Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom.
Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved cavity near the active site is involved in binding of the substrate. Surface epitopes and putative glycosylation sites have been compared with those of two other major group 2 allergens from Apis mellifera (honeybee) and Dolichovespula maculata (white-faced hornet). The analysis suggests that the harboured allergic IgE-mediated cross-reactivity between Ves v 2 and the allergen from D. maculata is much higher than that between Ves v 2 and the allergen from A. mellifera.
Skov
Lars K
LK
Biostructural Research, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100, Denmark.
Seppälä
Ulla
U
Coen
Jeremy J F
JJ
Crickmore
Neil
N
King
Te P
TP
Monsalve
Rafael
R
Kastrup
Jette S
JS
Spangfort
Michael D
MD
Gajhede
Michael
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014862:Wasp Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014862:Wasp Venoms; C101836:allergen Ves v 2; D006821:Hyaluronoglucosaminidase