Mass Spectrometry and X-ray Diffraction Analysis of Two Crystal Types of Dioclea virgata Lectin: An Antinociceptive Protein Candidate to Structure/Function Analysis

Abstract

The lectin from seeds of Dioclea virgata (DvirL) was purified in a single step affinity chromatography, sequenced by tandem mass spectrometry and submitted to crystallization and biological experiments. DvirL has a molecular mass of 25,412 ± 2 Da and the chains β and γ has 12,817 Da ± 2 and 12,612 Da ± 2, respectively. Primary sequence determination was assigned by tandem mass spectrometry and revealed a protein with 237 amino acids and 87% of identify with ConA. The protein crystals were obtained native and complexed with X-Man using vapor-diffusion method at a constant temperature of 293 K. A complete X-ray dataset was collected at 1.8 Å resolution. DvirL crystals were found to be orthorhombic, belonging to the space group I222, with a unit cell parameters a = 647.5 Å, b = 86.6 Å, c = 90.2 Å. Molecular replacement search found a solution with a correlation coefficient of 77.1% and an R(factor) of 44.6%. The present study also demonstrated that D. virgata lectin presents edematogenic and antinociceptive activities in rodents electing this protein as a candidate to structure/function analysis.

Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia

“…The effect of AAL demonstrated only in the inflammatory phase of this test indicates its selectivity for inflammatory nociception types. This feature had been pointed out for other leguminous lectins (Figueiredo et al , ; Delatorre et al , ; Pires et al , ).…”

“…Several studies had described the antinociceptive effect of lectins with binding specificity for glucose–mannose, isolated from leguminous belonging to the subtribe Diocleinae (tribe Phaseolae), such as Canavalia maritima , Canavalia grandiflora , Dioclea guianensis , Dioclea violacea (Holanda et al , ); Canavalia boliviana (Figueiredo et al , ); Dioclea virgata (Delatorre et al , ); and Canavalia brasiliensis (Pires et al , ). Most of these effects involved the lectin domain and peripheral nociception pathways.…”

ConA-Like Lectins: High Similarity Proteins as Models to Study Structure/Biological Activities Relationships

“…Edematogenic activity has been studied in ConA [31], ConGF [35], ConBr [29], Canavalia gladiata lectin (CGL) [29], C. maritima lectin (ConM) [29], C. virosa lectin (ConV) [36], C. oxyphylla lectin (CoxyL) [19], C. villosa lectin (CvilL) [20], Dioclea rostrata lectin (DRL) [37], D. wilsonii lectin (DWL) [38,39], D. virgata lectin (DvirL) [40], D. lasiophylla lectin (DlyL) [41], D. reflexa lectin (DrfL) [41] and Cymbosema roseum lectin (CRL1) [42] (Table 1). Anti-inflammatory effect has been studied in ConGF [43], DvirL [33], CRL1 [42], D. guianensis lectin (DguiL), D. violacea lectin (DVL) and Cratylia floribunda lectin (CFL) [28].…”

Homologous Canavalia Lectins Elicit Different Patterns of Antinociceptive Responses

“…As ConBr and ConM, but not CGL, demonstrated significant inhibitory responses essentially in the second phase of the formalin test, a strong relationship between these antinociceptive mechanisms and inflammatory pain is suggested. The unprecedented selectivity of CGL for the first phase, contrasts with that described for other Diocleinae lectins [3][4][5][6]. Carrageenan elicits inflammatory pain via stimulation of keratinocyte-derived chemokines and TNF- leading to release of IL-1 and prostanoids [10].…”

“…Diocleinae lectins elicit different degrees of antinociceptive responses. When administered by the intravenous route Canavalia grandiflora lectin inhibits inflammatory hypernociception in the Von Frey test [3], Dioclea virgata lectin inhibits paw licking in the formalin test [4], and Canavalia boliviana lectin central and peripheral nociception in several models [5]. When administered by the oral route, the lectins of C. maritima, C. grandiflora, Dioclea guianensis, D. violacea and Cratylia floribunda also showed antinociceptive responses in the model of abdominal constrictions induced by acetic acid [6].…”