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.
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.
Fuentes-Silva
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D
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510, Mexico.
Mendoza-Hernández
G
G
Stojanoff
V
V
Palomares
L A
LA
Zenteno
E
E
Torres-Larios
A
A
Rodríguez-Romero
A
A
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D028482:Hevea; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011110:Polymorphism, Genetic; D020033:Protein Isoforms / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014961:X-Ray Diffraction
D000485:Allergens; D052179:Antigens, Plant; D006023:Glycoproteins; C473105:Hev b 2 allergen, Hevea brasiliensis; D009005:Monosaccharides; D010446:Peptide Fragments; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins