Marine Bioprospecting: Enzymes and Stress Proteins from the Sea Anemones Anthopleura dowii and Lebrunia neglecta

Abstract

The bioprospecting of sea anemone tissues and secretions has revealed that they are natural libraries of polypeptides with diverse biological activities that can be utilized to develop of biotechnological tools with potential medical and industrial applications. This study conducted a proteomic analysis of crude venom extracts from Anthopleura dowii Verrill, 1869, and Lebrunia neglecta Duchassaing & Michelotti, 1860. The obtained data allowed us to identify 201 polypeptides, of which 39% were present in both extracts. Among the obtained sequences, hydrolase-type enzymes, oxidoreductases, transferases, heat shock proteins, adhesion proteins, and protease inhibitors, among others, were identified. Interaction analysis and functional annotation indicated that these proteins are primarily involved in endoplasmic reticulum metabolic processes such as carbon metabolism and protein processing. In addition, several proteins related to oxidative stress were identified, including superoxide dismutase, peroxiredoxins, thioredoxin, and glutathione oxidase. Our results provide novel information on the polypeptide composition of the crude venom extract from sea anemones, which can be utilized to develop molecules for therapeutic tools and industrial applications.

Keywords: Anthopleura dowii; Lebrunia neglecta; crude venom extract; enzymes; proteome; sea anemone.

Figure 1

Sea anemone collection area. The red diamond indicates El Sauzal Beach, Baja California Norte, Mexico, where the A. dowii specimens were collected. The blue circle indicates the area where the Puerto Morelos reef is located, Quintana Roo, Mexico, where L. neglecta was collected.

Figure 2

Proteomic comparison of crude venom extract from Anthopleura dowii and Lebrunia neglecta. (A) CVE electrophoretic profiles from A. dowii (Ad) and L. neglecta (Ln) samples analyzed with SDS–PAGE gel electrophoresis, 15% polyacrylamide, and Coomassie blue staining. (B–D) show Venn diagrams corresponding to the distribution of the number of unique spectra, unique peptides, and unique proteins, and the overlap between both samples. The distribution of the identified proteins with respect to their molecular weight, percentage of coverage, and the exponentially modified protein abundance index (emPAI) are shown in (E–G), respectively.

Figure 3

Annotation of the proteins identified in the crude venom extracts from A. dowii and L. neglecta by shotgun proteomics. They are classified into three main Gene Ontology (GO) categories (Supplementary Material S1): biological process, cellular component, and molecular function. The graphs show, on the x-axis, the sub-category of the identified proteins and, on the y-axis, the number of proteins identified for each sub-category. The annotation is based on amino acid sequence homology with respect to the proteins annotated in the UniProtKB database using BLASTP and QuickGO tools.

Figure 4

Differentially expressed proteins from proteomes of A. dowii and L. neglecta. (A) shows the volcano graph that represents the changes in the expression of 59 proteins present in both samples. The x-axis shows the log fold change of the L. neglecta/A. dowii ratio, while the y-axis shows the −log probability that was computed (p-value), associated with Student’s t-test. Orange dots indicate significantly downregulated proteins and blue dots indicate upregulated proteins. In (B,C), pie diagrams are shown with the lower and higher functional annotations, respectively, showing the differentially expressed proteins. In (D), protein–protein interaction network is shown. The circles indicate the interaction nodes and these are also indicated with parenthesis. a, proteins of sugar metabolism; b, proteins related to the stress response and the metabolism of reactive oxygen species; c, structural proteins; d, groups proteins that participate in protein folding; e and f, groups enzymes that participate in the modification of proteins and extracellular enzymes that act in the degradation of chitin, respectively.

Figure 5

Cathepsins-L. (A) Alignment of amino acid sequences of putative Cathepsin-L from A. dowii (Ad_Cathepsin (c29945_g1_i1)) with Cathepsins-L from humans (Uni-ProtKB: CATL2_HUMAN), dogs (UniProtKB: CATL1_CANLF) and flour beetles (REF-SEQ: XP_0315589703_1, NP_001163996.1. GenBank: ABC88768.1, ABC88768.1, BAK02675.1). The region corresponding to the signal peptide is underlined, the propeptide is indicated in italics, and the region of the mature protein is indicated in bold. The residues that make up the active site are shaded black and highlighted with white letters. The cysteines that participate in the formation of disulfide bridges are underlined, and the region covered by the tryptic peptides in the Ad_Cathepsin sequence is highlighted by a magenta background. (B) Structure models of Cathepsin. Ad_Cathepsin is blue, XP_031558765.1 is yellow, BAK02675.1 is navy blue, XP_970644.1 is green, CATL1-CANLF is medium gray, CATL2_HUMAN is purple, ABC88768.1 is light gray, NP_001163996.1 is red, and XP_970773.1 is pink. The comparison of the Cα chains presented RMSD values that oscillate between 0.483 and 0.558.

Figure 6

Chitinases. (A) Alignment of the amino acid sequences of the putative Exaiptasia pallida Chitotriosidase-1-like (XP_020909717.1) with chitinases from rats (Uni-ProtKB: CHIA_RAT), sea anemones (REFSEQ: XP_020902061.1) and humans (GenBank: AAM57182.1, AAE83140.1 y AAE83141.1). The magenta background shows the region covered by the tryptic peptides obtained from the proteomes of A. dowii and L. neglecta. The region corresponding to the signal peptide is underlined and the region of the mature protein is indicated in bold. The residues that make up the active and chitooligosaccharide binding sites are shaded black and highlighted with white letters. Cysteines involved in disulfide bond formation are underlined. (B) Structure models of chitinase: XP_020909717.1 is shown in green, XP_02092061.1 is in red, AAM57182.1 is yellow, AAE83140.1 is represented in gray, AAE83141.1 is blue, and CHIA_RAT is in purple. The comparison of the Cα chains presented RMSD values that oscillate between 0.127 and 0.616.

Figure 7

Peroxiredoxin-6 (Prx-6) alignment. (A) Alignment of the amino acid sequences of the putative Prx-6 from A. dowii (Ad_Prx6 (Transcriptome: c26896_g1_i1)) with Prx-6 from chickens (UniProtKB: PRDX6_CHICK), humans (UniProtKB: PRDX6_HUMAN), and from the sea anemone Actinia tenebrosa (REFSEQ: XP_031570487.1). The region covered by the tryptic peptides obtained in the A. dowii proteome is presented on a magenta background. On a black background with white letters, the residues related to Phospholipase A2 activity and peroxidative cysteine are highlighted. (B) Structure models of PRDX: Ad_Prx6 is gray, XP_031570487.1 is purple, PRDX6_CHICK is green, and PRDX6_HUMAN is blue. The comparison of the Cα chains presented RMSD values that oscillate between 0.032 and 0.342.

Figure 8

Superoxide dismutase [Mn] alignment. (A) Multiple alignment of the amino acid sequence of the putative mitochondrial superoxide dismutase from A. dowii (Ad_SOD [Mn] (Transcriptome: c27625_g1_i1)) with other human SOD2s (UniProtKB: SODM_HUMAN, GenBank: AWU17515.1 and AAE36440.1) and from pig-tailed macaques (UniProtKB: SODM_MACNE). The region covered by the tryptic peptides obtained from the proteomes of A. dowii and L. neglecta is presented on a magenta background. On a black background with white letters, the residues related to the binding to manganese are highlighted. The region corresponding to the transit peptide is underlined and the region of the mature protein is indicated in bold. (B) Structural models of SOD. Ad_SOD [Mn] is red, AWU17515.1 is blue, AAE36440.1 is gray, SODM_MACNE 551 is purple, and SODM_HUMAN is green. The comparison of the Cα chains presented RMSD values that oscillate between 0.014 and 0.536.

Figure 9

Hsp 70 kDa. (A) Multiple alignments of the amino acid sequence of putative A. dowii Hsp 70 (Ad_Hsp70 (Transcriptome: c27026_g1_i1)) with other proteins of the same family. In the alignment, the sequences Hsp70 from insects (UniProtKB: HSP7D_MANSE), from humans (UniProtKB: HSP72_HUMAN), from mice (UniProtKB: HSP7C_MOUSE), and several patented Hsp70s for different applications (GenBank: ACQ4089780.1, A QTV59613.1, QRJ96032.1, QKO41990.1). The region covered by the tryptic peptides obtained from the proteomes of A. dowii and L. neglecta is highlighted by a magenta background. The ATP binding site is underlined and the regions involved in the binding of nucleotide phosphates are highlighted with white letters on a black background. (B) Structure models of heat shock proteins: Ad_Hsp70 is green, HSP7D_MANSE is medium blue, ACQ40951.1 is blue, HSP7C_MOUSE is forest green, AAC89780.1 is gray, HSP72_HUMAN is pink, QTV59613.1 is yellow, QRJ96032.1 is red, and QKO41990.1 is purple. The comparison of the Cα chains presented RMSD values that oscillate between 0.007 and 0.77.

Figure 10

Serpin alignment. (A) Multiple alignment of the amino acid sequence of the putative serine protease inhibitor (Ad_Serpin (Transcriptome: c26903_g1_i1)) with the serpins of humans (UniProtKB: SPB6_HUMAN) and mice (UniProtKB: SPB6_MOUSE), and the elastase inhibitor identified in frogs (UniProtKB: ILEU_XENTR). The region covered by the tryptic peptides obtained from the A. dowii and L. neglecta proteomes is highlighted by a magenta background. (B) Structural models of protease inhibitors: Ad_Serpin is blue, ILEU_XENTR is gray, SPB6_MOUSE is green, and SPB6_HUMAN is purple. The comparison of the Cα chains presented RMSD values that oscillate between 0.361 and 0.863.