Nepenthes is a genus comprising carnivorous tropical pitcher plants that have evolved trapping organs at the tip of their leaves for nutrient acquisition from insect trapping. Recent studies have applied proteomics approaches to identify proteins in the pitcher fluids for better understanding the carnivory mechanism, but protein identification is hindered by limited species-specific transcriptomes for Nepenthes. In this study, the proteomics informed by transcriptomics (PIT) approach was utilized to identify and compare proteins in the pitcher fluids of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana through PacBio isoform sequencing (Iso-Seq) and liquid chromatography-mass spectrometry (LC-MS) proteomic profiling. We generated full-length transcriptomes from all three species of 80,791 consensus isoforms with an average length of 1,692 bp as a reference for protein identification. The comparative analysis found that transcripts and proteins identified in the hybrid N. × hookeriana were more resembling N. rafflesiana, both of which are insectivorous compared with omnivorous N. ampullaria that can derive nutrients from leaf litters. Previously reported hydrolytic proteins were detected, including proteases, glucanases, chitinases, phosphatases, nucleases, peroxidases, lipid transfer protein, thaumatin-like protein, pathogenesis-related protein, and disease resistance proteins. Many new proteins with diverse predicted functions were also identified, such as amylase, invertase, catalase, kinases, ligases, synthases, esterases, transferases, transporters, and transcription factors. Despite the discovery of a few unique enzymes in N. ampullaria, we found no strong evidence of adaptive evolution to produce endogenous enzymes for the breakdown of leaf litter. A more complete picture of digestive fluid protein composition in this study provides important insights on the molecular physiology of pitchers and carnivory mechanism of Nepenthes species with distinct dietary habits.

Neprosin is a novel prolyl endopeptidase (PEP) first discovered in the insectivorous tropical pitcher plants of Nepenthes species. Neprosin has two uncharacterized functional domains of neprosin activation peptide (PF14365) and neprosin domain (PF03080), which can be found in many uncharacterized plant proteases. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, an extensive in silico structure-function analysis of neprosins was conducted to investigate the catalytic mechanism of this protein family. Results from multiple sequence alignment and ConSurf showed neprosins lack the catalytic triad and motifs of common prolyl endopeptidase family S9. Protein models of neprosins from Nepenthes x ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated using four ab initio methods and comparatively assessed to get high-quality models. Structural alignment of neprosin models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal more resemblances to glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to molecular dynamics simulation, can be found at the substrate cleft and active site corresponding to a glutamic peptidase. Protein docking demonstrated that mature neprosins bind well with potent antigen αI-gliadin at the putative active cleft. Taken together, neprosins represent a new member of the glutamic peptidase family G3, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family.