*6.1. Homology of CST in Mammals*

Sequence alignment of CST in 53 mammalian species belonging to eight orders revealed >80% homology in 52 species, except in Platypus (lowest in the mammalian phylogenetic tree) where the homology with the primates (highest in the mammalian phylogenetic tree) was >58% (Figure 7), indicating that CST is highly conserved in mammals. The homology of individual amino acids is summarized in Figure 8. Aromatic amino acids such as phenylalanine, tyrosine and tryptophan are reported to exhibit a rigid, planar structure and possess added stability due to the π-electron cloud situated above and below the plane of the aromatic ring [131–133]. Therefore, F7, Y12, F14 conserved residues in CST can undergo aromatic–aromatic interactions such as hydrogen bonding coupled with attractive, non-covalent, dipole, and van der Walls interactions, and also pi-stacking of the benzene rings [134–137]. These interactions, in turn, can stabilize the overall structure of CST, as reported earlier for other proteins [138–141]. Analysis of the energetics of protein analyses revealed that the packing of non-polar groups in the protein interior is favorable owing to the favorable enthalpy of van der Walls interactions [142]. Therefore, it is reasonable to assume that van der Walls interactions of the aromatic amino acids (F7, Y12, F14) in association with van der Walls interactions of apolar (L5, G18) amino acids provided a global stability for CST [143,144]. In the course of evolution, with the change in interacting partners across species, we see a significant reduction in the conservation of charged residue. Interestingly, for the maintenance of structural framework, a 100% conservation of hydrophobic amino acid is maintained across the species through the mammalian evolutionary ladder.

#### *6.2. Single Nucleotide Polymorphisms (SNPs) in the CST Domain of Mammals*

Four non-synonymous SNPs have been identified in CST domain of CgA: Gly364Ser (US, Indian, and Japanese populations) [22,145,146], Gly367Val (only in Indian populations) [145], Pro370Leu (US and Indian populations) [22], and Arg374Gln (US populations only) [22]. Pro370Leu-CST has the highest potency of inhibiting catecholamine secretion and desensitizing catecholamine secretion, followed by WT-CST and Gly364-Ser-CST [22]. As a sharp contrast to catecholamine secretion [22], Gly364Ser was reported to be two-times more effective than Pro370Leu in exerting antibacterial activities [21].


**Figure 8.** Homology of the individual amino acid in catestatin sequence in 53 mammalian species belonging to seven orders.

#### **7. Conclusions**

(i) High conservation of CST in mammals: Alignment of CST sequences from 53 mammalian species belonging to eight orders revealed that CST sequence is highly conserved (>90% in 90% species) in mammals: Five (~24%) amino acids (M3, L5, F7, F14, and G18) are 100% conserved; nine (~43%) amino acids (S2, K4, S6, R8, R10, R15, P17, Q20 and L21) are 90–96% conserved; and three (~14%) amino acids (A9, A11, and G16) are >80% conserved. The least conserved sequences are G13 (>66%) and P19 (>58%), where human variants of CST were reported for G13 (G13S) and P19 (P19L), indicating that natural selection pressures still exist on those two amino acids [147–150].

(ii) CST as an immunomodulatory peptide: Existing literature (expression of CST in innate immune cells [21,98,127,151], inhibition of macrophage infiltration in tissues [97–99], decreased expression of pro-inflammatory cytokines by CST [97,98], and low plasma CST in fatal COVID-19 patients [129]) implicate CST as an immunomodulatory peptide.

(iii) CST as an antimicrobial peptide: Prominent effects of CST in the low micromolar range on inhibition of growth of Gram-positive and Gram-negative bacteria, fungi, and yeast establish CST as an antimicrobial peptide [21].

(iv) D-bCST1–15 as a potential therapy for microbial infection: D-bCST1–15 could be used as a monotherapy or as a combination therapy with cefotaxime, amoxicillin, and methicillin against the "superbugs" because it has more effective antibacterial activity compared to L-bCST1–15, penetration through the bacterial cell wall, resistance to bacterial proteases, undetectable susceptibility to resistance, and potentiation/synergic action of commonly prescribed antibiotics [23].

(v) CST as a cell permeable peptide: Penetration of CST (pI 12.03–12.48) in bacteria, fungus, yeast, and neutrophils [21,152], coupled with 70–75% homology with cell penetrating peptide Penetratin (pI 12.62), rightfully qualify CST as a cell permeable peptide.

(vi) Gut microbiome-mediated improvement in insulin sensitivity by CST: The increased ratio of Bacilotta to Bacteroidota, together with low levels of Verrucomicrobiota (e.g., *Akkermansia* spp.) in CST-KO mice [100], not only explains insulin resistance in CST-KO mice [97] but also implicates that CST is necessary for the maintenance of insulin sensitivity. A decreased ratio of *Bacilotta* to *Bacteroidota* coupled with increased abundance of *Verrucomicrobiota* after supplementation of CST-KO mice with CST [100] confirm that CST is necessary and sufficient to increase insulin sensitivity by modulating gut microbiota. Decreased population of *Akkermansia* and increased population of Proteobacteria in WTFMT-CST-KO coupled with increased population of butyrate producing *Intestimonas* in CST-KOFMT-WT [109] further substantiates regulation of obesity and insulin resistance by CST [97] via regulation of gut microbial population [100,109].

(vii) Improvement in antimicrobial effect of CST by cyclization: Based on the existing literature [39,41], we propose that cyclization of CST would markedly improve the antibacterial activity of CST.

**Author Contributions:** S.K.M. conceived the idea and wrote major portion of the manuscript. S.M. made all the figures. S.J., S.M., S.D., S.C. and S.K.M. performed literature search, interpreted data, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grants from the National Institutes of Health (1 R21 AG072487-01 and 1 R21 AG080246-01 to S.K.M.). S.J. is supported by AFTD Holloway Postdoctoral Fellowship (Award #2020-02).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** S.K.M. is a co-inventor of a patent on CST regulation of obesity.
