Characterization of natural antigen-specific antibodies from naïve sturgeon serum

Highlights

• The sturgeon natural serum antibodies bound to various antigens.

• The binding activities were increased from 3 to 7 months after hatching.

• The antigen specific natural antibodies were fractionated and evaluated.

Abstract

In this study, we isolated and characterized natural antibodies found in serum samples from Bester sturgeon (Huso huso × Acipenser ruthenus). Natural antibodies specifically detected hen egg lysozyme (HEL), keyhole limpet hemocyanin (KLH), and several species of pathogenic bacteria. Interestingly, we detected no antibodies with similar specificity in serum samples from rainbow trout (Oncorhynchus mykiss) or from Japanese flounder (Paralichthys olivaceus). Binding capacity of the sturgeon natural serum antibodies increased slightly at 7 months compared to 3 months after hatching. Antigen-specific antibodies against KLH, Aeromonas hydrophila and Streptococcus iniae were affinity-fractionated from naive sera of Bester sturgeon; specific detection of the corresponding antigens was observed. We conclude that Bester sturgeon are capable of generating unique natural antibodies including those that are pathogen-specific.

Introduction

Antibodies (also known as immunoglobulins, or Igs) are molecules that bind specifically to a target antigen. Igs are comprised of one or more heterodimeric subunits composed of two heavy chains (IgH) and two light chains (IgL) (SchroederJr and Cavacini, 2010). Evolutionary analysis has suggested that the genes encoding the Igs emerged in jawed-vertebrates after divergence from their jawless counterparts. Igs are critical components of the host defense system as they contribute to three major immunological functions, specifically, opsonization, neutralization and complement activation (Flajnik, 2002). Igs bind to antigens via their variable regions which are located at the N-termini of the aforementioned IgH and IgL chains. The C-termini of these polypeptides include the constant regions; this region of IgH determines Ig isotype and the nature of their effector function (Flajnik, 2002). Ig isotypes identified in cartilaginous fish include IgM, IgW, and IgNAR (Dooley and Flajnik, 2006); those in teleost fish include IgM, IgD, and IgT/Z (Ye et al., 2013; Mashoof and Criscitiello, 2016). IgM is a major circulating antibody in all fish species at levels as low as 0.8 mg/mL to more than 10 mg/mL in serum (Solem and Stenvik, 2006). Interestingly IgM represents nearly 50% of the total serum protein in cartilaginous fish (Clem et al., 1967); IgM is also a major constituent of the serum proteins identified in the sturgeon (Adkison et al., 1996).

Sturgeon is phylogenetically located between cartilaginous and teleost fish (LeBreton et al., 2004); their Ig genes display features of both groups. The gene encoding the constant region in the immunoglobulin light chain of the Siberian sturgeon, Acipenser baerii, is similar to that of the cartilaginous fish, while the gene encoding the variable region is similar to that of the teleost group (Lundqvist et al., 1996). Similarly, the gene encoding the constant region of the heavy chain is similar to that reported for teleost fish, while that encoding the variable region (VH2) specifically of the IgH-V family is similar to the VH1 identified in the cartilaginous Nurse shark Ginglymostoma cirratum (Zhu et al., 2014). Homologs of Igλ from cartilaginous fish have been found among the immunoglobulin light chains of the Chinese sturgeon, Acipenser sinensis (Zhu et al., 2016). These commonalities may relate to the fact that the sturgeon may be among the most primitive of the ray-finned fish and as such they are phylogenetically closer to cartilaginous fish than are most of the other known teleost fish.

Nonetheless, similar to what has been observed in studies featuring other teleost fish, sturgeon species are capable of mounting specific antibody responses that can be detected within three months after antigen injection (Drennan et al., 2007). Nurse sharks also require a prolonged period of time in order to develop specific antibody responses to antigen immunization (Dooley and Flajnik, 2005). By contrast, Atlantic cod (Gadus morhua) have little to no capacity to generate antibody responses (Pilström et al., 2005); whole genome sequencing revealed that cod lack genes encoding MHC II, CD4 and the immunoglobulin invariant chain (Ii), which are all elements that are required for effective antigen-specific antibody production (Star et al., 2011). Similarly, the elephant shark (Callorhinchus milii) lacks the genes encoding CD4 as well as most transcription factors (Venkatesh et al., 2014).

By contrast, cartilaginous fish and sturgeon generate natural antibodies which bind to a diverse array of antigens (Gonzalez et al., 1988); these antibodies are likely to be critical features of their respective biological defense mechanisms. However, the specificity of natural antibodies associated with sturgeon populations remains unclear. In this study, we evaluated the binding specificities of natural antibodies identified in sera from hybrid Bester sturgeon (Huso huso × Acipenser ruthenus) both in mature adults and at different stages of growth. We isolated unique antigen-specific antibodies from serum samples and evaluated their binding specificities.