Divergent disulfide bond architecture defines two IgY subclasses in snakes

Immunoglobulin Y (IgY) is the major serum antibody in reptiles and birds and is evolutionarily related to mammalian IgG and IgE, which likely arose by duplication and divergence from an IgY-like ancestral gene. While IgY diversification has been documented in several reptilian lineages, the structural basis underlying subclass divergence remains poorly understood. Here, I present a phylogenetic and structural analysis of IgY sequences from 20 snake species, revealing two distinct evolutionary lineages (A and B) that arose through gene duplication. Structural modeling of the constant regions from Arizona elegans uncovered a difference in the light chain-heavy chain (CL-CH1) inter-chain disulfide bond architecture between lineages: lineage B uses the CH1 cysteine at alignment column 13 (IMGT CH1-13) for the CL-CH1 disulfide linkage, whereas lineage A uses the CH1 cysteine at alignment column 99 (IMGT CH1-123), indicating alternative positioning of the light-chain attachment site within CH1. Analysis of 50 lineage-diagnostic amino-acid sites between lineages showed that changes are distributed across all constant domains (CH1-CH4), with 13 sites displaying radical substitutions affecting charge or polarity. Sliding-window dN/dS analysis indicated predominant purifying selection across both lineages, consistent with functional constraint following duplication. Together, these findings provide structural evidence consistent with subfunctionalization of snake IgY genes and suggest that alternative disulfide-bond configurations may confer distinct biophysical or functional properties to each antibody subclass.