Crystal structure of Echinococcus multilocularis cystatin B reveals a novel feature in classical stefins.

Alveolar echinococcosis (AE), caused by the metacestode larval of Echinococcus multilocularis, is one of the most lethal helminthic diseases in humans. Current treatment options, such as albendazole, are limited in their efficacy, highlighting the need for a deeper understanding of the parasite-host interaction to identify new therapeutic targets. One promising area of research involves helminth-derived cystatins, which are known to modulate host immune responses to facilitate parasite survival. A cystatin homologue from E. multilocularis (EmCystatin-B) was identified and analyzed. EmCystatin-B was cloned, expressed and purified. Its expression patterns were evaluated by western blot, qPCR and Immunohistochemistry The EmCystatin-B structure was solved by X-ray crystallography. EmCystatin-B was expressed in the mature protoscoleces as well as in the cytosol and nucleus of the metacestode vesicles. Moverover, EmCystatin-B adopts a conserved typical cystatin fold, but also exhibits unique structural features. Notably, a novel feature characterized by two intermolecular disulfide bridges between Cys4 in a EmCystatin-B molecular and Cys76 in adjacent molecule was discovered. Further investigation demonstrated this distinctive feature appears to be involved in the oligomerization of EmCystatin-B, facilitating a monomer-dimer-tetramer assembly pathway. The crystal structure of EmCystatin-B reveals a novel feature in classical stefins, and provides species-specific insights into the sequence, structure, and functional characteristics of EmCystatin-B.