Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Sep 1;63(Pt 9):787-91. doi: 10.1107/S1744309107039838. Epub 2007 Aug 31.

Abstract

Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a beta-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4(1) with unit-cell parameters a = b = 150.17, c = 77.41 A were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 A, beta = 113.6 degrees. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allergens / chemistry*
  • Allergens / genetics
  • Allergens / isolation & purification
  • Amino Acid Sequence
  • Antigens, Plant
  • Glycoproteins / chemistry
  • Glycoproteins / isolation & purification
  • Hevea
  • Molecular Sequence Data
  • Monosaccharides / analysis
  • Peptide Fragments / chemistry
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Polymorphism, Genetic
  • Protein Isoforms / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • X-Ray Diffraction

Substances

  • Allergens
  • Antigens, Plant
  • Glycoproteins
  • Hev b 2 allergen, Hevea brasiliensis
  • Monosaccharides
  • Peptide Fragments
  • Plant Proteins
  • Protein Isoforms
  • Recombinant Proteins