Evolution of g-type lysozymes in metazoa: insights into immunity and digestive adaptations

Abstract

Exploring the evolutionary dynamics of lysozymes is critical for advancing our knowledge of adaptations in immune and digestive systems. Here, we characterize the distribution of a unique class of lysozymes known as g-type, which hydrolyze key components of bacterial cell walls. Notably, ctenophores, and choanoflagellates (the sister group of Metazoa), lack g-type lysozymes. We reveal a mosaic distribution of these genes, particularly within lophotrochozoans/spiralians, suggesting the horizontal gene transfer events from predatory myxobacteria played a role in their acquisition, enabling specialized dietary and defensive adaptations. We further identify two major groups of g-type lysozymes based on their widespread distribution in gastropods. Despite their sequence diversity, these lysozymes maintain conserved structural integrity that is crucial for enzymatic activity, underscoring independent evolutionary pathways where g-type lysozymes have developed functionalities typically associated with different lysozyme types in other species. Specifically, using Aplysia californica as a reference species, we identified three distinct g-type lysozyme genes: two are expressed in organs linked to both feeding and defense, and the third exhibits broader distribution, likely associated with immune functions. These findings advance our understanding of the evolutionary dynamics shaping the recruitment and mosaic functional diversification of these enzymes across metazoans, offering new insights into ecological physiology and physiological evolution as emerging fields.

Keywords: Aplysia californica; ctenophores; gene retention; horizontal gene transfer; innate immunity; placozoa; porifera; spiralia.

FIGURE 1

Phylogenetic Tree Illustrating the Mosaic Distribution of g-Type Lysozyme Genes Across Metazoans. This phylogenetic tree depicts the evolutionary mosaic distribution of g-type lysozyme genes across animal taxa, with gene names displayed alongside their respective genus and species name and the taxonomic group they belong to. Bootstrap values below 80% have been excluded for clarity. Lysozymes from predatory myxobacteria, highlighted in black, exhibit strong sequence similarities with metazoan g-type lysozyme genes, suggesting potential horizontal gene transfer events. The tree distinguishes two groups of lysozymes: g-type1, representing more ancestral genes found in basal species of gastropods, and g-type2, which appears to have arisen from more recent gene duplication events and is primarily found in the more recently evolved Euthyneura group, including species like Elysia spp., Biomphalaria spp., Physella acuta, and the coenogastropod Littorina saxatilis

FIGURE 2

Comparative Analysis of g-Type Lysozyme Genes Across Diverse Species. (A) Multiple Sequence Alignment of g-type lysozyme genes from representative species, including humans, the soft coral Dendronephthya gigantea, one of the g-type lysozyme genes from the Aplysia californica genome, and a g-type lysozyme gene from the placozoan Trichoplax adhaerens. The alignment, enhanced by the secondary structure predictions from JPRED (Drozdetskiy et al., 2015), reveals alpha-helices marked by red bars and beta-sheets denoted by green and yellow arrows. These structural elements are essential for the enzyme’s lytic function, highlighting the evolutionary conservation despite sequence variability. Catalytic residues crucial to enzymatic activity are highlighted with stars above the alignment, emphasizing their conserved placement across species. (B) The 3D structural models of these proteins, generated by AlphaFold2 (Jumper et al., 2021), confirm the structural similarities in the regions noted in the sequence alignment. Their lysozymes also exhibit highly similar structural folds. The preservation is especially pronounced in the catalytic residues, Glutamic and Aspartic acids, which are crucial for the enzyme’s function (Malcolm et al., 1989). The consistent arrangement of these residues across representatives of different taxa underscores the structural fidelity of these proteins. It reflects evolutionary pressures to preserve key functional aspects of lysozymes crucial for antimicrobial defense. (C) Multiple sequence alignment of g-type lysozymes from a variety of species, focusing on the catalytic residues (Glutamic and Aspartic acids); these residues are marked with stars. Notably, deviations such as the non-conservation of these residues in the human Hs_LYG1 gene and two genes from Corticium candelabrum are indicated with black arrows. Additionally, a unique substitution in the gene Lg_LYZg3, where Tyrosine replaces Aspartic acid, is also highlighted, capturing the evolutionary dynamics of these residues.

FIGURE 3

Gene Expression Profiles of Aplysia californica g-Type Lysozyme Genes Across Various Contexts. This figure illustrates the expression profiles of g-type lysozyme genes from Aplysia californica across multiple biological contexts. (A) Tissue-specific expression patterns of LYZg1 and LYZg3, normalized to Transcripts Per Million (TPM). (B) Tissue-specific expression patterns of LYZg2, highlighting its activity in immune-related tissues. (C) Developmental stage-specific expression patterns of LYZg1 and LYZg3, showing notable peaks during early post-metamorphic stages. (D) Expression levels of LYZg2 across developmental stages, indicating significant increases during post-metamorphic transitions. (E) Expression profiles of LYZg1, LYZg2, and LYZg3 in individual neurons, emphasizing their roles in neural function. All expression data are normalized to TPM. (See Supplementary Material for additional details).