Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom

TY - JOUR

T1 - Structure of recombinant Ves v 2 at 2.0 Angstrom resolution

T2 - structural analysis of an allergenic hyaluronidase from wasp venom

AU - Skov, Lars K

AU - Seppälä, Ulla

AU - Coen, Jeremy J F

AU - Crickmore, Neil

AU - King, Te P

AU - Monsalve, Rafael

AU - Kastrup, Jette S

AU - Spangfort, Michael D

AU - Gajhede, Michael

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Binding Sites

KW - Crystallography, X-Ray

KW - Glycosylation

KW - Hyaluronoglucosaminidase

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Folding

KW - Recombinant Proteins

KW - Sequence Alignment

KW - Sequence Analysis, Protein

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Wasp Venoms

U2 - 10.1107/S0907444906010687

DO - 10.1107/S0907444906010687

M3 - Journal article

C2 - 16699186

SN - 2059-7983

VL - 62

SP - 595

EP - 604

JO - Acta Crystallographica Section D: Structural Biology

JF - Acta Crystallographica Section D: Structural Biology

IS - Pt 6

ER -