Five Silkworm 30K Proteins Are Involved in the Cellular Immunity against Fungi

SIMPLE SUMMARY: The molecular mechanism of 30K proteins in anti-fungal immunity remains unclear. Here, we examined the mRNA levels of 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7, and found that all of these proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, implying their involvement in immune response. The binding assay results showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. In vitro, the encapsulation of hemocytes on day 5 of the fifth instar larval stage was promoted by the coating of agarose beads with recombinant BmLP1 and BmLP4. Therefore, these results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface. We believe that our study makes a significant contribution to the literature because it provides insights into the 30K-mediated cellular immunity in silkworms. ABSTRACT: Background: 30K proteins are a major group of nutrient storage proteins in the silkworm hemolymph. Previous studies have shown that 30K proteins are involved in the anti-fungal immunity; however, the molecular mechanism involved in this immunity remains unclear. Methods: We investigated the transcriptional expression of five 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7. The five recombinant 30K proteins were expressed in an Escherichia coli expression system, and used for binding assays with fungal cells and hemocytes. Results: The transcriptional expression showed that the five 30K proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, indicating the possibility of their involvement in immune response. The binding assay showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. Furthermore, we found that BmLP1-coated and BmLP4-coated agarose beads promote encapsulation of hemocytes in vitro. The hemocyte encapsulation was blocked when the BmLP1-coated beads were preincubated with BmLP1 specific polyclonal antibodies. Conclusions: These results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface.