title: Proteoforms of the platelet-aggregating enzyme PA-BJ, a serine proteinase from Bothrops jararaca venom.
abstract: 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 pIs 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.
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
authors.forename: Edson T
authors.initials: ET
authors.lastname: Yamashiro
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
authors.forename: Ana K
authors.initials: AK
authors.lastname: Oliveira
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
authors.forename: Eduardo S
authors.initials: ES
authors.lastname: Kitano
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
authors.forename: Milene C
authors.initials: MC
authors.lastname: Menezes
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.
authors.forename: Inácio L
authors.initials: IL
authors.lastname: Junqueira-de-Azevedo
authors.affiliation: Laboratório Nacional de Biociências (LNBio), Campinas, Brazil.
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Paes Leme
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil. Electronic address: solange.serrano@butantan.gov.br.
authors.forename: Solange M T
authors.initials: SM
authors.lastname: Serrano
keywords: Glycosylation; PAR1; Platelet-aggregation; Proteoform; Proteome; Snake venom serine proteinase