Proteoforms of the platelet-aggregating enzyme PA-BJ, a serine proteinase from Bothrops jararaca venom

Autor(es)

Yamashiro, Edson T. ^[1] ; Oliveira, Ana K. ^[1] ; Kitano, Eduardo S. ^[1] ; Menezes, Milene C. ^[1] ; Junqueira-de-Azevedo, Inacio L. ^[1] ; Leme, Adriana F. Paes ^[2] ; Serrano, Solange M. T. ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Ctr Toxins Immune Response & Cell Signaling CeTIC, BR-05503000 Sao Paulo - Brazil

^[2] Lab Nacl Biociencias LNBio, Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1844, n. 12, p. 2068-2076, DEC 2014.
• Citações Web of Science: 5

Resumo

Snake venoms contain serine proteinases that are functionally similar to thrombin and specifically cleave fibrinogen to convert it into fibrin or activate platelets to aggregation. PA-BJ is a serine proteinase from Bothrops jararaca venom that promotes platelet aggregation and this effect is mediated by the G-coupled protein receptors PAR1 and PAR4. In this study we describe an improved procedure to obtain PA-BJ from B. jararaca venom that uses less chromatographic steps, and, interestingly, results in the isolation of eight proteoforms showing slightly different pls and molecular masses due to variations in their glycosylation levels. The identity of the isolated PA-BJ forms (1-8) was confirmed by mass spectrometry, and they showed similar platelet-activating activity on washed platelet suspensions. N- and O-deglycosylation of PA-BJ 1-8 under denaturing conditions generated variable electrophoretic profiles and showed that some forms were resistant to complete deglycosylation. Furthermore, N- and O-deglycosylation under non-denaturing conditions also showed different electrophoretic profiles between the PA-BJ forms and caused partial loss of their ability to cleave a recombinant exodomain of PAR1 receptor. In parallel, three cDNAs encoding PA-BJ-like enzymes were identified by pyrosequencing of a B. jararaca venom gland library constructed with RNA from a single specimen. Taken together, our results suggest that PA-BJ occurs in the B. jararaca venom in multiple proteoforms displaying similar properties upon platelets regardless of their variable isoelectric points, molecular masses, carbohydrate moieties and susceptibility to the activity of glycosidases, and highlight that variability of specific venom components contributes to venom proteome complexity. (C) 2014 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 11/11308-0 - Estudo do proteoma de folhas de cultivares e genótipos de cana-de-açúcar
• Beneficiário: Eduardo Shigueo Kitano
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 11/08514-8 - Estudo do degradoma do HF3, uma metaloproteinase hemorrágica da classe P-III do veneno da serpente Bothrops jararaca, sobre fibroblastos em cultura
• Beneficiário: André Zelanis Palitot Pereira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 98/14307-9 - Center for Applied Toxinology
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs