A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex

Abstract

Three novel and closely related leukocyte immune-type receptors (IpLITR) have been identified in channel catfish (Ictalurus punctatus). These receptors belong to a large polymorphic and polygenic subset of the Ig superfamily with members located on at least three independently segregating loci. Like mammalian and avian innate immune regulatory receptors, IpLITRs have both putative inhibitory and stimulatory forms, with multiple types coexpressed in various lymphoid tissues and clonal leukocyte cell lines. IpLITRs have an unusual and novel relationship to mammalian and avian innate immune receptors: the membrane distal Ig domains of an individual IpLITR are related to fragment crystallizable receptors (FcRs) and FcR-like proteins, whereas the membrane proximal Ig domains are related to several leukocyte receptor complex encoded receptors. This unique composition of Ig domains within individual receptors supports the hypothesis that functionally and genomically distinct immune receptor families found in tetrapods may have evolved from such ancestral genes by duplication and recombination events. Furthermore, the discovery of a large heterogeneous family of immunoregulatory receptors in teleosts, reminiscent of amphibian, avian, and mammalian Ig-like receptors, suggests that complex innate immune receptor networks have been conserved during vertebrate evolution.

Fig. 1

Predicted amino acid sequence, domain comparisons, and schematic representation of IpLITR1, IpLITR2, and IpLITR3. a Alignment of the extracellular and b TM/CYT regions of IpLITR1, IpLITR2, and IpLITR3. Signal peptide (SP), and immunoglobulin (Ig) domains are labeled; cysteine residues predicted to be involved in intrachain disulfide bonds are marked with asterisks; gray shaded residues represent differences from IpLITR1 in a and differences from IpLITR2 in b. TMs are underlined, ITIM-like motifs are boxed, an overlapping ITSM within the CYT of IpLITR1 is indicated by a bracket, and TM charged residues are shaded black and marked (+). c Phylogenetic analysis of Ig domains in IpLITRs. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions and a scale bar are shown below the trees. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as boxes, N-linked glycosylation sites are marked as ballpoint lines. Individual IpLITR domains are shaded according to their relatedness between IpLITRs and percent amino acid identity with IpLITR1 Ig domains indicated to the left of IpLITR2 and IpLITR3. IpLITR2 D3 and IpLITR3 D5 and D6 are 15.2–39.3% identical to all IpLITR1 Ig domains

Fig. 2

Southern blot analyses. Genomic DNA from 16 sibling catfish was digested, separated, transferred to nylon membranes, and hybridized with a MHCIIβ, b IpLITR1 D1, c IpLITR1 D4, and d IpLITR3 D6-specific probes. Letters a–i indicate different segregation patterns (b′, e′, and h′ represent variant segregation patterns that may be the result of recombination). Representative RFLP bands illustrating different linkage groups are indicated by ●, ▲, and ■. Kilobase size markers are indicated to the left of each blot

Fig. 3

Northern blot and RT-PCR analyses of IpLITR tissue expression. Catfish LITR tissue expression was performed by Northern blot analysis using a an IpLITR1 D1-specific probe, and b an IpLITR2 D3-specific probe. Total RNA from pronephros (head kidney), mesonephros (trunk kidney), spleen, heart, liver, gill, and muscle were examined. Kilobase markers are on the left margin and arrows indicate the major hybridizing bands observed. RNA integrity and load levels were determined by hybridization using a catfish EF1α probe as a housekeeping gene. c RT-PCR analyses of IpLITR1 and IpLITR2 in various catfish tissues. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin

Fig. 4

a RT-PCR analyses of IpLITR1 and IpLITR2 expression in various catfish clonal cell lines and a polyclonal MLC. Total RNA was obtained from the catfish 42TA macrophages, 3B11 B cells, TS32.15 and TS32.17 nonautonomous cytotoxic T cells, autonomous G14D T cells, a MLC, and 1F3 NK-like cells. RT-PCR was performed using primers specific for IpLITR1, IpLITR2, and IpEF1α. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin. b Schematic representation of IpLITR-types identified by sequencing of RT-PCR products obtained using IpLITR1 and IpLITR2-specific primers. The originally identified prototype IpLITRs are boxed, and arrows indicate the relative positions of the primer pairs used in RT-PCR reactions. Sizes in base pairs corresponding to the bands observed in a are indicated above each schematic. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as hatched boxes and noncanonical immunotyrosine-based activation motif-like motifs shown as white boxes within the CYT. Individual Ig domains are shaded according to relatedness as described for Fig. 1, and percentages above each receptor represent percentage of amino acid identities of the predicted extracellular region of the IpLITR-like sequences when compared with the prototype IpLITRs

Fig. 5

Phylogenetic analyses reveal unique Ig domain composition within individual IpLITRs. The Ig domains compared are indicated by gray shading in the schematics to the left of each phylogenetic tree. a Comparisons of IpLITR, representative FcR and FCRL, and human LRC D1 and D2 sequences. b Comparisons of IpLITR1 D3 and D4 with representative FcR, FCRL, KIR, LILR, and PIRA1 D2 and D3 sequences, as well as NKp46 and CHIRA2 D1 and D2 sequences. c Comparison of IpLITR3 D5 and D6 with D1 and D2 of representative mammalian FcRs, FCRLs, and LILRs/KIRs. The accession numbers for the various Ig domain sequences used are: human (hu)FcγRI(CAI12557), huFcγRII (CAA35642), huFcγRIII (CAA36870), huFcεRI (AAH05912), huFCRL3 (AAH28933), huFCRL4 (AAK93970), huFCRL5 (NP_112571), huFcαR/CD89 (AAH27953), huLILRB1 (AAH15731), huLILRB2 (AAB87662), huKIR3DL1 (AAC83928), huKIR3DS1 (AAV32446), huSIRP (CAA71404), human NKp46 (AJ001383), mouse (mo)FcγRI (AAD34931), moFcγRII (AAA37608), moFcγRIII (NP_034318), moFcεRI (NP_034314), moFCRL3 (AAS91578), moNKp46 (AJ223765), rat (ra) FcγRIII (AAA42050), raPIRA1 (XP_341773), raNKp46 (AF082533), guinea pig (gp) FcγRII (D13692), macaque (mac)FcγRI (AAL92095), bovine (bo)FCRL5 (XP_595289), boKIR3DS1 (AAP33626), boSIRP (CAA71943) chimpanzee (cp)LILRA2 (NP_001009044), orangutan (or)KIR (AAM78473), monkey (mk)KIR3DL20 (AAU50562), CHIRA2 (CAG33731), catfish (Ip)NITR1 (AF397454), and IpNITR2 (AF397455). For all analyses, SIRPs and/or IpNITRs were included as outgroups to root the trees. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees

Fig. 6

IpLITR2 contains Ig domains related to both FcRs/FCRLs and CHIRs. a Amino acid alignment of IpLITR2 D1 and D2 sequences with representative mammalian FcRs and FCRLs. Accession numbers are as in Fig. 5. Gray shading: residues similar/identical to IpLITR2. Boxed residues: contacts for Ig Fc in huFcεRI. Black and gray arrows represent the predicted β-strands for IpLITR2 and huFcεRI, respectively. Hatched boxes indicate conserved cyteines and dashes represent gaps. b Phylogenetic analysis of IpLITR2 D1 and D3 sequences (gray shaded) compared with representative mammalian FcR sequences (as in Fig. 5) and D1 sequences of representative CHIRs. The accession numbers for the various Ig domain sequences used are: CHIRB2 (XP_422905), CHIRB1 (CAH55757), CHIRAB2 (CAG33733), CHIRA2 (CAG33731), CHIRB5 (AJ879908), CHIR A1 (AF306851), CHIRB4 (XP_428342), CHIRB6 (CAI53861), and CHIRAB3 (AJ879909). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees. c Alignment of IpLITR2 D3 and CHIR D1. Gray shading indicates residues that are similar/identical to IpLITR2, black and gray arrows represent predicted β-strands for IpLITR2 and CHIRA2, respectively; “dotted” lines indicate predicted helices and hatched boxes indicate conserved cyteines. Gaps in alignment are indicated by dashes

Fig. 7

Annotation of zebrafish LITR-like proteins. a Phylogenetic analysis of ZfLITR and IpLITR3 Ig domains. Ig domains were predicted using SMART (Letunic et al. 2004) and the amino acid sequences aligned using CLUSTALW (Thompson et al. 1997). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001). Only branch values >50 are shown, and the major Ig domain groups are designated by brackets and shaded according to their relatedness with IpLTR3 Ig domain. b Schematic representation of ZfLITR-like proteins compared with IpLITR3. Domains are shaded according to their phylogenetic relationship with IpLITR3 domains. Hatched bars on IpLITR3 and Zf XP_692267 indicate signal peptides, TM segments are solid black lines, and ITIMs are shown as gray boxes for XP_694305. Amino acid identity vs corresponding IpLITR3 Ig domains are indicated as percentage value above each domain, and only IpLITR3 D3, D4, D5, and D6 were related to the Ig domains of the representative ZfLITR analyzed, as indicated by the dashed box. Predicted size in amino acids for each molecule is indicated on the left