Primary digestive cathepsins L of Tribolium castaneum larvae: Proteomic identification, properties, comparison with human lysosomal cathepsin L

We previously described the most highly expressed enzymes from the gut of the red flour beetle, Tribolium castaneum, as cathepsins L. In the present study, two C1 family-specific cysteine cathepsin L enzymes from the larval midgut were isolated and identified using MALDI-TOF MS analysis. The isolated T. castaneum cathepsins were characterized according to their specificity against chromogenic and fluorogenic peptide substrates, and the most efficiently hydrolyzed substrate was Z-FR-pNA with Arg in the P1 subsite. The specificity of insect digestive cathepsins was compared with human lysosomal cathepsin L, the well-studied peptidase of the C1 family ca-thepsins. T. castaneum digestive cathepsins efficiently hydrolyzed substrates with small and uncharged amino acid residues at P1 (Ala, Gln) more than human cathepsin L. In particular, these insect digestive cathepsins cleaved with higher efficiency the analogs of immunogenic peptides of gliadins, which contribute to autoimmune celiac disease in susceptible people, and thus insect enzymes may be useful in enzymatic treatments for this disease. A bioinformatic study supported by the proteomic analysis of the primary structures of the isolated cathepsins was used to compare tertiary models. The phylogenetic analysis of coleopteran and human cathepsins from the L subfamily indicated that insect digestive cathepsins grouped separately from lysosomal cathepsins.

Automated summary

This study identified two C1 family-specific cysteine cathepsin L enzymes from the larval midgut of the red flour beetle, showing their higher efficiency in hydrolyzing substrates compared to human cathepsin L. Insect digestive cathepsins exhibited higher hydrolysis efficiency towards analogs of immunogenic peptides, which may contribute to therapeutic treatments for autoimmune celiac disease. Bioinformatic and phylogenetic analyses suggest that insect digestive cathepsins are separated from lysosomal cathepsins in the L subfamily.