Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis

Tania Barth, Samuel Coelho Mandacaru, Sébastien Charneau, Marcelo Valle de Souza, Carlos André Ornelas Ricart, Eliane Ferreira Noronha, Amanda Araújo Souza, Sonia Maria de Freitas, Peter Roepstorff*, Wagner Fontes, Mariana S. Castro, Osmindo Rodrigues Pires Júnior

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Abstrakt

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 μg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

OriginalsprogEngelsk
TidsskriftJournal of Proteomics
Vol/bind192
Sider (fra-til)102-113
ISSN1874-3919
DOI
StatusUdgivet - 10. feb. 2019

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Citationsformater

Barth, T., Mandacaru, S. C., Charneau, S., Souza, M. V. D., Ricart, C. A. O., Noronha, E. F., Souza, A. A., Freitas, S. M. D., Roepstorff, P., Fontes, W., Castro, M. S., & Pires Júnior, O. R. (2019). Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis. Journal of Proteomics, 192, 102-113. https://doi.org/10.1016/j.jprot.2018.08.012