Unveiling Putative Functions of Mucus Proteins and Their Tryptic Peptides in Seven Gastropod Species Using Comparative Proteomics and Machine Learning-Based Bioinformatics Predictions

Abstract

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.

Keywords: bioactive peptides; gastropod; machine-learning prediction; mucus; proteomics.

Figure 1

Mucus samples from seven gastropod species separated on 1D-SDS polyacrylamide gels. The gels were stained with Coomassie brilliant blue (A) and silver nitrate (B). By lane: 1, MW = protein molecular marker; 2, AF = Achatina fulica; 3, PC = Pomacea canaliculata; 4, CS = Cryptozona siamensis; 5, SS = Semperula siamensis; 6, HD = Hemiplecta distincta; 7, CF = Cyclophorus fulguratus; and 8, and HP = Helix pomatia.

Figure 2

Comparative proteomes and peptides of mucus from seven gastropod species. (A) Number of protein bands of the mucus proteins from seven gastropod species separated by 1D-SDS-PAGE and stained by Coomassie brilliant blue (blue) and silver nitrate (red) as counted by the GelAnalyzer program. The number of protein bands is shown on the x-axis. The gastropod species are shown on the y-axis. (B) Functional annotation terms of the mucus proteome in three Gene Ontology categories (y-axis) biological process (BP), molecular function (MF), and cellular component (CC). The x-axis represents the number of proteins. (C) The number of different peptides identified in the mucus of seven gastropod species. These peptide data were obtained from the mass spectrometric analysis. The number of peptides is shown on the x-axis. AF = Achatina fulica; PC = Pomacea canaliculata; CS = Cryptozona siamensis; SS = Semperula siamensis; HD = Hemiplecta distincta; CF = Cyclophorus fulguratus; and HP = Helix pomatia.

Figure 3

Clustering of peptides identified in the mucus of seven gastropod species. The peptides were clustered according to their presence (A), relative abundance (B), molecular mass (C), number of amino acids or length (D), hydrophobicity score (E), numbers of positively charged amino acids (F), and negatively charged amino acids (G). Columns represent gastropod species, and rows represent peptides. AF = Achatina fulica; PC = Pomacea canaliculata; CS = Cryptozona siamensis; SS = Semperula siamensis; HD = Hemiplecta distincta; CF = Cyclophorus fulguratus; and HP = Helix pomatia.

Figure 4

Correspondence analysis of 28 patterns of variable peptides in the mucus of seven gastropod species. Blue squares in the heatmap (A) indicate the presence of the peptide, and white squares indicate the absence of a peptide. The sizes of the red circles in the balloon plot (B) indicate the number of peptides within each pattern. The biplot (C) shows global patterns of the variable peptides (blue circles) and the gastropod species (triangles) according to the first two dimensions (Dim1 and Dim2). Colors of the square cosine scores indicate the gastropod species. The scores for the first five dimensions (Dim1 to Dim5) are shown in (D), and the sizes of the green circles represent the score level. AF = Achatina fulica; PC = Pomacea canaliculata; CS = Cryptozona siamensis; SS = Semperula siamensis; HD = Hemiplecta distincta; CF = Cyclophorus fulguratus; and HP = Helix pomatia.

Figure 5

Prediction of 20 bioactive properties from the mucous peptides of seven gastropod species. The predictions were based on two machine-learning algorithms (k-nearest neighbor and random forest). (A) The prediction results show peptides that were predicted by either method in light blue, whereas those predicted by both methods are colored in dark blue. (B) Distribution of the bioactive peptides across seven gastropod species. Sizes of the red circles represent the number of peptides. (C) Number of the bioactive peptides classified by the properties are presented in the same order as the properties in (A). (D) Number of bioactive peptides (indicated by numbers on top of the bars) that had multiple bioactive properties (from one to seven). AP: antiparasitic, DD: drug-delivering, AC: anticancer, AV: antiviral, CC: cell-communicating, TH: tumor-homing, AF: antifungal, AI: anti-inflammatory, CP: cell-penetrating, AB: antibacterial, Abi: antibiofilm, QS: quorum-sensing, and AH: antihypertensive properties. The abbreviations of the gastropod species are the same as those presented in Figure 4.