A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG

Abstract

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Figure 1

IgM_1gj immunoprecipitated from neonatal plasma, neonatal spleen and epigonal organ, and adult epigonal organ has a structure similar to mammalian IgG. (A) Radio-iodinated IgM_1gj in plasma exists as monomers (lower IgM_1gj band in lane 3) and dimers (upper IgM_1gj band in lane 3) in nonreduced 5% PAGE. Under reducing conditions in 9% PAGE, the H_1gj M_r is ≈55 kDa, associated covalently with an L chain (see text and supplementary Fig. 5). The 80-kDa H chain is derived from 19S and 7S IgM. Lanes 1, NK11 mAb (negative control), lanes 2, CB16 mAb (IgM-specific but noncrossreactive on IgM_1gj), lanes 3, GA15 (IgM-specific and crossreactive on IgM_1gj). (B) Neonatal spleen and epigonal organ cells preferentially secrete IgM_1gj. Cells were metabolically labeled with ³⁵S met/cys, and secreted IgM and IgM_1gj were immunoprecipitated with the cross-reactive GA15, GA16, and LK14 mAbs and run on reducing 9% reducing PAGE. (C) Adult epigonal organ and spleen cells were metabolically labeled with ³⁵S met/cys, and secreted IgM_1gj was immunoprecipitated and run in 9% reducing PAGE. Relative to IgM, IgM_1gj is maintained at high levels in the epigonal organ. Note that GA15, GA16, and LK14 mAbs recognize both IgM_1gj and IgM, whereas the other IgM-specific mAbs do not cross-react. Additionally, putative IgW was immunoprecipitated from the epigonal organ supernatants by using the LK14 and GA16 mAbs (14).

Figure 2

IgM_1gj is germline joined, and the C_H2 domain exon has been deleted from the H_1gj gene cluster. (A) Alignment of IgM_1gj with conventional nurse shark IgM (Accession no. M92851). The CDRs in the V_H domain are boxed. Note that the short CDR3 uses only one D gene (22). The C_H1 cys used for L chain association in IgM is substituted by asn (N) in H_1gj (bolded, asterisk). The ancestral IgM C_H2 domain is missing. Putative C_H glycosylation sites are boxed–an additional glycosylation site occurs in C_H3 (H_1gjC_H2) that is not found in conventional IgM. The arrowheads at the beginning of the V and C_H1 domains and at the end of the J and C_H1 regions denote the primers used for amplification of exon-specific probes. (B) Cartoon comparing the V and C domains of IgM and IgM_1gj H chains. The ancestral IgM C_H2 domain is missing in H_1gj. Putative glycosylation sites (black lollipops) and interchain disulfide bonds (—) are noted. (C) IgM_1gj germline V-D-J join. The VDJ junction nucleotide sequence aligned to a fully sequenced horn shark Ig cluster (ref. , see Fig. 2D) suggests the presence of only one D gene, D2, in the join. D2 nucleotides matching to the horn shark D2 segment have one underline (see Fig. 2D); D2 nucleotides possibly derived from a P-nucleotide addition are double underlined (we propose that the GA dinucleotide was part of the original D2 region.) (D) Coding ends and RSS (heptamers and nonamers bolded) of rearranging segments (V, D2, and J) of a representative horned shark variable region gene (ref. ; accession no. X13447). If a similar gene gave rise to the IgM_1gj join (Fig. 2C), a cleavage would have occurred between the last base of the V and D2 3′ coding ends and the heptamers (bold), and two bases (italics) would have been trimmed from the J coding end. In the C legend, we describe a potential scenario for processing of the IgM_1gj 5′ D2 junction.

Figure 3

The IgM_1gj gene cluster is single copy. Southern blot analysis of V_H1gj and C_H1-1gj genes under high stringency conditions reveals only one band with four different enzymes. A canonical IgM V_H probe hybridizes to the V_H-1gj gene only under low stringency conditions. The white diamond shapes show the positions of V_H-1gj in the three V_H blots. B, BamHI; E1, EcoRI; EV, EcoRV; H, HindIII.

Figure 4

IgM_1gj is expressed in neonatal spleen and epigonal organ and perpetuated in adult epigonal organ. (A) Northern blot with neonatal and adult tissue RNA showing relative expression of IgM_1gj, bona fide IgM, RAG1, and positive control nucleotide diphosphate kinase (NDPK). IgM_1gj is highly expressed in the spleen and epigonal organ of newborn pups and maintained in adult epigonal organ. Canonical IgM RNA in the newborn spleen (exposed 80 h) is at much lower levels than in the adult (exposed 17 h) and is enriched for transmembrane (Tm) over secretory (Sec) Ig. RAG1 is expressed in the thymus and epigonal organ throughout life. (B) Northern blot by using mRNA from 1-day-old (1), 1-week-old (2), and 41-day-old (3) pups. The same probes in Fig. 4A were used, except TdT was included.