Abstract
Cell–cell and cell-substrate based adhesion of yeasts are major determinants of their adoption of different life styles. Genome-mining of ascomycetous GPI-anchored cell wall proteins with lectin-like PA14 domains identified a unique class of putative adhesins in the clade of methylotrophic Komagataella yeasts, many of which are known to colonize plants and insects involving yet unknown adhesion mechanisms. Here, we report the functional and structural analysis of two of its members: KpFlo1 (=Cea1), that is highly specific for terminal N-acetylglucosamine moieties, and KpFlo2, which represents an orphan lectin with intact binding site but unknown specificity. Crystal structures of the Cea1 adhesion domain complexed to N-acetylglucosamine and N,N′-diacetylchitobiose reveal a Ca2+-dependent binding mode that differs from other members of the PA14/Flo5 adhesin family. Heterologous expression of Cea1A in Saccharomyces cerevisiae promotes cellular adhesion to non-reducing ends of non-crystalline chitin. Overall, our data suggest that high-affinity recognition of β-GlcNAc-capped glycans by Cea1 enable Komagataella species to interact with surface cues present in fungi and insects.
Highlights
Among them Cea1A represents a novel subgroup of adhesive PA14 domains, which mediate high affinity recognition of chitinous poly- and oligomers
By using the sequences of ScFlo5A and Epa1A as initial search templates, we identified by PSIBLAST (Altschul et al, 1997) nine orthologs in Komagataella pastoris DSMZ70382 (Figure 1A), which belong to PA14 (Pfam-ID: PF07691) or PA14_2 (=GLEYA domain; Pfam-ID: PF10528) domain-containing GPI-cell wall-associated protein (CWP) adhesins
These data show that while Cea1A shares some of the structural characteristics found in Flos and epithelial adhesins, it confers ligand binding by a clearly distinct mode not observed in other PA14/Flo5-like adhesins so far
Summary
Fungi colonize most known ecological niches and are nearly ubiquitously found on Earth. Despite revealing the common PA14-like β-sandwich and DcisD motif these structures showed different modes of carbohydrate recognition (Petosa et al, 1997; Veelders et al, 2010; Ielasi et al, 2012; Maestre-Reyna et al, 2012; Sim et al, 2013; Goossens et al, 2015) These adhesins confer either self-recognition (Flo) or host-recognition (Epa), and can be divided into two different structural subgroups. We mined the genomes of ascomycetous fungi for hitherto unknown PA14/Flo5-like adhesin domains and identified such domains in several putative GPI-CWP adhesins of methylotrophic yeasts from the Komagataella genus Despite their biotechnological application for heterologous protein production, the natural lifestyles and adhesion properties of Komagataella species like Komagataella pastoris and K. phaffii are largely unknown (Ogata et al, 1969; Mbawala et al, 1990; Daly and Hearn, 2005). Among them Cea1A represents a novel subgroup of adhesive PA14 domains, which mediate high affinity recognition of chitinous poly- and oligomers
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