The modular origin and evolutionary expansion of the enigmatic DGF-1 protein family in trypanosomatids

Abstract How novel proteins and protein architectures evolve is a central question in evolutionary biology. Early-branching trypanosomatids provide a valuable system to investigate this process because they retain ancestral eukaryotic features while also encoding lineage-specific proteins with unknown evolutionary origins. Among these, the Dispersed Gene Family 1 (DGF-1) is one of the largest and most enigmatic multigene families, characterized by its exceptional size, repetitive organization, and poorly understood biological function. We performed a comprehensive structural and evolutionary analysis of DGF-1 and identified a previously unrecognized modular architecture composed of 7 tandemly repeated structural domains, here termed ribs, that form a huge extracellular protrusion, followed by a conserved C-terminal membrane-associated region containing a hat domain and multiple transmembrane helices. To investigate the origin of this architecture, each structural module, as well as the complete protein, was searched across the Discoba supergroup using both sequence- and structure-based remote homology searches. These analyses revealed that the DGF-1 architecture did not originate in free-living kinetoplastids. Instead, the free-living kinetoplastid Bodo saltans contains proteins harboring only rib-like domains and other proteins containing the membrane-associated region, whereas the first complete DGF-1 architecture appears in the early-branching trypanosomatid Paratrypanosoma confusum. Within trypanosomatids, the 7 rib modules are highly conserved across paralogous copies and cluster according to their position within the protein, indicating that the canonical 7-rib architecture predates the emergence of bona fide DGF-1 proteins. Comparative genomic analyses further show that DGF-1 genes were retained in some species, independently expanded in others with its largest expansion occurring in Trypanosoma cruzi, or secondarily lost in different lineages (leishmania and African trypanosomes). Together, these findings reconstruct the evolutionary origin of a previously unknown protein architecture and provide a framework for understanding the emergence of lineage-specific protein families in trypanosomatids.