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IgA Fc receptors in cattle and horses
Veterinary Immunology and Immunopathology 108 (2005) 139–143 www.elsevier.com/locate/vetimm
IgA Fc receptors in cattle and horses H. Craig Morton * Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, Rikshospitalet University Hospital, N-0027 Oslo, Norway
Abstract The biological role of IgA depends, at least partly, on the interaction with specific receptors (FcaRs) on the surface of leukocytes. The human FcaR, CD89, was the first IgA Fc receptor to be identified, and binding of IgA-coated particles to CD89 triggers numerous cellular effector functions including phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and release of inflammatory mediators. Recently, CD89 orthologs have been identified in a number of other species, including cows and horses. This brief review will summarize our current knowledge regarding the structure and function of bovine and equine CD89. # 2005 Elsevier B.V. All rights reserved. Keywords: Immunoglobulin A; Fc receptor; CD89; Bovine; Equine
1. Introduction Receptors for the Fc regions of immunoglobulin (Ig) molecules (Fc receptors, FcRs) are expressed by many cell types, especially phagocytes. Ligation of FcRs by Ig-coated targets can trigger numerous cellular effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and secretion of cytokines and other inflammatory mediators (Raghavan and Bjorkman, 1996; Daeron, 1997; Ravetch, 1997). Thus, FcRs provide a crucial link between the humoral and cellular arms of the immune system. The FcRs for IgG (FcgRs) and IgE (FceRI) from humans and mice are the best characterized, and data * Tel.: +47 23 07 14 89; fax: +47 23 07 15 11. E-mail address: [email protected].
from murine models has shown that these FcRs are important for triggering inflammatory reactions and also for protection against infectious microorganisms and parasites. However, animal models have also revealed that inappropriate inflammation triggered by FcRs can also contribute to the generation and potentiation of some allergic and autoimmune diseases (Hogarth, 2002; Takai, 2002; Gould et al., 2003). Homologs of many of these FcgRs and FceRs have also been identified in a number of livestock species, and readers are referred to an excellent recent review for further information (Kacskovics, 2004). FcRs for IgA (FcaRs) have been shown to be expressed on the surface of leukocytes from many different species, but until relatively recently only the human myeloid FcaR, CD89, has been molecularly characterized (Maliszewski et al., 1990). More recently, we have identified FcaRs with high
0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.07.008
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H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
Table 1 Accession numbers and references for all CD89 proteins described till now Species
Accession no. Reference
Human Chimpanzee Rhesus macaque Cynomolgus macaque Cow Horse Rat
X54150 BK005386a AY386684 AY386690 AY247821 AY587560 AB109767
Maliszewski et al. (1990) Morton et al. (2005) Rogers et al. (2004) Rogers et al. (2004) Morton et al. (2004a) Morton et al. (2005) Maruoka et al. (2004)
a
The chimpanzee CD89 sequence was deduced from draft genomic sequence and has not been confirmed at the cDNA level. This sequence is thus available from the Third Party Annotation Section of the DDJB/EMBL/GenBank databases.
homology to human CD89 in cattle (Morton et al., 2004a) and horses (Morton et al., 2005). In addition, CD89 sequences from rats (Maruoka et al., 2004), macaques (Rogers et al., 2004), and chimpanzees (Morton et al., 2005) have also been recently reported (Table 1). Since it is by far the best characterized FcaR from any species, this review will first briefly summarize what is presently known concerning the structure and function of human CD89. Recent data describing the identification and characterization of bovine and equine CD89 will then be discussed.
2. Human CD89 (HuCD89) The HuCD89 cDNA was first cloned in 1990 (Maliszewski et al., 1990), and numerous mAbs recognizing this receptor exist (Shen et al., 1989; Monteiro et al., 1992; Zhang et al., 2000). HuCD89 is expressed at high levels on neutrophils (PMNs), and monocytes, and at lower levels on eosinophils (Monteiro and van de Winkel, 2003). Recently, the detection of functional HuCD89 on liver Kupffer cells has led to the suggestion that this receptor is important for immunity against bacteria that have managed to penetrate the mucosal barrier (van Egmond et al., 2000). Furthermore, HuCD89 has proved to be an extremely effective trigger molecule on PMNs and may in fact be more efficient than FcgRs (Monteiro and van de Winkel, 2003). Moreover, HuCD89 has been shown to play an important role in IgA-mediated protection against several pathogens including Can-
dida albicans, Bordetella pertussis, Streptococcus pneumoniae, and Plasmodium yoelii (Monteiro and van de Winkel, 2003; Pleass et al., 2003). The HuCD89 gene has been mapped to the human leukocyte receptor complex (LRC) on chromosome 19q13.4. Other genes found in the LRC include those for the killer cell immunoglobulin-like receptors (KIRs), the leukocyte immunoglobulin-like receptors (LILRs), and NKp46. Interestingly, HuCD89 has been shown to be more closely related to these proteins than to the other human FcRs. Only one other FcR, bovine Fcg2R (BoFcg2R), has been shown to belong to this gene family, and characterization of this receptor has suggested that it shares a number of similarities with HuCD89 (Morton et al., 1999, 2001). Structurally, HuCD89 is composed of two extracellular Ig-like domains, the three-dimensional structure of which has recently been solved (Herr et al., 2003) (Fig. 1). The cytoplasmic tail of HuCD89 is relatively short and contains no recognized signaling motifs. However, HuCD89 has been shown to associate with a specialized signaling molecule, the FcR g chain, via a positively charged arginine residue in its transmembrane domain (Morton et al., 1995). Several other LRC-encoded proteins also possess charged residues in their transmembrane domains and current evidence suggests that this is an important characteristic of activatory receptors. Uniquely for FcRs, HuCD89 binds IgA via the membrane-distal domain (D1, see Fig. 1). The related BoFcg2R also binds its ligand, BoIgG2, via this domain. Numerous mutational studies have identified residues in D1 of HuCD89, which are important for
Fig. 1. Schematic depiction of HuCD89 generated from X-ray crystal coordinates (Protein Data Bank accession number 1OW0).
H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
the interaction with IgA. Similarly, the HuCD89 binding site within IgA has been mapped and has been shown to encompass residues in both the Ca2 and Ca3 domains. The elucidation of the three-dimensional structure of HuCD89 in complex with an IgA Fc fragment also revealed that one Fc can bind two receptors simultaneously (Herr et al., 2003). Since previous crystallization studies have shown that the stoichiometry of the IgG–FcgR and IgE–FceRI interaction is 1:1, this may be an extremely interesting finding that may have implications for the downstream signaling events triggered via HuCD89. It is also important to note that the current crystallographic data only show HuCD89 in complex with monomeric IgA Fc. Thus, we still do not fully understand how HuCD89 interacts with the various other molecular forms of IgA which exist (i.e. dimeric, polymeric, and secretory IgA).
3. Bovine CD89 (BoCD89) BoCD89 was originally identified by screening translated bovine EST sequences with the protein sequence of HuCD89 (Morton et al., 2004a). This allowed for the design of PCR primers to amplify the complete cDNA sequence from bovine RNA by RT-
141
PCR. The amplified BoCD89 cDNA contains an 870 bp open reading frame encoding a 290 amino acid protein (Fig. 2). The first 21 amino acids are predicted to code for the N-terminal leader sequence. Thus, the mature receptor begins with Gln (Q) 22 and is composed of a 204 amino acid extracellular region which folds into two Ig-like domains with four potential N-glycosylation sites (Asn-21, Asn-44, Asn-118, and Asn-161). This is followed by a 19 amino acid transmembrane domain, containing a positively charged arginine residue, and a 46 amino acid cytoplasmic tail devoid of known signaling motifs. Despite the relatively high level of homology with HuCD89, none of the anti-HuCD89 mAbs which were tested were able to cross-react with BoCD89. Thus, the expression pattern of BoCD89 was only examined at the cDNA level using RT-PCR. This analysis showed that the BoCD89 cDNA was expressed in PMNs, but not in T cells, NK cells, B cells, or monocytes/macrophages. In light of what is known about the expression pattern or HuCD89, the apparent lack of BoCD89 expression on bovine monocytes is somewhat puzzling, but true confirmation of the presence or lack of protein expression at the cell surface must wait until an anti-BoCD89 specific mAb becomes available.
Fig. 2. Alignment of the deduced protein sequences of bovine (BoCD89), equine (EqCD89), and human CD89 (HuCD89). Dots (. . .) signify identical amino acids. A dash (-) denotes a gap introduced to optimize similarity. The predicted start site of the mature proteins is indicated by a filled triangle (!). Conserved cysteine residues are indicated by a plus sign (+). Tyrosine (Y)/phenylalanine (F) 35 is marked with a filled diamond (^). The transmembrane domains are underlined and the conserved arginine (R) residue, important for association of HuCD89 with FcR g chain, is indicated by a filled circle (*).
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The chromosomal location of the BoCD89 gene has also been determined by RH mapping. The BoCD89 gene was mapped to bovine chromosome 18 close to KIR2DL1 and NKp46 (Storset et al., 2003). These results suggested that BoCD89, KIR2DL1, and NKp46 are clustered in a bovine LRC similar to the situation in humans. Rosetting analysis with BoCD89 transfectants confirmed that this receptor was able to bind both bovine and human IgA, and a more recent study mapped the interaction site in greater detail (Morton et al., 2004b). Previously, mutational analysis of HuCD89 and BoFcg2R, identified several residues within their membrane-distal domains important for ligand binding (Wines et al., 1999, 2001; Morton et al., 2001). For HuCD89, these studies have shown that residues Y35 (in the B–C loop) and R82 (in the F–G loop) are essential for IgA binding, while H85 (also in the F–G loop) also contributes to the interaction. Similarly, two residues in the F–G loop of D1 of BoFcg2R, specifically F82 and W87, have been shown to be critical for the binding of bovine IgG2 (Morton et al., 2001). Amino acid alignment of the protein sequence of BoCD89 with HuCD89 showed that the tyrosine residue at position 35 is conserved (Fig. 2). In addition, the arginine and histidine residues within the F–G loop of BoCD89 are also conserved, although due to a two amino acid deletion earlier in the domain they are designated R80 and H83, respectively. Therefore, to map the IgA binding site a panel of four mutants was generated in which Y35, R80, H83, and W85 were each replaced with alanine. Analysis of these mutants showed mutation of R80, H83, and W85 had no apparent effect on IgA binding. In contrast, however, mutation of Y35 completely abolished IgA binding suggesting that this residue is essential for IgA binding.
4. Equine CD89 (EqCD89) Like BoCD89, EqCD89 was initially identified from available equine EST sequences (Morton et al., 2005). The EqCD89 cDNA contains an 891 bp open reading frame, encoding a 271 amino acid protein (Fig. 2). Like HuCD89 and BoCD89 the first 21 amino acids are predicted to encode the N-terminal leader sequence, and the first residue of the mature receptor is Q22. Thus,
the two extracellular Ig-like domains of EqCD89 are made up of 204 amino acids and include four potential N-glycosylation sites (Asn-21, Asn-44, Asn-118, and Asn-154). The transmembrane domain of EqCD89 is 19 amino acids in length and includes the characteristic positively charged arginine residue common to other CD89 proteins (Morton et al., 2005). The cytoplasmic tail of EqCD89 is 27 amino acids long, and like the other CD89s is devoid of known signaling motifs. Due to the lack of mAbs specific for EqCD89, the expression pattern of the mature protein is still unknown; however, the cDNA is readily amplifiable by RT-PCR from equine PMN RNA. The chromosome location of the EqCD89 gene is also unknown, but it is tempting to speculate that it may be located in an equine LRC-like region on chromosome 10 which has been shown to contain the KIR and LILR genes of the horse (Takahashi et al., 2004). Binding studies showed that EqCD89 was able to bind to equine, bovine, and human IgA. Interestingly, these studies also revealed that HuCD89 and BoCD89 were unable to bind equine IgA. Previous studies (see above) revealed that the tyrosine residue at position 35 (Y35) is critical for IgA binding in both HuCD89 and BoCD89. However, EqCD89 has phenylalanine at the corresponding position (F35). Subsequent mutational studies showed that while mutating F35 to tyrosine had no apparent affect on the IgA-binding characteristics of EqCD89, when F35 was mutated to alanine, IgA-binding was abolished.
5. Conclusions An IgA Fc receptor highly homologous to HuCD89 is present in both cattle and horses. However, due to a lack of specific mAbs the expression pattern of these proteins has not been analysed. However, the high level of homology between BoCD89, EqCD89 and HuCD89 suggests that their three-dimensional structures are similar (Fig. 1). In addition, the presence of a charged residue in the transmembrane domain of BoCD89 and EqCD89 suggests that they are able to interact with signaling molecules such as the FcR g chain, and efficiently trigger cellular effector functions. A greater understanding of the function of CD89 in cattle and horses may reveal much about the biological role of IgA in these species.
H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
Acknowledgements The author would like to thank A.K. Storset (Norwegian School of Veterinary Science, Oslo, Norway), R.J. Pleass, J.M. Woof (University of Dundee, Dundee, UK), E. Dissen (University of Oslo, Oslo, Norway), J.L. Williams (Roslin Institute, Roslin, UK), and P. Brandtzaeg (Rikshospitalet University Hospital, Oslo, Norway) for their contributions to the work described in this review.
References Daeron, M., 1997. Fc receptor biology. Annu. Rev. Immunol. 15, 203–234. Gould, H.J., Sutton, B.J., Beavil, A.J., Beavil, R.L., McCloskey, N., Coker, H.A., Fear, D., Smurthwaite, L., 2003. The biology of IgE and the basis of allergic disease. Annu. Rev. Immunol. 21, 579– 628. Herr, A.B., Ballister, E.R., Bjorkman, P.J., 2003. Insights into IgAmediated immune responses from the crystal structures of human FcaRI and its complex with IgA1–Fc. Nature 423, 614–620. Hogarth, P.M., 2002. Fc receptors are major mediators of antibody based inflammation in autoimmunity. Curr. Opin. Immunol. 14, 798–802. Kacskovics, I., 2004. Fc receptors in livestock species. Vet. Immunol. Immunopathol. 102, 351–362. Maliszewski, C.R., March, C.J., Schoenborn, M.A., Gimpel, S., Shen, L., 1990. Expression cloning of a human Fc receptor for IgA. J. Exp. Med. 172, 1665–1672. Maruoka, T., Nagata, T., Kasahara, M., 2004. Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex. Immunogenetics 55, 712–716. Monteiro, R.C., Cooper, M.D., Kubagawa, H., 1992. Molecular heterogeneity of Fca receptors detected by receptor- specific monoclonal antibodies. J. Immunol. 148, 1764–1770. Monteiro, R.C., van de Winkel, J.G., 2003. IgA Fc receptors. Annu. Rev. Immunol. 21, 177–204. Morton, H.C., van den Herik-Oudijk, I.E., Vossebeld, P., Snijders, A., Verhoeven, A.J., Capel, P.J., van de Winkel, J.G., 1995. Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR g chain. Molecular basis for CD89/FcR g chain association. J. Biol. Chem. 270, 29781– 29787. Morton, H.C., van Zandbergen, G., van Kooten, C., Howard, C.J., van de Winkel, J.G., Brandtzaeg, P., 1999. Immunoglobulinbinding sites of human FcaRI (CD89) and bovine Fcg2R are located in their membrane-distal extracellular domains. J. Exp. Med. 189, 1715–1722. Morton, H.C., Howard, C.J., Storset, A.K., Brandtzaeg, P., 2001. Identification of residues within the extracellular domain 1 of
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bovine Fcg2R essential for binding bovine IgG2. J. Biol. Chem. 276, 47794–47800. Morton, H.C., Pleass, R.J., Storset, A.K., Dissen, E., Williams, J.L., Brandtzaeg, P., Woof, J.M., 2004a. Cloning and characterization of an immunoglobulin A Fc receptor from cattle. Immunology 111, 204–211. Morton, H.C., Pleass, R.J., Woof, J.M., Brandtzaeg, P., 2004b. Characterization of the ligand binding site of the bovine IgA Fc receptor (bFcaR). J. Biol. Chem. 279, 54018–54022. Morton, H.C., Pleass, R.J., Storset, A.K., Brandtzaeg, P., Woof, J.M., 2005. Cloning and characterization of equine CD89 and identification of the CD89 gene in chimpanzees and rhesus macaques. Immunology 115, 74–84. Pleass, R.J., Ogun, S.A., McGuinness, D.H., van de Winkel, J.G., Holder, A.A., Woof, J.M., 2003. Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection. Blood 102, 4424–4430. Raghavan, M., Bjorkman, P.J., 1996. Fc receptors and their interactions with immunoglobulins. Annu. Rev. Cell Dev. Biol. 12, 181–220. Ravetch, J.V., 1997. Fc receptors. Curr. Opin. Immunol. 9, 121–125. Rogers, K.A., Scinicariello, F., Attanasio, R., 2004. Identification and characterization of macaque CD89 (immunoglobulin A Fc receptor). Immunology 113, 178–186. Shen, L., Lasser, R., Fanger, M.W., 1989. My 43, a monoclonal antibody that reacts with human myeloid cells inhibits monocyte IgA binding and triggers function. J. Immunol. 143, 4117–4122. Storset, A.K., Slettedal, I.O., Williams, J.L., Law, A., Dissen, E., 2003. Natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor (KIR) multigene family contains members with divergent signaling motifs. Eur. J. Immunol. 33, 980–990. Takahashi, T., Yawata, M., Raudsepp, T., Lear, T.L., Chowdhary, B.P., Antczak, D.F., Kasahara, M., 2004. Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes. Eur. J. Immunol. 34, 773–784. Takai, T., 2002. Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 2, 580–592. van Egmond, M., van Garderen, E., van Spriel, A.B., Damen, C.A., van Amersfoort, E.S., van Zandbergen, G., van Hattum, J., Kuiper, J., van de Winkel, J.G., 2000. FcaRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity. Nat. Med. 6, 680–685. Wines, B.D., Hulett, M.D., Jamieson, G.P., Trist, H.M., Spratt, J.M., Hogarth, P.M., 1999. Identification of residues in the first domain of human Fca receptor essential for interaction with IgA. J. Immunol. 162, 2146–2153. Wines, B.D., Sardjono, C.T., Trist, H.H., Lay, C.S., Hogarth, P.M., 2001. The interaction of FcaRI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster. J. Immunol. 166, 1781–1789. Zhang, W., Bi, B., Oldroyd, R.G., Lachmann, P.J., 2000. Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of Fca receptors (FcaR) with a monoclonal antibody, MIP8a. Clin. Exp. Immunol. 121, 106–111.
IgA Fc receptors in cattle and horses H. Craig Morton * Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, Rikshospitalet University Hospital, N-0027 Oslo, Norway
Abstract The biological role of IgA depends, at least partly, on the interaction with specific receptors (FcaRs) on the surface of leukocytes. The human FcaR, CD89, was the first IgA Fc receptor to be identified, and binding of IgA-coated particles to CD89 triggers numerous cellular effector functions including phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and release of inflammatory mediators. Recently, CD89 orthologs have been identified in a number of other species, including cows and horses. This brief review will summarize our current knowledge regarding the structure and function of bovine and equine CD89. # 2005 Elsevier B.V. All rights reserved. Keywords: Immunoglobulin A; Fc receptor; CD89; Bovine; Equine
1. Introduction Receptors for the Fc regions of immunoglobulin (Ig) molecules (Fc receptors, FcRs) are expressed by many cell types, especially phagocytes. Ligation of FcRs by Ig-coated targets can trigger numerous cellular effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and secretion of cytokines and other inflammatory mediators (Raghavan and Bjorkman, 1996; Daeron, 1997; Ravetch, 1997). Thus, FcRs provide a crucial link between the humoral and cellular arms of the immune system. The FcRs for IgG (FcgRs) and IgE (FceRI) from humans and mice are the best characterized, and data * Tel.: +47 23 07 14 89; fax: +47 23 07 15 11. E-mail address: [email protected].
from murine models has shown that these FcRs are important for triggering inflammatory reactions and also for protection against infectious microorganisms and parasites. However, animal models have also revealed that inappropriate inflammation triggered by FcRs can also contribute to the generation and potentiation of some allergic and autoimmune diseases (Hogarth, 2002; Takai, 2002; Gould et al., 2003). Homologs of many of these FcgRs and FceRs have also been identified in a number of livestock species, and readers are referred to an excellent recent review for further information (Kacskovics, 2004). FcRs for IgA (FcaRs) have been shown to be expressed on the surface of leukocytes from many different species, but until relatively recently only the human myeloid FcaR, CD89, has been molecularly characterized (Maliszewski et al., 1990). More recently, we have identified FcaRs with high
0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.07.008
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H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
Table 1 Accession numbers and references for all CD89 proteins described till now Species
Accession no. Reference
Human Chimpanzee Rhesus macaque Cynomolgus macaque Cow Horse Rat
X54150 BK005386a AY386684 AY386690 AY247821 AY587560 AB109767
Maliszewski et al. (1990) Morton et al. (2005) Rogers et al. (2004) Rogers et al. (2004) Morton et al. (2004a) Morton et al. (2005) Maruoka et al. (2004)
a
The chimpanzee CD89 sequence was deduced from draft genomic sequence and has not been confirmed at the cDNA level. This sequence is thus available from the Third Party Annotation Section of the DDJB/EMBL/GenBank databases.
homology to human CD89 in cattle (Morton et al., 2004a) and horses (Morton et al., 2005). In addition, CD89 sequences from rats (Maruoka et al., 2004), macaques (Rogers et al., 2004), and chimpanzees (Morton et al., 2005) have also been recently reported (Table 1). Since it is by far the best characterized FcaR from any species, this review will first briefly summarize what is presently known concerning the structure and function of human CD89. Recent data describing the identification and characterization of bovine and equine CD89 will then be discussed.
2. Human CD89 (HuCD89) The HuCD89 cDNA was first cloned in 1990 (Maliszewski et al., 1990), and numerous mAbs recognizing this receptor exist (Shen et al., 1989; Monteiro et al., 1992; Zhang et al., 2000). HuCD89 is expressed at high levels on neutrophils (PMNs), and monocytes, and at lower levels on eosinophils (Monteiro and van de Winkel, 2003). Recently, the detection of functional HuCD89 on liver Kupffer cells has led to the suggestion that this receptor is important for immunity against bacteria that have managed to penetrate the mucosal barrier (van Egmond et al., 2000). Furthermore, HuCD89 has proved to be an extremely effective trigger molecule on PMNs and may in fact be more efficient than FcgRs (Monteiro and van de Winkel, 2003). Moreover, HuCD89 has been shown to play an important role in IgA-mediated protection against several pathogens including Can-
dida albicans, Bordetella pertussis, Streptococcus pneumoniae, and Plasmodium yoelii (Monteiro and van de Winkel, 2003; Pleass et al., 2003). The HuCD89 gene has been mapped to the human leukocyte receptor complex (LRC) on chromosome 19q13.4. Other genes found in the LRC include those for the killer cell immunoglobulin-like receptors (KIRs), the leukocyte immunoglobulin-like receptors (LILRs), and NKp46. Interestingly, HuCD89 has been shown to be more closely related to these proteins than to the other human FcRs. Only one other FcR, bovine Fcg2R (BoFcg2R), has been shown to belong to this gene family, and characterization of this receptor has suggested that it shares a number of similarities with HuCD89 (Morton et al., 1999, 2001). Structurally, HuCD89 is composed of two extracellular Ig-like domains, the three-dimensional structure of which has recently been solved (Herr et al., 2003) (Fig. 1). The cytoplasmic tail of HuCD89 is relatively short and contains no recognized signaling motifs. However, HuCD89 has been shown to associate with a specialized signaling molecule, the FcR g chain, via a positively charged arginine residue in its transmembrane domain (Morton et al., 1995). Several other LRC-encoded proteins also possess charged residues in their transmembrane domains and current evidence suggests that this is an important characteristic of activatory receptors. Uniquely for FcRs, HuCD89 binds IgA via the membrane-distal domain (D1, see Fig. 1). The related BoFcg2R also binds its ligand, BoIgG2, via this domain. Numerous mutational studies have identified residues in D1 of HuCD89, which are important for
Fig. 1. Schematic depiction of HuCD89 generated from X-ray crystal coordinates (Protein Data Bank accession number 1OW0).
H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
the interaction with IgA. Similarly, the HuCD89 binding site within IgA has been mapped and has been shown to encompass residues in both the Ca2 and Ca3 domains. The elucidation of the three-dimensional structure of HuCD89 in complex with an IgA Fc fragment also revealed that one Fc can bind two receptors simultaneously (Herr et al., 2003). Since previous crystallization studies have shown that the stoichiometry of the IgG–FcgR and IgE–FceRI interaction is 1:1, this may be an extremely interesting finding that may have implications for the downstream signaling events triggered via HuCD89. It is also important to note that the current crystallographic data only show HuCD89 in complex with monomeric IgA Fc. Thus, we still do not fully understand how HuCD89 interacts with the various other molecular forms of IgA which exist (i.e. dimeric, polymeric, and secretory IgA).
3. Bovine CD89 (BoCD89) BoCD89 was originally identified by screening translated bovine EST sequences with the protein sequence of HuCD89 (Morton et al., 2004a). This allowed for the design of PCR primers to amplify the complete cDNA sequence from bovine RNA by RT-
141
PCR. The amplified BoCD89 cDNA contains an 870 bp open reading frame encoding a 290 amino acid protein (Fig. 2). The first 21 amino acids are predicted to code for the N-terminal leader sequence. Thus, the mature receptor begins with Gln (Q) 22 and is composed of a 204 amino acid extracellular region which folds into two Ig-like domains with four potential N-glycosylation sites (Asn-21, Asn-44, Asn-118, and Asn-161). This is followed by a 19 amino acid transmembrane domain, containing a positively charged arginine residue, and a 46 amino acid cytoplasmic tail devoid of known signaling motifs. Despite the relatively high level of homology with HuCD89, none of the anti-HuCD89 mAbs which were tested were able to cross-react with BoCD89. Thus, the expression pattern of BoCD89 was only examined at the cDNA level using RT-PCR. This analysis showed that the BoCD89 cDNA was expressed in PMNs, but not in T cells, NK cells, B cells, or monocytes/macrophages. In light of what is known about the expression pattern or HuCD89, the apparent lack of BoCD89 expression on bovine monocytes is somewhat puzzling, but true confirmation of the presence or lack of protein expression at the cell surface must wait until an anti-BoCD89 specific mAb becomes available.
Fig. 2. Alignment of the deduced protein sequences of bovine (BoCD89), equine (EqCD89), and human CD89 (HuCD89). Dots (. . .) signify identical amino acids. A dash (-) denotes a gap introduced to optimize similarity. The predicted start site of the mature proteins is indicated by a filled triangle (!). Conserved cysteine residues are indicated by a plus sign (+). Tyrosine (Y)/phenylalanine (F) 35 is marked with a filled diamond (^). The transmembrane domains are underlined and the conserved arginine (R) residue, important for association of HuCD89 with FcR g chain, is indicated by a filled circle (*).
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The chromosomal location of the BoCD89 gene has also been determined by RH mapping. The BoCD89 gene was mapped to bovine chromosome 18 close to KIR2DL1 and NKp46 (Storset et al., 2003). These results suggested that BoCD89, KIR2DL1, and NKp46 are clustered in a bovine LRC similar to the situation in humans. Rosetting analysis with BoCD89 transfectants confirmed that this receptor was able to bind both bovine and human IgA, and a more recent study mapped the interaction site in greater detail (Morton et al., 2004b). Previously, mutational analysis of HuCD89 and BoFcg2R, identified several residues within their membrane-distal domains important for ligand binding (Wines et al., 1999, 2001; Morton et al., 2001). For HuCD89, these studies have shown that residues Y35 (in the B–C loop) and R82 (in the F–G loop) are essential for IgA binding, while H85 (also in the F–G loop) also contributes to the interaction. Similarly, two residues in the F–G loop of D1 of BoFcg2R, specifically F82 and W87, have been shown to be critical for the binding of bovine IgG2 (Morton et al., 2001). Amino acid alignment of the protein sequence of BoCD89 with HuCD89 showed that the tyrosine residue at position 35 is conserved (Fig. 2). In addition, the arginine and histidine residues within the F–G loop of BoCD89 are also conserved, although due to a two amino acid deletion earlier in the domain they are designated R80 and H83, respectively. Therefore, to map the IgA binding site a panel of four mutants was generated in which Y35, R80, H83, and W85 were each replaced with alanine. Analysis of these mutants showed mutation of R80, H83, and W85 had no apparent effect on IgA binding. In contrast, however, mutation of Y35 completely abolished IgA binding suggesting that this residue is essential for IgA binding.
4. Equine CD89 (EqCD89) Like BoCD89, EqCD89 was initially identified from available equine EST sequences (Morton et al., 2005). The EqCD89 cDNA contains an 891 bp open reading frame, encoding a 271 amino acid protein (Fig. 2). Like HuCD89 and BoCD89 the first 21 amino acids are predicted to encode the N-terminal leader sequence, and the first residue of the mature receptor is Q22. Thus,
the two extracellular Ig-like domains of EqCD89 are made up of 204 amino acids and include four potential N-glycosylation sites (Asn-21, Asn-44, Asn-118, and Asn-154). The transmembrane domain of EqCD89 is 19 amino acids in length and includes the characteristic positively charged arginine residue common to other CD89 proteins (Morton et al., 2005). The cytoplasmic tail of EqCD89 is 27 amino acids long, and like the other CD89s is devoid of known signaling motifs. Due to the lack of mAbs specific for EqCD89, the expression pattern of the mature protein is still unknown; however, the cDNA is readily amplifiable by RT-PCR from equine PMN RNA. The chromosome location of the EqCD89 gene is also unknown, but it is tempting to speculate that it may be located in an equine LRC-like region on chromosome 10 which has been shown to contain the KIR and LILR genes of the horse (Takahashi et al., 2004). Binding studies showed that EqCD89 was able to bind to equine, bovine, and human IgA. Interestingly, these studies also revealed that HuCD89 and BoCD89 were unable to bind equine IgA. Previous studies (see above) revealed that the tyrosine residue at position 35 (Y35) is critical for IgA binding in both HuCD89 and BoCD89. However, EqCD89 has phenylalanine at the corresponding position (F35). Subsequent mutational studies showed that while mutating F35 to tyrosine had no apparent affect on the IgA-binding characteristics of EqCD89, when F35 was mutated to alanine, IgA-binding was abolished.
5. Conclusions An IgA Fc receptor highly homologous to HuCD89 is present in both cattle and horses. However, due to a lack of specific mAbs the expression pattern of these proteins has not been analysed. However, the high level of homology between BoCD89, EqCD89 and HuCD89 suggests that their three-dimensional structures are similar (Fig. 1). In addition, the presence of a charged residue in the transmembrane domain of BoCD89 and EqCD89 suggests that they are able to interact with signaling molecules such as the FcR g chain, and efficiently trigger cellular effector functions. A greater understanding of the function of CD89 in cattle and horses may reveal much about the biological role of IgA in these species.
H.C. Morton / Veterinary Immunology and Immunopathology 108 (2005) 139–143
Acknowledgements The author would like to thank A.K. Storset (Norwegian School of Veterinary Science, Oslo, Norway), R.J. Pleass, J.M. Woof (University of Dundee, Dundee, UK), E. Dissen (University of Oslo, Oslo, Norway), J.L. Williams (Roslin Institute, Roslin, UK), and P. Brandtzaeg (Rikshospitalet University Hospital, Oslo, Norway) for their contributions to the work described in this review.
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