Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERß2 and are functionally modulated by estrogens.

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERß2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERß2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17ß-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

Identification of two IgD+ B cell populations in channel catfish, Ictalurus punctatus.

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

BOB.1 of the channel catfish, Ictalurus punctatus: not a transcriptional coactivator?

Expression of the immunoglobulin heavy chain (IGH) locus of the channel catfish (Ictalurus punctatus) is driven by the Emu3' enhancer, whose core region contains two octamer motifs and a muE5 site. Orthologues of the Oct1 and Oct2 transcription factors have been cloned in the channel catfish and shown to bind to the octamer motifs within the core enhancer. While catfish Oct2 is an activator of transcription, catfish Oct1 failed to drive transcription and may act as a negative regulator of IGH transcription. In mammals, the Oct co-activator BOB.1 (B cell Oct-binding protein1, also known as OCA-B and OBF-1) greatly enhances the transcriptional activity of Oct factors and plays an important role in the development of the immune system. An orthologue of BOB.1 has been cloned in the catfish, and its function characterized. The POU binding domain of the catfish BOB.1 was found to be 95% identical at the amino acid level with the binding domain of human BOB.1, and all the residues directly involved in binding to the Oct-DNA complex were conserved. Despite this conservation, catfish BOB.1 failed to enhance transcriptional activation mediated by endogenous or co-transfected catfish Oct2, and failed to rescue the activity of the inactive catfish Oct1. Electrophoretic mobility shift assays showed that catfish BOB.1 was capable of binding both catfish Oct1 and Oct2 when they formed a complex with the Oct motif. Analysis of recombinant chimeric catfish and human BOB.1 proteins demonstrated that the failure to drive transcription was due to the lack of a functional activation domain within the catfish BOB.1.

Identification of Igsigma and Iglambda in channel catfish, Ictalurus punctatus, and Iglambda in Atlantic cod, Gadus morhua.

Immunoglobulin light (IGL) chain genes encoding sigma and lambda from channel catfish, Ictalurus punctatus, and lambda from Atlantic cod, Gadus morhua, were identified by mining of expressed sequence tag databases, 5'-RACE and RT-PCR protocols. cDNAs for each of these IGL chains encode typical variable (V), joining (J), and constant (C) regions and Southern blot analyses and genomic sequencing show that genes encoding these isotypes, like other teleost IGL genes, are found in a cluster organization of one or two V gene segments, followed by single J and C gene segments, all in the same transcriptional orientation. However, unlike the teleost kappa genes, genes encoding catfish sigma and lambda are few in number and the two isotypes are each encoded by only two clusters. Similarly, Atlantic cod lambda genes are predicted to be encoded by two or three clusters. As expected, sequence and phylogenetic analyses comparisons demonstrate that catfish Vsigma and Csigma genes are most similar to Vsigma and Csigma genes of other ectothermic vertebrates. Although catfish and Atlantic cod Vlambda genes cluster with other vertebrate Vlambda genes, their Clambda sequences cluster in a distinct group separate from other vertebrate IGL C sequences. However, support for classifying these sequences as lambda, is their V and J recombination signal sequence (RSS) organization. The catfish and Atlantic cod genes have typical lambda-like RSS with the Vlambda RSS consisting of heptamer-23 bp spacer-nonamer and the Jlambda RSS consisting of heptamer-12 bp spacer-nonamer. This is the first report demonstrating the presence of Iglambda in teleosts.

Function of E-protein dimers expressed in catfish lymphocytes.

E-proteins are essential class I bHLH transcription factors that play a role in lymphocyte development. In catfish lymphocytes the predominant E-proteins expressed are CFEB (a homologue of HEB) and E2A1, which both strongly drive transcription. In this study the role of homodimerization versus heterodimerization in the function of these catfish E-proteins was addressed through the use of expression constructs encoding forced dimers. Constructs expressing homo- and heterodimers were transfected into catfish B cells and shown to drive transcription from the catfish IGH enhancer. Expression from an artificial promoter containing a trimer of muE5 motifs clearly demonstrated that the homodimer of E2A1 drove transcription more strongly (by a factor of 10-25) than the CFEB homodimer in catfish B and T cells, while the heterodimer showed intermediate levels of transcriptional activation. Both CFEB1 and E2A1 proteins dimerized in vitro, and the heterodimer CFEB1-E2A1 was shown to bind the canonical muE5 motif.

B cell receptor accessory molecules in the channel catfish, Ictalurus punctatus.

B cell receptor (BCR) accessory molecules CD79a and CD79b homologs were identified in the channel catfish, Ictalurus punctatus. Both are found as single copy genes that encode proteins containing a signal peptide, an extracellular immunoglobulin domain, a transmembrane region and a cytoplasmic tail containing an immune-receptor tyrosine-dased activation motif (ITAM). IpCD79a and IpCD79b transcripts correlate well with IgM message expression. They are highly expressed in peripheral blood leukocytes (PBL) enriched in membrane (m) IgM+ cells and catfish clonal B cell lines, but not in catfish clonal T cells, indicating that IpCD79a and IpCD79b expression is B cell restricted. Studies using catfish clonal B cells (3B11) transfected with constructs encoding epitope-tagged IpCD79a and IpCD79b revealed that IpCD79a was expressed as a 45 kDa protein and IpCD79b was expressed as a 32 kDa protein. Furthermore, co-immunoprecipitations of epitope-tagged CD79 proteins demonstrate that these molecules are non-covalently associated with mIgM. These data correlate with some of the previous immunoprecipitation data demonstrating that catfish mIgM associates with proteins of 45 and 32 kDa.

Interaction between E-protein and Oct transcription factors in the function of the catfish IGH enhancer.

Transcriptional control of the immunoglobulin heavy chain (IGH) locus in the channel catfish, Ictalurus punctatus, is incompletely understood. It is, however, known that 2 variant octamer motifs and a microE5 motif in the core region of the enhancer (Emicro3') are important in driving transcription, and it has been suggested that interaction between transcription factors (Oct factors and E-proteins) bound to these sites contributes to enhancer function. In this study, the functional relationships between the microE5 motif, the proximal octamer motif, and the factors that bind them have been examined. The results of mutational analysis of these motifs showed that their interaction is important to driving transcription from the enhancer. Furthermore, the catfish Oct transcription factors were capable of a physical interaction with the catfish E-proteins. These results support a role for interaction between transcription factors bound to the octamer and microE5 motifs in the function of the Emicro3' enhancer.

Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies.

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.

Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: a negative regulator of immunoglobulin gene transcription?

BACKGROUND: The enhancer (Emu3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding muE5 site. An orthologue to the Oct2 transcription factor has previously been cloned in catfish and is a functionally active transcription factor. This study was undertaken to clone and characterize the Oct1 transcription factor, which has also been shown to be important in driving immunoglobulin gene transcription in mammals. RESULTS: An orthologue of Oct1, a POU family transcription factor, was cloned from a catfish macrophage cDNA library. The inferred amino acid sequence of the catfish Oct1, when aligned with other vertebrate Oct1 sequences, revealed clear conservation of structure, with the POU specific subdomain of catfish Oct1 showing 96% identity to that of mouse Oct1. Expression of Oct1 was observed in clonal T and B cell lines and in all tissues examined. Catfish Oct1, when transfected into both mammalian (mouse) and catfish B cell lines, unexpectedly failed to drive transcription from three different octamer-containing reporter constructs. These contained a trimer of octamer motifs, a fish VH promoter, and the core region of the catfish Emu3' IGH enhancer, respectively. This failure of catfish Oct1 to drive transcription was not rescued by human BOB.1, a co-activator of Oct transcription factors that stimulates transcription driven by catfish Oct2. When co-transfected with catfish Oct2, Oct1 reduced Oct2 driven transcriptional activation. Electrophoretic mobility shift assays showed that catfish Oct1 (native or expressed in vitro) bound both consensus and variant octamer motifs. Putative N- and C-terminal activation domains of Oct1, when fused to a Gal4 DNA binding domain and co-transfected with Gal4-dependent reporter constructs were transcriptionally inactive, which may be due in part to a lack of residues associated with activation domain function. CONCLUSION: An orthologue to mammalian Oct1 has been found in the catfish. It is similar to mammalian Oct1 in structure and expression. However, these results indicate that the physiological functions of catfish Oct1 differ from those of mammalian Oct1 and include negative regulation of transcription.

Channel catfish, Ictalurus punctatus, CD4-like molecules.

Two CD4-like (CD4L) molecules have been identified in channel catfish, Ictalurus punctatus. Although phylogenetically related to other vertebrate CD4 molecules, they exhibit only 19% amino acid identity to each other. IpCD4L-1 encodes a predicted protein containing four immunoglobulin domains, a transmembrane region and a cytoplasmic tail containing a p56(lck) binding site. In contrast, IpCD4L-2 encodes for a similar protein with three immunoglobulin domains. The gene organization of IpCD4L-1 is very similar to that of other vertebrate CD4 genes, while the genomic organization of IpCD4L-2 is different. Southern blots indicate both catfish molecules are likely single copy genes and mapping studies show that both are found on a single Bacterial Artificial Chromosome suggesting close linkage. At the message level, IpCD4L-1 and -2 are expressed in various catfish lymphoid tissues and in non-B-cell peripheral blood leukocytes (PBL). Both messages are upregulated in concanavalin A (ConA) and alloantigen stimulated PBL, but not in lipopolysaccharide (LPS)-stimulated cultures. In contrast, they are differentially expressed among the catfish clonal T cell lines. While both molecules appear to be T cell specific, their functional significance in catfish is unknown.

Regulation of the immunoglobulin heavy chain locus expression at the phylogenetic level of a bony fish: transcription factor interaction with two variant octamer motifs.

Transcriptional control of the IGH locus in teleosts is not fully understood, but evidence from catfish and zebrafish indicates major roles for octamer-binding (Oct) and E-protein transcription factors. A pair of variant octamer motifs in the Emu3' enhancer of the catfish has been shown to be particularly important in driving expression, justifying detailed study of their function. These octamer motifs were examined to determine if they bound Oct2 POU domains in monomeric or dimeric (PORE and MORE) configurations. While catfish Oct2 was shown to be capable of binding PORE and MORE motifs in dimeric conformation, the two octamer motifs in Emu3' bound Oct2 POU domains only in monomeric configuration. Catfish Oct2, when bound in this monomeric conformation, was shown to bend the DNA helix. The relative position of the two octamer motifs in Emu3' affected the activity of the enhancer, and moving the octamer motifs closer together by 5 bp greatly reduced the activity of the enhancer. This effect was not due to steric hindrance preventing the binding of Oct transcription factors to the two motifs, but rather was shown to be due to the disruption of an additional transcription factor binding site lying between the two octamer motifs.

Channel catfish immunoglobulins: repertoire and expression.

The channel catfish, Ictalurus punctatus, is widely recognized as an important model for studying immune responses in ectothermic vertebrates. It is one of the few fish species for which defined viable in vitro culture systems have been established and is currently the only fish species from which a variety of functionally distinct clonal leukocyte lines are available. Moreover, there is a large basis of biochemical and molecular information on the structure and function of catfish immunoglobulins (Igs). Catfish, as other teleosts, have a tetrameric homolog of IgM as their predominant serum Ig plus a homolog of IgD. They also have genetic elements basically similar to those of mammals, which encode and regulate their expression. The catfish Ig heavy (H) chain locus is a translocon-type locus with three Igdelta genes linked to an Igmu gene or pseudogene. The catfish IgH locus is estimated to contain approximately 200 variable (V) region genes representing 13 families as well as at least three diversity (D) and 11 joining (JH) genes. The catfish has two light (L) chain isotypes, F and G, both encoded by loci organized in multiple cassettes of VL-JL-CL with the VL in the opposite transcriptional orientation. Hence, all requisite components for encoding antibodies are present in the catfish, albeit with certain variations. In the future, whether or not additional unique features of Ig function and expression will be found remains to be determined.

Evolution of vertebrate E protein transcription factors: comparative analysis of the E protein gene family in Takifugu rubripes and humans.

E proteins are essential for B lymphocyte development and function, including immunoglobulin (Ig) gene rearrangement and expression. Previous studies of B cells in the channel catfish (Ictalurus punctatus) identified E protein homologs that are capable of binding the muE5 motif and driving a strong transcriptional response. There are three E protein genes in mammals, HEB (TCF12), E2A (TCF3), and E2-2 (TCF4). The major expressed E proteins found in catfish B cells are homologs of HEB and of E2A. Here we sought to define the complete family of E protein genes in a teleost fish, Takifugu rubripes, taking advantage of the completed genome sequence. The catfish CFEB (HEB homolog) sequence identified homologous E-protein-encoding sequences in five scaffolds in the Takifugu genome database. Detailed comparative analysis with the human genome revealed the presence of five E protein homologs in Takifugu. Single genes orthologous to HEB and to E2-2 were identified. In contrast, two members of the E2A gene family were identified in Takifugu; one of these shows the alternative processing of transcripts that identifies it as the ortholog of the E12/E47-encoding mammalian E2A gene, whereas the second Takifugu E2A gene has no predicted alternative splice products. A novel fifth E protein gene (EX) was identified in Takifugu. Phylogenetic analysis revealed four E protein branches among vertebrates: EX, E2A, HEB, and E2-2.

MHC RFLP analyses in channel catfish full-sibling families: identification of the role of MHC molecules in spontaneous allogeneic cytotoxic responses.

Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.

Channel catfish NK-like cells are armed with IgM via a putative FcmicroR.

Two-color flow cytometry demonstrated that 4-8% of channel catfish PBL are positive for both F and G IgL chain isotypes, suggesting that they passively acquire serum IgM via a putative FcmicroR. These cells show spontaneous killing toward allogeneic targets, and in vitro stimulation of PBL with allogeneic cells results in an increase of double IgL chain positive cells with a concomitant increase in nonspecific cytotoxicity. Long-term cultures of alloantigen-stimulated PBL contain both sIgM(+) and sIgM(-) cytotoxic cells that transcribe message for the catfish homolog of the FcepsilonR gamma chain, but not for Igmicro and TCR-alpha,-beta, or -gamma chains. Immunoprecipitation of lysates from sIgM(+) NK-like cells with anti-IgM co-immunoprecipitated a putative FcmicroR of approximately 64 kDa. Finally, removal of IgM from sIgM(+) NK-like cells and replacement with anti-hapten antibody enabled antibody-armed effectors to kill haptenated targets that were refractory to killing by effectors armed with normal IgM. This is the first report suggesting that teleost NK-like cells express a putative FcmicroR which participates in antibody-dependent cell-mediated cytotoxicity.

Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus).

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

Channel catfish cytotoxic cells: a mini-review.

The use of allogeneic and autologous lymphoid cell lines has facilitated studies of cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cells in channel catfish. Naïve catfish leukocytes were shown to spontaneously kill allogeneic cells and virally-infected autologous cells without the need for prior sensitization, and allogeneic cytotoxic responses were greatly enhanced by in vitro alloantigen stimulation. Both catfish CTL and NK-like cells have been successfully cloned from these alloantigen-stimulated cultures, and represent the first cytotoxic cell lines derived from any ectothermic vertebrate. These cloned cytotoxic cells contain granules and likely induce apoptosis in sensitive targets via a putative perforin/granzyme mechanism. In addition, some catfish CTL clones may also kill targets by an additional mechanism, possibly by Fas/FasL-like interactions. Importantly, these cytotoxic cells do not express the marker for catfish nonspecific cytotoxic cells (NCCs), and thus represent cell types distinct from NCCs. The use of monoclonal antibodies against the catfish F and G immunoglobulin light chain isotypes revealed the presence of a putative Fc receptor for IgM (Fc mu R) on some catfish NK-like cells that appears to 'arm' these cells with surface IgM. In addition, a potentially important monoclonal antibody (CC41) developed against catfish NK-like cells was found to recognize an approximately 150kDa molecule on the surface of catfish cytotoxic cells. These studies clearly demonstrate that catfish possess an array of different cytotoxic cells. The availability of various cloned cytotoxic cell lines should enable unambiguous functional studies to be performed in ways not currently possible with any other fish species.

Expression of alternatively spliced CD45 isoforms by channel catfish clonal T and B cells is dependent on activation state of the cell and regulated by protein synthesis and degradation.

In mammals, expression of the three alternatively spliced exons of the tyrosine phosphatase CD45 is regulated by the developmental and activation state of the cell. In comparison, the channel catfish, Ictalurus punctatus, CD45 homolog contains 18 functional alternatively spliced exons. Since very little is known about CD45 regulation in ectothermic vertebrates, this study examines the regulation of catfish CD45 mRNA isoform expression in clonal T and B cells in response to stimulation. Results show that mitogenic stimulation using catfish serum or concanavalin A induced expression of mRNAs for small CD45 isoforms, and isoform message expression was growth curve dependent, i.e. cells in logarithmic phase express message for smaller CD45 isoforms, whereas stationary phase cells express message for longer CD45 isoforms. In addition, cells treated with the protein synthesis inhibitor cycloheximide expressed message for longer CD45 isoforms, and treatment with lactacystin, which blocks protein degradation, rescued smaller isoform message expression. Collectively these data suggested that expression of CD45 isoforms, in catfish, at least at the mRNA level, is "constitutively dynamic" and highly dependent on extracellular stimuli.

Channel catfish soluble FcmuR binds conserved linear epitopes present on Cmu3 and Cmu4.

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcmuR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cmu3 and Cmu4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cmu3 and Cmu4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igmu), chicken (Ignu), mouse (Igmu, Iggamma1, Iggamma2a, Iggamma2b, and Igalpha), rabbit (Igmu and Iggamma) and goat (Iggamma) IgH chains, and mouse Igkappa and Iglambda, and chicken Iglambda IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.