Through data mining of the Cordyceps militaris genome, a lectin-encoding gene, CMLec3, was identified. In this study, the CMLec3 sequence was analyzed using bioinformatics approaches, and the gene was heterologously expressed in Escherichia coli BL21 cells. The biological activity of the product was examined. In addition, CMLec3 gene expression levels were assessed. The results showed that the CMLec3 protein contained a lectin domain structure and was successfully expressed. The CMLec3 protein partly inhibited HeLa cell proliferation. CMLec3 exhibited the highest gene expression in the primordium at a level 5.19 times that of the mycelium and 1.35 times that of the fruiting body. This suggests that the gene may be related to fruiting body development.

Lentinus tigrinus (L. tigrinus), a white-rot fungus that grows naturally on rotten hardwood during spring and summer in China, is an edible and medicinal mushroom containing a valuable combination of nutrients including high amino acid concentrations and low sugar levels. However, no reports have isolated and characterized FIP genes from L. tigrinus to date. In our study, two novel fungal immunomodulatory proteins (FIPs) from Lentinus tigrinus were identified and named Fip-lti1 and Fip-lti2. The bioactive characteristics of Fip-lti1 and Fip-lti2 were compared to a well-known FIP (LZ-8 from Ganoderma lucidum) to investigate the effect of Fip-lti1 and Fip-lti2 expression on Concanavalin A (ConA)-induced liver injury. Both Fip-lti1 and Fip-lti2 protected livers from ConA-induced necrosis, as evidenced by decreased serum aminotransferase levels (AST, ALT) and relieved liver histology. Levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress (SOD, MDA) were shown to be reduced by expressing Fip-lti1 and Fip-lti2. In addition, the hepatoprotective effect of Fip-lti1, Fip-lti2, and LZ-8 correlated with ameliorating the imbalance of Th1/Th2 (IFN-γ/IL-4). The observed liver protection of Fip-lti1 and Fip-lti2 was mechanistically explored. Treatments with Fip-lti1 and Fip-lti2 regulated GATA3/T-bet expression, activated the decreased Nrf-2/HO-1 pathway, and countered the upregulated NLRP3/ASC/NF-κBp65 signaling in ConA-stimulated liver injury. In conclusion, we identified two fungal proteins (Fip-lti1 and Fip-lti2) that can protect liver from ConA-induced liver injury.

In Volvariella volvacea, a species of edible mushroom, cryogenic autolysis is a typical part of abnormal metabolism. Previous functional annotation cluster analyses of cold-induced gene expression profiles have shown that the ubiquitin-conjugating enzyme E2 (UBE2) and the cyclin-like F-box domain (FBDC) form the functional clusters. Among them, a specific FBDC gene has been found to be in response to cold stress and correlated with the cryogenic autolysis of V. volvacea. In this study, analysis of gene expression profiling showed that only one type of UBE2 in V. volvacea (UBEV2) was significantly up-regulated. Further quantitative real-time PCR analysis confirmed that the expression of UBEV2 was significantly up-regulated (P < 0.05) after cold-treatment lasting 4, 6, and 8 h. Western-bolt analyses confirmed that the protein expression of UBEV2 was in response to cold stress. This provided evidence that UBEV2 was a cold stress-responsive gene and closely correlated with cryogenic autolysis. The specific distribution of UBEV2 in recently diverged herb decay fungi indicated that UBEV2 was not evolutionarily correlated with early diverging fungi. Phylogenetic analysis indicated that UBEV2 was generated by horizontal gene transfer (HGT) from the ancestry of Selaginella moellendorffii UBE2. Further relative time estimation and detection of natural selection showed that there has been recent positive selection after HGT in UBEV2. Molecular modeling and logo analysis showed that the cysteine-cysteine motif is the characteristic of the UBEV2 family. These observations indicate that UBEV2 and FBDV1 are the newly discovered cold stress-responsive genes correlated with the cryogenic autolysis of V. volvacea.