Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians

SIMPLE SUMMARY: Cysteine protease inhibitors (cystatins) are molecules that play key protective roles in protein degradation and are involved in the immunomodulation of host responses to parasites. Little is known about the cystatin gene repertoire, evolution, and lineage-specific adaptations of early-emerging metazoans. Using bioinformatics searches, we identified orthologues of cystatins in basal animal lineages including free-living and parasite taxa. We aimed to explore whether their cystatin gene repertoire and evolution follow similar patterns recognized for derived metazoans and whether the modifications are linked to the organism’s life history. We revealed that cysteine protease inhibitors from early-emerging animal groups are highly diverse, with modifications in gene organization and protein architecture. A new subtype of cystatins was discovered in the parasitic cnidarians, the Myxozoa, which has so far been only reported for a group of derived animals: trematode flukes. We set out hypotheses to describe the driving forces for the origins of this unique cystatin subtype and propose evolutionary scenarios elucidating the current existence of cystatins in the Metazoa, especially in their early-emerging lineages. Our research identified molecules for which future functional studies may help to identify their roles in host–parasite interactions and for the parasite itself. ABSTRACT: The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.