Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity

Abstract

Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus. Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants

“…We are also unable to detect any binding of other water-soluble cell wall-derived carbohydrates to the PDLP5 ectodomain (Supplementary Figure 13b). The PDLP5 DUF26 domains share strong structural homology also with two fungal lectins, the α-galactosyl-binding Lyophyllum decastes lectin (LDL) 45 and a glycan-binding Y3 lectin from Coprinus comatus 46 . Both proteins closely align with the plant DUF26 domain, and share one of the three disulfide bridges (Fig.…”

The two faces of Coprinus comatus—Functional properties and potential hazards

“…Assays using 7-aminoactinomycin D and Annexin V double staining indicated induction dose-dependent mannereffect of Y3 on early and also late apoptosis of Jurkat cells (90% apoptotic cells of total cells). In this study, Y3 shows only weak effect on cells viability against cervical cancer HeLa cells, pancreas carcinoma Dan-G cells, and liver carcinoma HepG2 cells(Zhang et al, 2017).accase from mycelia of CC may have antiproliferative properties. In nature, laccase takes a part in various physiological processes, because of its important role in lignin degradation(Baldrian, 2006;Brijwani, Rigdon, & Vadlani, 2010).…”

Mechanistic insights into the evolution of DUF26-containing proteins in land plants

“…We also tested other water soluble cell wall derived carbohydrates, but were not able to detect any binding to the PDLP5 ectodomain (Figure S13b). The PDLP5 DUF26 domains share significant structural homology not only with the plant Gnk2, but also with two fungal lectins, the α-galactosyl-binding Lyophyllum decastes lectin (LDL) 45 and a glycan-binding Y3 lectin from Coprinus comatus 46 . Both proteins closely align with the plant DUF26 domain, and share one of the three disulfide bridges (Figure 4e-f).…”