title: Protein crystal content analysis by mass spectrometry and preliminary X-ray diffraction of a lectin from Canavalia grandiflora seeds with modulatory role in inflammation.
abstract: BACKGROUND
  Lectins are a family of proteins capable of deciphering the glycan code. Several authors have published works about crystallization and mass spectrometry analyses of ConA-like lectins. However, mass spectrometry has never been used to characterize lectin crystal content. In this study, Canavalia grandiflora lectin (ConGF), a ConA-like lectin, was crystallized, part of its primary structure sequenced and the pro-inflammatory activity evaluated. In addition, the crystal content was analyzed by mass spectrometry.
  
  
  METHODS
  ConGF was crystallized in the presence of X-Man by hanging-drop vapor diffusion at 293 K and the protein crystal content was analyzed by electrospray ionization in a SYNAPT HDMS mass spectrometer. Partial sequence was obtained by protein digestion with several proteolytic enzymes and the peptides sequenced by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The pro-inflammatory potential of ConGF was also evaluated in the model of rat paw edema.
  
  
  RESULTS
  The protein crystals consist of mature α chain and β and γ fragments measuring 25 612 ± 2 Da, 12 962 ± 2 Da and 12 667 ± 2 Da, respectively. The crystal belongs to the orthorhombic space group I222 (unit cell parameters: a = 67.70, b = 55.90, c = 107.46 Å), assuming a monomer in the asymmetric unit. The solvent content was calculated as 43.50% and the protein content as 2.5 µg. Furthermore, a significant part of the primary structure (65.8%) was determined by mass spectrometry.
  
  
  CONCLUSIONS
  As far as we know this is the first report of lectin crystal content characterized by mass spectrometry. Like other ConA-like lectins, GonGF induced paw edema however differing in potency and duration. The observed pro-inflammatory activity suggests that ConGF might be a useful tool in the study of inflammation processes and structure/function relationships.
authors.affiliation: Laboratório de Moléculas Biologicamente Ativas, Universidade Federal do Ceará, Fortaleza, Brazil.
authors.forename: Rafael Conceição
authors.initials: RC
authors.lastname: Simões
authors.forename: Bruno Anderson Matias
authors.initials: BA
authors.lastname: Rocha
authors.forename: Maria Julia Barbosa
authors.initials: MJ
authors.lastname: Bezerra
authors.forename: Ito Liberato
authors.initials: IL
authors.lastname: Barroso-Neto
authors.forename: Francisco Nascimento
authors.initials: FN
authors.lastname: Pereira-Junior
authors.forename: Raniere
authors.initials: R
authors.lastname: da Mata Moura
authors.forename: Kyria Santiago
authors.initials: KS
authors.lastname: do Nascimento
authors.forename: Celso Shiniti
authors.initials: CS
authors.lastname: Nagano
authors.forename: Plinio
authors.initials: P
authors.lastname: Delatorre
authors.forename: Alana
authors.initials: A
authors.lastname: de Freitas Pires
authors.forename: Ana Maria Sampaio
authors.initials: AM
authors.lastname: Assreuy
authors.forename: Alexandre Holanda
authors.initials: AH
authors.lastname: Sampaio
authors.forename: Benildo Sousa
authors.initials: BS
authors.lastname: Cavada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004487:Edema / Q000139:chemically induced*; D006614:Hindlimb; D007249:Inflammation / Q000139:chemically induced*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010802:Phylogeny; D037121:Plant Lectins / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012639:Seeds / Q000737:chemistry; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D037121:Plant Lectins; C039268:lectin, Canavalia