A novel multigene family encodes diversified variable regions

Abstract

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an approximately 113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.

Figure 1

(a) cDNA structures of SN193 and SN6A indicating functional boundaries. The relative scale is indicated. (b) Alignments of the putative V regions of NITR genes SN6A and SN193 with the V regions of: human TCRα (M13725), Xenopus CTX (XLU43330), nurse shark new antigen receptor (ECU18713), mouse VpreB (MMVPRE83), mouse V_H (AF045029), and human V_κ (PIR S46374). Residues shared by half or more of the sequences and/or by both NITR genes and one other sequence are enclosed. Alignments are based on clustal w (14) and visual optimization. Numbers over conserved amino acid residues correspond to International Immunogenetics Database (IMGT) designations (http://imgt.cnusc.fr:8104/). Sequence identity relationships to nucleotide and protein databases were based on blastn and blastx analyses, respectively. (c Upper) Alignment of the putative J region-located C-terminal of the V Ig domain of SN193 with the J regions of: human TCRα (PIR S57872); mouse TCRα (M64239); rat TCRα precursor (PIR F27579); and rat Igκ (M62828). Mammalian J regions were selected on the basis of the highest alignment scores (blastx) with SN193. (c Lower) Alignment of the putative J region-located C-terminal to the V/C2 Ig domain of SN6A with the same three mammalian prototypes, selected in the same manner as above. Identical residues shared between the NITR gene segments and the mammalian prototypes are enclosed. (d–f Upper) Northern blot analyses of poly(A)⁺ mRNA (1.5 μg per track) recovered from intestine, cranial kidney, and spleen hybridized with probes complementing S. nephelus: (d) IgL; (e) NITR cDNA SN6A V and V/C2 domains (NITR6A); and (f) TCRα. (d–f Lower) Following removal of bound probe, blots were rehybridized with a probe complementing Spheroides S26. Probes were labeled to equivalent specific activity. Exposure times were 7 days for IgL and TCRα, 18 days for NITR6A, and 2 days for S26. Reduced exposure and overexposure are entirely consistent with the patterns presented. An RNA size standard is indicated (1.35 kb). Equivalent expression patterns were obtained when referenced to the expression of β actin and when total RNA was used and referenced to 18S RNA.

Figure 2

(a) Contig of PACs containing NITR genes based on the overlap of end sequences and the complete nucleotide sequence of PAC 19B20. Clones are oriented 5′ to 3′ starting from PAC 54H22, relative to PAC 19B20. This orientation is based on partial sequences of other clones and is consistent with partial restriction analyses. (b) Organization of the NITR genes encoded in the PAC 19B20 insert. Each gene consists of five exons, encoding: (I) a (split) leader peptide; (II) putative extracellular V and V/C2 Ig domains; (III) transmembrane region; (IV) N-terminal cytoplasmic region; and (V) C-terminal cytoplasmic region. Genes are designated on the basis of the sequence families of the V (–13) and V/C2 (–5) Ig domains, respectively, as well as the presence (+) or absence (−) of an ITIM in exon V. For example, NITR 1 (6.2⁺) contains a type-6 V domain and type-2 V/C2 domain, and possesses an ITIM in exon V. fs, frameshift. (c) Variation in the organization of exons and introns in three NITR genes; classification is the same as in b.

Figure 3

The predicted peptide structures of the V, V/C2, TM, Cyt1, and Cyt2 of the 24 NITR genes encoded in PAC 19B20; pseudogenes 6 and 8 are not included. Genes are listed in an order that facilitates intergenic comparisons; vertical brackets in V and V/C2 enclose the 10 members of the SN6A type-1 gene family. ▵, Uncertainties with regard to the mature start site of NITR genes, referenced to gene 10; alternative start sites are predicted, as described (18). The basis for the designation of V and V/C2 domains in exon II is based largely on the homologous relationship of the NITR genes to Ig and TCR V domains (Fig. 1b), the shorter lengths of V/C2 domains and the relative locations of V/C2 exon/intron boundaries (▴) in two other species of bony fish, in which an intron separates the (two) extracellular Ig domains. The outlier genes (5, 9, and 10) are separated. Note the interspersed color bands and extensive regions of nonidentity in the three designated outliers, particularly in V/C2. For reference purposes, positions that are (highly) conserved between V_H, V_κ, V_λ, TCRVα, and TCRVβ are designated in one-letter code by using International Immunogenetics Database (IMGT) numbering (Fig. 1b legend); conserved cysteine is circled. The location (by reference to Ig/TCR designations) of sequence regions corresponding to CDR1 and CDR2 as well as the boundaries of the glycine bulge (J homology) regions, [(F)GXG], are shown by brackets above and below the alignments. Absolute identity is shown in red (up to one difference is allowed); substitutions that result in changes which retain functional groups, defined conservatively as: G or A; I, L, M, or V; K or R; S or T; F or Y; D or E; and N or Q, are shown in yellow. Recognized TM and Cyt1 families are designated by Roman numerals to the left of the alignments. ITIMs in Cyt2 are enclosed. Variation among the members of the SN6A type-1 gene family (enclosed in a vertical bracket) are shown in the continuous horizontal color bars below gene 26. Positions at which no variation occurs are shown in red; a single amino acid difference in one family member is shown in pink; one amino acid substitution in two or more family members is shown in orange; two or more different substitutions in multiple family members are shown in blue. The comparison is referenced to the predicted start site of gene 10.

Figure 4

Unrooted minimum phylogenetic trees comparing (a) V and (b) V/C2 domains of NITR genes. Trees are based on amino acid sequences by using the proportion of difference as an estimate of distance (15). The bar at the bottom indicates the number of amino acid changes per site. Positions containing gaps were excluded for pairwise distance estimates. Groups and subgroups are indicated by I or II and A or B, respectively. Numbers to the left of nodes indicate estimates of percentage probability that the length of the branch separating the adjacent cluster is greater than zero (i.e., that the cluster is statistically significant). Only values ≥70% are shown. Six statistically similar trees were obtained for the V domain and four for the V/C2 domain comparisons; however, the trees differed only in minor aspects of topology within the groups.