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

Abstract

Introduction: The evolution of adaptive immunity in Camelidae resulted in the concurrent expression of classic heterotetrameric and unconventional homodimeric heavy chain-only IgG antibodies. Heavy chain-only IgG bears a single variable domain and lacks the constant heavy (C_H) γ1 domain required for pairing with the light chain. It has not been reported whether this distinctive feature of IgG is also observed in the IgA isotype.

Methods: Gene-specific primers were used to generate an IgA heavy chain cDNA library derived from RNA extracted from the dromedary's third eyelid where isolated lymphoid follicles and plasma cells abound at inductive and effector sites, respectively.

Results: Majority of the cDNA clones revealed 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 C_Hα1 domain. In a few clones, however, the cDNA of the canonical IgA heavy chain was amplified which included the C_Hα1 domain, analogous to C_Hγ1 domain in IgG1 subclass. Moreover, we noticed a short, proline-rich hinge, and, at the N-terminal end of the C_Hα3 domain, a unique, camelid-specific pentapeptide of undetermined function, designated as the inter-α region. Immunoblots using rabbit anti-camel IgA antibodies raised against C_Hα2 and C_Hα3 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 the third eyelid, trachea, small and large intestine. In contrast, the leporine anti-C_Hα1 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-C_Hα domain antibodies confirmed the expression of both variants of IgA heavy chains in plasma cells in the third eyelid's lacrimal gland, conjunctiva, tracheal and intestinal mucosa.

Conclusion: We found that in the dromedary, the IgA isotype has expanded the immunoglobulin repertoire by co-expressing unconventional and canonical IgA heavy chains, comparable to the IgG class, thus underscoring the crucial role of heavy chain-only antibodies not only in circulation but also at the mucosal frontiers.

Keywords: Arabian camel immunoglobulin A; IgA variable heavy heavy (VHH) domain; heavy chain-only IgA; mucosal immunity; third eyelid; unconventional IgA heavy chain.

Figure 1

Histomorphology of conjunctiva-associated lymphoid tissue and the Harderian gland (HG) in the third eyelid of the dromedary. In (A), posterior (bulbar) to the HG, numerous isolated lymphoid follicles (ILF) beneath subepithelial domes (*) and the specialized follicle-associated epithelium (arrow) of the conjunctiva represent the inductive site of mucosal B cell maturation. In (B), numerous plasma cells (arrows) cluster between the acini of the HG and form the principal effector site of the mucosal humoral immune response. Asterisks (*) mark the lumen of acini. Magnifications and scale bar lengths are as follows: in (A) 10x and 100 μm, respectively; in (B), 100x and 10 μm, respectively. Sections were stained with H&E.

Figure 2

Rapid amplification of 5’ and 3’ cDNA ends (RACE) of dromedary IgA heavy chain and primary structure of classic and unconventional IgA heavy chain. In (A), PCR products of 5’ RACE, and in (B), 3’ RACE are shown after agarose gel electrophoresis, with a DNA ladder loaded in the first well. The majority of 5’ PCR species were ~800 bp, a few were ~1.1 kbp. In contrast, 3’ RACE PCR species consisted uniformly of ~450 bp. There were some minor variations in the length of the ~800 kbp 5’ PCR products, reflecting the heterogeneity of CDR in V_HH domains. The difference of ~300 bp among the two 5’ PCR species, however, was explained by identification of the C_Hα1 domain in classic IgA heavy chain. In (C), two representative amino acid sequences of dromedary IgA heavy chains chosen from our library were aligned, with conventional IgA heavy chain (IgA classic) at the top and unconventional IgA heavy chain (IgA HC) at the bottom. Variable domains of both heavy chains (V_H and V_HH), preceded by a 19-mer leader peptide, comprised four framework regions (FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3), whereas constant domains of classic IgA heavy chain encompassed three (C_Hα1, C_Hα2 and C_Hα3), in contrast to only two domains (C_Hα2 and C_Hα3) in the unconventional format. A short hinge region, consisting of valine and six consecutive prolines, was located between C_Hα1 and C_Hα2 domains in conventional IgA heavy chain, in contrast to the hinge region in heavy chain-only IgA which separated FR4 of the V_HH domain from the C_Hα2 domain. The tailpiece (Tp) represented the C-terminal end of all IgA heavy chains variants. Dashes indicate deficient single amino acid residues as well as lack of the entire C_Hα1 domain in heavy chain-only IgA. Definition of the constituents of the variable and constant domains (marked by alternating normal font and bold letters) was accomplished according to IMGT^® delimitations (42) and the numbering scheme of human IgA1 and IgA2 chains, respectively (43, 44). Underlined residues in FR1 and FR2 indicate amino acid changes considered critical for conformation and solubility of the V_HH domain of heavy chain-only IgA. Further downstream, a unique pentapeptide (SEDAI) marked the N-terminus of the C_Hα3 domain.

Figure 3

Amino acid sequences of the C_Hα2-C_Hα3 boundary in mammals. Amino acid sequences at the 3’ terminal end of C_Hα2 and the 5’ terminus of C_Hα3 domains of the dromedary, Bactrian camel and alpaca were compared with those of other mammals. Names in Latin are to the left. The gap between C_Hα2 and C_Hα3 domains is deliberate. A distinct pentapeptide (bold letters) consisting of serine (leucine in alpaca), glutamic acid, aspartic acid, alanine (proline in alpaca) and isoleucine was only found in camelids, upstream of a highly conserved arginine followed by an invariant proline at the N-terminus of the C_Hα3 domain. Glutamic acid at the 5’ end of C_Hα3 was also present in gelada baboon, horse, donkey, guinea pig, red squirrel, and California grey whale, whereas aspartic acid at this position was identified only in the greater bamboo lemur. In contrast, the most frequently encountered motif in the interdomain region of other mammals at the N-terminal end of the C_Hα3 domain was a tetrapeptide consisting of glycine, asparagine, threonine (or methionine, isoleucine, and valine) and phenylalanine.

Figure 4

IgA heavy chain variants in the intestine, trachea, third eyelid, milk, and serum. One-humped camel proteins (~20 μg) from small (lane 1) and large intestine (lane 2), trachea (lane 3), third eyelid (lane 4), milk (lane 5), female (lane 6) and male (lane 7) serum and human male serum (lane 8) were separated under reducing conditions by 10% (w/v) SDS-PAGE, transferred to nitrocellulose membranes, probed with anti-C_Hα1 (A), anti-C_Hα2 (B), anti-C_Hα3 (C), and anti-IAR (D) antibodies, and visualized with HRP-conjugated anti-rabbit goat antibodies. In (A), the anti-C_Hα1 antibody reacted with a ~60 kDa species, indicating the expression of canonical IgA heavy chains, with cross-reactivity to two protein species of ~60 kDa and ~65 kDa in human serum (lane 8). In camel milk (lane 5) and sera (lanes 6 and 7), an additional prominent protein species of ~45 kDa was identified by the anti-C_Hα1, but not by other antibodies, most likely representing a proteolytic fragment of canonical IgA heavy chain. In (B–D), two protein bands of ~52 kDa and ~60 kDa of comparable intensity were identified in tissues (lanes 1, 2, 3 and 4), but not in milk (lane 5) nor serum (lanes 6 and lane 7) where the higher molecular weight species corresponding to canonical IgA heavy chains prevailed. Level of IgA expression in the trachea (lane 3) appeared low, likely reflecting lower plasma cell density in the mucosa of the upper respiratory tract. Cross-reactivity of circulating human IgA species was observed with all anti-C_H domain antibodies, except the camel-specific anti-IAR antibody. Molecular weight markers in kDa are indicated to the left of each panel.

Figure 5

Expression of IgA heavy chain variants in the Harderian gland. Immunohistochemistry was performed on a section through the lacrimal (Harderian) gland (HG) of the third eyelid of the dromedary. Anti-C_Hα1 domain antibodies were used in (B, E) and anti-C_Hα2 domain antibodies in (C) and (F) and visualized by goat anti-rabbit HRP conjugated antibodies and hydrogen peroxide/DAB as substrates. The anti-C_Hα1 antibody detected canonical IgA heavy chains, whereas canonical and unconventional IgA heavy chains were both reactive to the anti-C_Hα2 antibody. In (A), no IgA was identified with secondary antibodies only. In (B, C), immunoreactivity was observed in the periacinar spaces and within epithelial cells throughout the lobule with anti-C_Hα1 and anti-C_Hα2 antibodies, respectively. In (E, F), two rectangular areas selected in (B, C) show, at higher power, intense immunostaining primarily in plasma cells in the periacinar spaces (horizontal arrows), and at the luminal apices of epithelial cells (short vertical arrows). In (D), an H&E-stained section demonstrates an agglomerate of typical plasma cells (horizontal arrows) and red blood cells within capillaries (V) adjacent to the basal membranes of acini. Magnifications and scale bar lengths were as follows: in (A–C), 20x and 50μm, respectively; in (D), 100x and 10 μm, respectively; in (E, F), 60x and 20μm, respectively.

Figure 6

Expression of IgA heavy chain variants in MALT of the third eyelid and trachea. Immunostaining was performed on sections through the conjunctiva of the third eyelid (top panels) and on sections through the trachea (bottom panels) using antibodies raised against C_Hα1 domain in (A, D), C_Hα2 domain in (B, E), and IAR in (C, F), visualized by goat anti-rabbit HRP conjugated antibodies and hydrogen peroxide/DAB as substrates. The anti-C_Hα1 antibody detected canonical IgA heavy chain only. By contrast, both canonical and unconventional IgA heavy chains were identified by the anti-C_Hα2 as well as the anti-IAR antibody. In (A–C), IgA heavy chain immunoreactivity to all three antibodies was identified in plasma cells within the stratified epithelial layer of the conjunctiva and in the subconjunctival stroma (horizontal arrows) as well as within an isolated lymphoid follicle (ILF). IgA heavy chain immunoreactivity was also observed along the surface of the conjunctiva. In (D–F), IgA⁺ plasma cells (vertical arrows) were noticed alongside the basal membrane of a subepithelial mucous gland lined by columnar epithelium and in the subepithelial stroma of the pseudostratified tracheal epithelium. The lumen of the gland is marked by (*). Immunoreactivity was also present within the glandular epithelial layer, reflecting transcytosis. Of note, the brown color generated with the anti-C_Hα2 and the anti-IAR antibody was more intense. In this series of microphotographs, magnification and scale bar length was 40x and 20μm, respectively.

Figure 7

Histomorphology, expression of IgA heavy chains and tissue eosinophilia in the small intestine. In (A), villi and crypts characterized the mucosa of the small intestine, which was separated from the submucosa (SM) by the muscularis mucosae (MM). In (B), aggregates of submucosal lymphoid follicles with germinal centers (GC) surrounded by numerous lymphocytes, typical of Peyer’s patches, were present. In (C), details of crypts revealed interspersed goblet cells filled with mucin (G). In the adjacent lamina propria, numerous eosinophils (vertical arrows), plasma cells (horizontal arrows), histiocytes (H) and small vessels (V) were observed. Sections in (A–C) were stained with H&E. In (D), strong IgA heavy chain immunoreactivity to the anti-IAR antibody was detected in plasma cells (horizontal arrows) and within enterocytes while sparing mucin in goblet cells (G). Magnifications and scale bare lengths were as follows: in (A, B), 10x and 100μm, respectively; in (C), 100x and 10μm, respectively; in (D), 60x and 20μm, respectively.

Figure 8

Histomorphology and expression of IgA heavy chain variants in the large intestine. In (A, D), the hallmarks of colon mucosa are shown in H&E-stained sections. Upper row panels depict longitudinal, lower row panels transverse sections. Crypts were lined primarily by mucin-enriched epithelial cells (goblet cells) and surrounded by lamina propria, where plasma cells (arrows) were found. In (B, E), plasma cells showed IgA heavy chain immunoreactivity to the anti-C_Hα1 (arrows), and in (C, F) to the anti-C_Hα2 antibody (yellow arrows). Immunoreactivity was also observed in the enterocytes within crypts, indicative of transcytosis. In (A), a capillary (V) was identified by the presence of red blood cells. In contrast to the lamina propria of the small intestine ( Figure 7C ) eosinophils were absent in this area of the colon. In all microphotographs magnification was 60x, and scale bar length was 20μm.