Green Turtles (Chelonia mydas) Have Novel Asymmetrical Antibodies

Abstract

Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain–only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.

Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida

“…Cell-mediated immunity in Hawaiian (9) and Floridian (8) green turtles declines with an increase in the tumor score, so both populations would presumably have equivalent susceptibility to tumor formation. Finally, evidence at the protein expression level suggests that 7S IgY and 5.7S IgY expression is identical in turtles from both regions (40).…”

“…Inherent differences in immune systems between Floridian turtles and Hawaiian turtles also do not explain the differences in the serological responses to ChHV5. The role of different IgYs in green turtles is not fully understood, but 7S IgY responses arise earlier than 5.7S IgY responses in Hawaiian (40) and Floridian (41) green turtles experimentally exposed to antigens. The roles of these IgYs are better known in ducks, where 5.7S IgY is important in skin sensitization and dominates in chronic immunity (54), while increased ratios of 5.7S/7S IgY dampen the phagocytic response (55).…”

“…A key aspect of understanding and managing viral diseases in animals is the early detection of infection, ideally, in the form of a serological test that can detect exposure to the pathogen prior to the development of clinical signs. The generation of mono-clonal antibodies (MAbs) against the two major types of turtle immunoglobulins, 7S IgY and 5.7S IgY, which has a truncated Fc region (40,41), was a significant step in allowing the development of serological assays. There have since been two attempts to develop serological assays for the detection of antibodies against ChHV5.…”

Quantifying Circulating IgY Antibody Responses against Select Opportunistic Bacterial Pathogens and Correlations with Body Condition Factors in Wild American Alligators, Alligator mississippiensis

“…Strong banding of proteins under denaturing and reducing conditions was observed by SDS-PAGE, revealing a heavy chain of approximately 65 kDa, corresponding to the heavy chain of typical full length IgY. The IgY heavy chain eluted as a slight double band, corresponding to either degraded heavy chain under reducing conditions, or because of asymmetry in IgY as reported for green sea turtles [ 29 ]. Purified alligator IgY also contained a protein of approximately 34 kDa corresponding to the truncated isoform of IgY [ 37 ].…”

“…Previous work demonstrated that IgY can be purified from reptile serum using the immunoglobulin-binding protein-G from Streptococcal bacteria [ 29 , 30 ]. To purify IgY from alligator serum in this study, a Protein-G column (Thermo Fisher) was equilibrated by running 10 mL of 0.10 M Tris pH 8.0 through the columns followed by 10 mL of 0.01 M Tris pH 8.0.…”