2.6.1. Metalloproteinases

Metalloproteinases are enzymes characterized by a catalytic zinc ion in its active site [25]. They have been described as the toxic components responsible for the induction of tissue damage, necrosis and hemorrhage [26,27] in various venoms [28–30]. Eleven metalloproteases with four subgroups—zinc metalloproteinase, matrix metalloproteinase (MMP) [31], A disintegrin and metalloproteinase domain-containing protein (ADAM) [32,33], and A disintegrin and metalloproteinase with thrombospondin motifs (ADMTMS) [34,35]—are predicted with their similarity 30.6–42.6% in mucus-enriched proteins of *A. coerulea* (Table 2). All three predicted zinc metalloproteinases display a relatively conserved astacin-like subfamily segmen<sup>t</sup> with a HEXXH or even more strict HEXXHXXGXXH zinc-binding site/active site [36,37]. A model metalloproteinase (pdb ID: 3LQB) is utilized to do the sequence alignment (Figure 6A) and 3D modeling (Figure 6B). With the exception of the highly conserved HEXXHXXGXXH motif, four parallel β-sheets and three α-helixes are also structurally similar. The differences include an initial coil and turn in all three sequences, an extra α-helix and a turn in 28DILKEVGS35 of TRINITY\_DN45838\_c0\_g1|m.27904, an α-helix in 90GPRCY95, a changeable area with an α-helix in 139RKYSHGQ145 of TRINITY\_DN44124\_c13\_g11|m.23323, a turn in 143PGGASTLGA152 of TRINITY\_DN35212\_c0\_g1|m.10040 and an α-helix and turn in TRINITY\_DN45838\_c0\_g1|m.27904, an extra coil between 169 and 173 of all three predicted sequences, and a turn in 169PEIT183 of TRINITY\_DN45838\_c0\_g1|m.27904. Interestingly, two sequences—TRINITY\_DN45838\_c0\_g1|m.27904 and TRINITY\_DN44124\_c13\_g11|m.23323—aligned to the same metalloproteinase, nas-13, display a large molecular evolutional distance, and are close to XP 022799178.1 from the coral *Stylophora pistillata* and AAX09930.1 from the same jellyfish *A. coerulea*, respectively. Meanwhile, the remaining sequence, TRINITY\_DN35212\_c0\_g1|m.10040, is evolutionally close to a sequence JAC85096 from *Clytia hemisphaerica* (Figure 6C).

**Figure 6.** Sequence alignment, 3D modeling and phylogenetic analysis of putative zinc metalloproteinases from *A. coerulea*. (**A**) Three putative sequences TRINITY\_DN45838\_c0\_g1|m.27904, TRINITY\_DN44124\_c13\_g11|m.23323, and TRINITY\_DN35212\_c0\_g1|m.10040 in mucus-enriched proteins are aligned with a model metalloproteinase (pdb ID: 3LQB). At the bottom of columns, asterisks (\*) show conserved positions, colons (:) show conserved substitutions and points (.) show non-conserved substitutions. Grey line, green bend, blue banded arrowhead and red solenoid represent coil, turn, sheet and helix, respectively. Different fragments are framed by red lines. (**B**) 3D modeling was simulated using the template metalloproteinase (pdb ID: 3LQB) by SWISS-MODEL and viewed by Discovery Studio 4.5. The colors grey, green, blue and red represent coils, turns, sheets and helices, respectively. Different fragments are indicated by red arrows. (**C**) Phylogenetic tree constructed using three putative zinc metalloproteinases and 11 other sequences from different species using MEGA 7 with the Neighbor-Joining method.

### 2.6.2. Serine Protease Inhibitors

Serine protease inhibitors or serpins [38] are the largest and most diverse superfamily of protease inhibitors [39], with inhibitory or non-inhibitory functions in blood coagulation, fibrinolysis, host defense, and impairment of motility of human glioblastoma cells [29,40,41]. They have widely been found in the venoms of many poisonous animals such as sea anemones, snakes, scorpions, spiders, anurans and hymenopterans [42]. Four sequences have been successfully scanned with their similarities identified at 36.6–56.4% to mucus-enriched proteins of *A. coerulea* (Table 2). Similar to zinc metalloproteinases, serpins are more similar in spatial structure than amino acid sequences simulated with a model serpin (pdb ID: 5CDZ) (Figure 7A,B). Two clusters of β-sheets are distributed in the central and tail areas, whereas 11 α-helixes are scattered in the periphery of proteins. The main structural variations contain an extended loop in 86HPNDPLEP93 of TRINITY\_DN29892\_c0\_g1|m.6537, 82KHTAD86 of TRINITY\_DN45322\_c11\_g3|m.26476 and 63DLKEDIRGSSAFGFVGSEAALE84 of TRINITY\_DN9397\_c0\_g3|m.37796, an extended coil and turn in 367FPSLRFDE374 of TRINITY\_DN29892\_c0\_g1|m.6537, and an extended coil in 368RCAIPIPLV376 of TRINITY\_DN9397\_c0\_g3|m.37796. Phylogenetic analysis shows that TRINITY\_DN45322\_c11\_g3|m.26476 and TRINITY\_DN29892\_c0\_g1|m.6537 are close to AHC98669 in *Rhipicephalus microplus* while TRINITY\_DN9397\_c0\_g3|m.37796 is near XP\_015915455 in *Parasteatoda tepidariorum* (Figure 7C).

**Figure 7.** Sequence alignment, 3D modeling and phylogenetic analysis of the putative serpins from *A. coerulea*. (**A**) Three putative sequences TRINITY\_DN45322\_c11\_g3|m.26476, TRINITY\_DN9397\_c0\_g3|m.37796, and TRINITY\_DN29892\_c0\_g1|m.6537 in mucus-enriched proteins are aligned with a model serpin (pdb ID: 5CDZ). At the bottom of columns, asterisks (\*) show conserved positions, colons (:) show conserved substitutions and points (.) show non-conserved substitutions. Grey line, green bend, blue banded arrowhead and red solenoid represent coil, turn, sheet and helix, respectively. Different fragments are framed by red lines. (**B**) 3D modeling was simulated using the template serpin (pdb ID: 5CDZ) by SWISS-MODEL and viewed by Discovery Studio 4.5. The colors grey, green, blue and red represent coils, turns, sheets and helices, respectively. Different fragments are indicated by red arrows. (**C**) Phylogenetic tree constructed by three putative serpins and 10 other sequences from different species using MEGA 7 with the Neighbor-Joining method.
