Original Chinese medicinal centipedes include S. mutilans, S. multidens, S. mojiangica, and S. negrocapitis (Wang et al., 1997). Here, we studied the novel substitutional pharmaceutical centipede, S. mojiangica, with comparative analysis of active molecules. Scolopendra mojiangica showed a relatively close relationship to S. negrocapitis, S. mutilans, and S. multidens (Figure 1A), though a smaller body size than S. mutilans, S. dehaani, and S. multidens. Similar to other species, it also uses venom to attack prey and in defense.
The protocol for isolating venom glands from S. mojiangica was described in our previous study (Liu et al., 2012). Healthy adult centipedes (n=280) without injury were selected, and the venom glands were dissected from their first pair of limbs. After that, 3 V AC was used to stimulate the venom gland and ensure that more toxins were included, so that proteome coverage could be improved. The isolated venom glands were then further processed (Figure 1B). A portion of each sample was used to obtain the proteome by SDS-PAGE analysis. Protein bands from the venom gland were excised for in-gel digestion and subjected to ESI-MS/MS analysis. The remaining portion of each sample was used to extract RNA, followed by RNA-Seq analysis of the transcriptome.
A total of 246 proteins were identified in S. mojiangica at 95% coverage by ESI-MS/MS analysis (Supplementary Table S1; Figure 2A). In the proteome, 73.6% of proteins (n=181) were cellular components and 19.1% of proteins (n=47) were unknown functional proteins, which were putative venom toxins. Only 18 proteins were identified as toxin-like proteins, including neurotoxins, K+ channel inhibitors, and blarina toxins (Figure 2B; Table 1). Although we obtained more proteins in S. mojiangica than in S. mutilans and S. viridis with proteomic analysis, the detected toxin-like proteins in S. mojiangica represented a lower percentage of total proteins than those identified in S. mutilans in our previous study (Figure 2C). In the venom proteome, most of the identified proteins showed a molecular weight of less than 50 kDa, similar to the proteome of S. mutilans (Figure 2D). Thus, the centipedes contained notably small functional molecules for potential pharmaceutical use, as expected. Based on peptide detection, 23.2% of proteins consisted of six or more unique peptides (Supplementary Figure S2). In addition, the more enriched the peptides assembled into proteins, the more comprehensive was the proteome obtained.
Sequence ID GenBank accession No. Sequence description Category Peptides E-Value MW (kD) Calc. pI FPKM ScoMo_singlet48841 AT0003236 Blarina toxin precursor (EC 3.4.21.-) Blarina toxin 9 1.00E-37 21.61 4.15 92.54 ScoMo_singlet50899 AT0003766 Mucrofibrase-5 precursor (EC 3.4.21.-) Mucrofibrase-5 11 4.00E-16 14.40 9.93 3 454.74 ScoMo_singlet71394 AT0002263 Pseudechetoxin-like protein precursor Pseudechetoxin 276 9.00E-42 28.74 9.86 7 195.57 ScoMo_contig2076 gi|429840589 K+ channel inhibitor Channel inhibitor 617 4.00E-164 62.76 9.15 1.37 ScoMo_singlet78309 AT0000117 Latisemin precursor Latisemin 412 2.00E-22 20.89 7.96 0.00 ScoMo_contig4762 AT0003236 Blarina toxin precursor (EC 3.4.21.-) Blarina toxin 108 1.00E-44 28.58 6.5 15 173.32 ScoMo_singlet45908 AT0003741 Thrombin-like enzyme contortrixobin (EC 3.4.21.-) Serine proteinase 109 1.00E-41 44.94 5.08 1 685.57 ScoMo_singlet67462 AT0000120 Pseudecin precursor Pseudechetoxin 66 5.00E-32 23.71 8.91 14 111.58 ScoMo_singlet72573 AT0000552 Hopsarin-D (EC 188.8.131.52) Hopsarin-D 93 1.00E-121 85.15 6.53 132.70 ScoMo_singlet76606 AT0000554 Trocarin precursor (EC 184.108.40.206) Trocarin 38 3.00E-138 84.92 6.17 60.34 ScoMo_singlet25641 AT0000552 Hopsarin-D (EC 220.127.116.11) Hopsarin-D 46 5.00E-20 27.21 4.6 184.53 ScoMo_singlet69905 AT0000554 Trocarin precursor (EC 18.104.22.168) Trocarin 14 4.00E-107 40.69 5.28 1 245.366 ScoMo_singlet57737 AT0003404 Zinc metalloproteinase fibrolase (EC 22.214.171.124) Metalloproteinase 20 4.00E-16 35.21 8.13 48.71 ScoMo_singlet8256 AT0000762 Alpha-latrocrustotoxin Alpha-latrocrustotoxin 10 0 50.48 6.79 136.27 ScoMo_singlet68890 AT0000552 Hopsarin-D (EC 126.96.36.199) Hopsarin-D 13 5.00E-75 42.03 7.88 161.84 ScoMo_singlet7846 gi|392295725 Omega-slptx-ssm2a neurotoxin precursor Neurotoxin 11 8.00E-36 8.56 4.93 16 647.01 ScoMo_singlet55496 gi|501293796 Cathepsin L Cathepsin L 180 1.00E-155 37.30 6.35 2.83 ScoMo_singlet39956 AT0000554 Trocarin precursor (EC 188.8.131.52) Trocarin 12 4E-09 4.64 3.79 5.55 MW: Molecular Weight; Calc. pI: The calculated isoelectric point (pI); FPKM: Fragments Per Kilobase of exon model per Million mapped fragments.
Table 1. Toxin-like proteins/peptides identified from venom proteome of S. mojiangica centipede
We acquired 43 381 437 clean reads assembled into 132 597 contigs from the venom gland using the Trinity program. As a result, the transcriptome data consisted of 107 642 putative gene objects (all unigenes) ranging from 101 bp to 9 184 bp, with an average length of 423 bp. The number of unigenes larger than 500 bp was 24 219. The largest unigenes were 9 184 bp in size, and the N50 of the unigenes was 214 bp (Supplementary Figure S3 and Table S2).
For comparative analysis, the venom gland transcriptome from S. mojiangica showed many transcripts (n=46 571) with high similarity to those of S. mutilans. Notably, however, most transcripts showed low similarity between the two centipede species (Figure 3A). In the transcriptomic expression analysis, the read count of each transcript in S. mojiangica and S. mutilans showed biases for gene expression, with higher expressed transcripts in S. mojiangica (Figure 3B). Functional annotation analyses of these transcripts were combined with Blast searching and phylogenetic analyses to obtain toxin-like unigenes. In total, 410 toxin-like transcripts were identified in the transcriptome of S. mojiangica, more than that identified in S. mutilans (342 transcripts). Furthermore, these transcripts were divided into 34 categories, mainly consisting of alpha-latrocrustotoxin, delta-latroinsectotoxin, ion channel inhibitors, and alpha-latrotoxin (Figure 3C).
As expected, we identified 34 kinds of toxin-like unigenes (n=342) from the transcriptome of S. mutilans using the same annotation method as that of S. mojiangica (Figure 4A). In total, 11 of these toxin-like unigenes encoded the most transcripts in the two centipedes. With gene expression analyses, most toxin-like unigenes showed no differential expression between S. mojiangica and S. mutilans, except for four toxin-like unigenes (i.e., alpha-latrotoxin, hopsarin-D, metalloproteinase, and trocarin) (Figure 4B).
Figure 4. Comparison of toxin-like molecules distributed in centipedes S. mojiangica and S. mutilans
Finally, we determined the toxicity and performed crude isolation of the centipede venom. The crude centipede venom exhibited strong insecticidal action (Figure 5A), and the crude venom had a similar potency as the venom of S. mutilans. The crude venom and its fractions eluted from the S-100HR column (Supplementary Figure S1; Figure 5B) showed hemolytic activity. The elution of peak 1 (P1) showed high hemolytic activity on human RBCs when 1 mg/mL protein/peptide was incubated for 4 h. In contrast, peaks 3, 5, and 6 (P3, P5, and P6) had lower hemolytic activity than that of P1 and crude venom.
Phylogeny of scolopendrid centipedes and isolation of venom gland
Proteomic analysis of venom components
Transcriptomic analysis of venom components
Comparative determination of centipede toxins
|ZR-2019-163 Supplementary Tables and Figures.zip|