic acid (Gla)1 have been identified. It remains the sole source of structural information of invertebrate γ-carboxylase subtrates. Four novel γ- carboxyglutamic acid (Gla)1 containing peptides were purified from the venom of Conus textile and characterized by biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have 6 Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L-bromotryptophan. Gla(2)-TxVI/A and Gla(2)- TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids –46 to –27) were nearly identical and highly hydrophobic. The predicted propeptide region (–20 to –1) that contains the γ-carboxylation recognition site (γ-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus γ-carboxylase and synthetic peptide substrates localized the γ-CRS of Gla(1)-TxVI to the region –14 to –1 of the polypeptide precursor: the Km was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 μM when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km 10-fold.This work was supported by grants K2001-03X-04487-27A and K2001- 03GX-04487-27, 08647, 13147 from the Swedish Medical Research Council, the European Union Cono-Euro-Pain (QLK3-CT-2000-00204), the Swedish Foundation for Strategic Research, the Kock Foundation, the Påhlsson Foundation and the Foundation of University Hospital, Malmö

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

<p>Abstract</p> <p>Background</p> <p>Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from <it>M. bovis </it>BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.</p> <p>Results</p> <p>The 2DE proteomic map of <it>M. bovis </it>BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.</p> <p>Conclusions</p> <p>Here we report the detailed 2DE profile of CFPs from <it>M. bovis </it>BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.</p

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites

Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut

Made available in DSpace on 2015-06-17T12:05:19Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) dario_kalume2etal_IOC_2014.pdf: 1677709 bytes, checksum: 6625aae3bddb1dfdb18a3dd1fde714e5 (MD5) Previous issue date: 2014Johns Hopkins University. Bloomberg School of Public Health. Malaria Research Institute. The W. Harry Feinstone Department of Molecular Microbiology and Immunology. Baltimore, MD, USA.Johns Hopkins University. Bloomberg School of Public Health. Malaria Research Institute. The W. Harry Feinstone Department of Molecular Microbiology and Immunology. Baltimore, MD, USA / Case Western Reserve University. Center for Global Health and Diseases. Cleveland, OH, USA.Loyola University. Department of Biology. Chicago, IL, USA.Johns Hopkins University. Bloomberg School of Public Health. Malaria Research Institute. The W. Harry Feinstone Department of Molecular Microbiology and Immunology. Baltimore, MD, USA.Chinese Academy of Sciences. Shanghai Institutes for Biological Sciences. Institute of Plant Physiology and Ecology. cKey Laboratory of Insect Developmental and Evolutionary Biology. Shangai, China.Duquesne University. Department of Biological Sciences. Pittsburgh, PA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. fLaboratory of Malaria and Vector Research. Rockville, MD, USA.National Institutes of Health. National Institute of Allergy and Infectious Diseases. fLaboratory of Malaria and Vector Research. Rockville, MD, USA.Johns Hopkins University. The Johns Hopkins University School of Medicine. McKusick–Nathans Institute of Genetic Medicine. Departments of Biological Chemistry, Pathology, and Oncology. Baltimore, MD, USA.Johns Hopkins University. Bloomberg School of Public Health. Malaria Research Institute. The W. Harry Feinstone Department of Molecular Microbiology and Immunology. Baltimore, MD, USA.Plasmodium ookinete invasion of the mosquito midgut is a crucial step of the parasite life cycle but little is known about the molecular mechanisms involved. Previously, a phage display peptide library screen identified SM1, a peptide that binds to the mosquito midgut epithelium and inhibits ookinete invasion. SM1 was characterized as a mimotope of an ookinete surface enolase and SM1 presumably competes with enolase, the presumed ligand, for binding to a putative midgut receptor. Here we identify a mosquito midgut receptor that binds both SM1 and ookinete surface enolase, termed “enolase-binding protein” (EBP). Moreover, we determined that Plasmodium berghei parasites are heterogeneous for midgut invasion, as some parasite clones are strongly inhibited by SM1 whereas others are not. The SM1-sensitive parasites required the mosquito EBP receptor for midgut invasion whereas the SM1- resistant parasites invaded the mosquito midgut independently of EBP. These experiments provide evidence that Plasmodium ookinetes can invade the mosquito midgut by alternate pathways. Furthermore, another peptide from the original phage display screen, midgut peptide 2 (MP2), strongly inhibited midgut invasion by P. berghei (SM1-sensitive and SM1-resistant) and Plasmodium falciparum ookinetes, suggesting that MP2 binds to a separate, universal receptor for midgut invasion

Protein profile of blood monocytes is altered in HTLV-1 infected patients: implications for HAM/TSP disease

Submitted by Janaína Nascimento ([email protected]) on 2019-01-14T11:38:11Z No. of bitstreams: 1 ve_Echevarria-Lima_Juliana_etal_INI_2018.pdf: 3502915 bytes, checksum: de470bb9f80e4cb38a644e81b9c2d215 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-01-17T22:30:44Z (GMT) No. of bitstreams: 1 ve_Echevarria-Lima_Juliana_etal_INI_2018.pdf: 3502915 bytes, checksum: de470bb9f80e4cb38a644e81b9c2d215 (MD5)Made available in DSpace on 2019-01-17T22:30:44Z (GMT). No. of bitstreams: 1 ve_Echevarria-Lima_Juliana_etal_INI_2018.pdf: 3502915 bytes, checksum: de470bb9f80e4cb38a644e81b9c2d215 (MD5) Previous issue date: 2018Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Departamento de Imunologia. Laboratório de Imunologia Básica e Aplicada. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro.Instituto de Bioquímica Médica Leopoldo de Meis. Unidade de Espectrometria de Massas e Proteômica. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto Nacional de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Câncer. Coordenação Geral de Ensino e Pesquisa. Programa de Oncobiologia Celular e Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Departamento de Imunologia. Laboratório de Imunologia Básica e Aplicada. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Neuroinfecções. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Neuroinfecções. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Neuroinfecções. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro.Instituto de Bioquímica Médica Leopoldo de Meis. Unidade de Espectrometria de Massas e Proteômica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Imunologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro.Instituto de Bioquímica Médica Leopoldo de Meis. Unidade de Espectrometria de Massas e Proteômica. Rio de Janeiro, RJ, Brasil.Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The endothelial breakdown and migration of leukocytes, including monocytes, to the spinal cord are involved in HAM/TSP development. Monocytes from HTLV-1-infected individuals exhibit important functional diferences when compared to cells from uninfected donors. Using proteomic shot gun strategy, performed by nanoACQUITY-UPLC system, we analyzed monocytes isolated from peripheral blood of asymptomatic carriers (AC), HAM/TSP and uninfected individuals. 534 proteins were identifed among which 376 were quantifed by ExpressionE software. Our study revealed a panel of changes in protein expression linked to HTLV-1 infection. Upregulation of heat shock proteins and downregulation of canonical histone expression were observed in monocytes from HTLV-1-infected patients. Moreover, expression of cytoskeleton proteins was increased in monocytes from HTLV-1-infected patients, mainly in those from HAM/TSP, which was confrmed by fow cytometry and fuorescence microscopy. Importantly, functional assays demonstrated that monocytes from HAM/TSP patients present higher ability for adhesion and transmigration thought endothelium than those from AC and uninfected individuals. The major changes on monocyte protein profle were detected in HAM/TSP patients, suggesting that these alterations exert a relevant role in the establishment of HAM/TSP