The immunoglobulin A isotype of the Arabian camel (Camelus dromedarius) preserves the dualistic structure of unconventional single-domain and canonical heavy chains

Abstract

AbstractEvolutionary processes of adaptive immunity inCamelidaeand some cartilaginous fishes resulted in the concurrent expression of classic heavy-light chain heterotetrameric antibodies together with unconventional heavy chain-only homodimeric antibodies of the IgG class. Heavy chain-only IgG bears a single variable domain and lacks the constant heavy (CH) γ1 domain required for the covalent pairing with the light chain. It has so far not been reported whether this distinctive feature of IgG is also observed in the IgA isotype. Therefore, gene-specific primers were used to generate a library of IgA heavy chain cDNA derived from RNA extracted from the third eyelid of the dromedary where isolated lymphoid follicles and plasma cells abound at inductive and effector sites, respectively. Majority of the cDNA clones revealed the hallmarks of heavy chain-only antibodies,i.e.camelid-specific amino acid substitutions in framework region 1 and 2, broad length distribution of complementarity determining region 3, and the absence of the CHα1 domain. In a few clones, however, the cDNA of canonical IgA heavy chain was amplified which included the CHα1 domain, analogous to CHγ1 domain in dromedary IgG1 subclass. Moreover, we noticed a short, proline-rich hinge, similar to that in human IgA2, and, at the N-terminal end of the CHα3 domain, a unique, camelid-specific pentapeptide consisting of serine, glutamic acid, aspartic acid, alanine or threonine, and isoleucine, of undetermined function, designated as the inter-α region. Immunoblots using rabbit anti-camel IgA antibodies raised against CHα2 and CHα3 domains as well as the inter-α region revealed the expression of a ∼52 kDa and a ∼60 kDa IgA species, corresponding to unconventional and canonical IgA heavy chain, respectively, in tissue extracts of the third eyelid,trachea, small and large intestine. In contrast, the leporine anti-CHα1 antibody detected canonical, but not unconventional IgA heavy chain, in all the examined tissues, milk, and serum, in addition to another hitherto unexplored species of ∼45 kDa in milk and serum. Immunohistology using anti-CHα domain antibodies confirmed the expression of both variants of IgA heavy chains in plasma cells in the third eyelid’s lacrimal gland, and in thelamina propriaofconjunctiva, bronchial and intestinalmucosa. We conclude that in the dromedary, the IgA isotype has expanded the immunoglobulin repertoire by co-expressing single-domain unconventional as well as canonical IgA heavy chains, comparable to the IgG class, thus underscoring the crucial role of heavy chain-only antibodies in immune defense not only in circulation but also at the mucosal frontiers.