- Email: [email protected]
Identification of allelic polymorphism in the caprine IGHA gene
ARTICLE IN PRESS
Developmental & Comparative Immunology
Developmental and Comparative Immunology 30 (2006) 741–745
www.elsevier.com/locate/devcompimm
Short communication
Identification of allelic polymorphism in the caprine IGHA gene Huitong Zhou, Jon G.H. Hickford, Qian Fang Cell Biology Group, Agriculture and Life Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand Received 14 July 2005; received in revised form 10 October 2005; accepted 30 October 2005 Available online 22 November 2005
Abstract Variation in the immunoglobulin heavy alpha chain (IGHA) constant region has been reported in a number of species. In this study, the IGHA gene was investigated in goats using PCR-single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences were identified from 111 Boer and Angora goats. Either one or two sequences were detected in individual goats, and all the sequences shared high homology to the published ovine and bovine IGHA sequences. These sequences were predicted to encode three amino acid sequences, two with a longer hinge region and one with a shorter hinge region. The variation reported here may affect the structure of the hinge and hence the function of IgA. r 2005 Elsevier Ltd. All rights reserved. Keywords: Goat IgA; IGHA gene; Hinge; Polymorphism
Immunoglobulin A (IgA) is the major antibody class present in the mucosal secretions of most mammals and is also found at measurable concentrations in the serum of many species. On mucosal surfaces, IgA may represent a key first line of defense by binding to invading pathogens; whereas in the serum, IgA can mediate the elimination of pathogens that have breached the mucosal surface and thus function as a second line of defense. IgA appears to be genetically heterogeneous, and genetic variation in the constant region of the heavy chain of IgA (IGHA) has been documented in a number of species. Humans, chimpanzees, gorillas and gibbons have two IGHA genes which differ mainly in the hinge-coding region [1,2], giving rise to two IgA subtypes, IgA1 and IgA2 [3]. Corresponding author. Tel.: +64 3 325 2811; fax: +64 3 325 3851. E-mail address: [email protected] (J.G.H. Hickford).
Rabbits have 13 IGHA genes that differ mainly in the hinge-coding region, of which 11 appear to be expressed [3,4]. In all other species examined, there is only a single IGHA gene, but a number of species including mouse [5], macaque [6], pig [7], dog [8] and sheep [9] have variation in the constant region nucleotide sequence and length of the hinge-coding region. Variation in the hinge region may have an impact on the flexibility of IgA and antigen binding [10], and may affect the sensitivity of IgA to cleavage by bacterial or parasitic proteases [11]. In parasitized ruminants, IgA is probably the most intensively investigated immunoglobulin, but investigations have been focused on the quantity of IgA and little attention has been paid to the IgA heterogeneity. In this study, we describe the search for IGHA polymorphism in goats using PCR-single-strand conformational polymorphism (SSCP) analysis.
0145-305X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.10.012
ARTICLE IN PRESS 742
H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
Twenty-three Boer goats and 88 Angora goats were investigated. Genomic DNA was isolated from blood samples collected on FTAs cards (Whatman, Middlesex, UK) following the manufacturer’s protocol. A fragment of the caprine IGHA gene containing the entire hinge-coding region was amplified using PCR primers designed for the ovine IGHA gene [9]. These primers were IGHA-up (50 CCAAAGCCAGCAAGACCGT-30 ) and IGHA-dn (50 -ACTCAGGAGCAGATCCTCGA-30 ). Primers were synthesized by Proligo, Boulder, CO, USA. Amplification was performed in a 20-ml reaction containing the genomic DNA on one 1.2-mm punch of FTA paper, 0.25 mM of each primer, 150 mM dNTPs (Eppendorf, Hamburg, Germany), 2.5 mM Mg2+, 0.5 U Taq DNA polymerase (Qiagen, Hilden, Germany) and 1 reaction buffer supplied. Amplification was carried out in an iCycler (BioRad Laboratories, Hercules, CA, USA), and the thermal profile consisted of denaturation at 94 1C for 2 min, followed by 32 cycles of 94 1C for 30 s, 60 1C for 30 s and 72 1C for 30 s, with a final extension step at 72 1C for 5 min. PCR amplimers of two different lengths were visualized by electrophoresis in 1% Seakems LE agarose (BioWhittaker) gels using 1 TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA), containing 200 ng ml1 ethidium bromide. To screen for polymorphism in the caprine IGHA gene, all amplimers were subjected to SSCP analysis. A 0.7-ml aliquot of each amplimer was mixed with 7 ml of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol), and after denaturation at 95 1C for 5 min, samples were rapidly cooled on wet ice and then loaded on 16 cm 18 cm, 14.5% acrylamide:bisacrylamide (37.5:1) (Bio-Rad) gels containing 1% glycerol. Electrophoresis was performed using Protean II xi cells (Bio-Rad), at 410 V for 18 h at 17 1C in 0.5 TBE buffer, and gels were silverstained according to the method of Bassam et al. [12]. Under these conditions, three unique SSCP banding patterns could be detected (Fig. 1). One to two patterns were observed for each animal, which was consistent with there either being homozygous or heterozygous genotypes. To determine the nucleotide sequences corresponding to the different SSCP patterns, genomic DNA samples representative of different SSCP patterns were selected for amplification using the proofreading enzyme ProofStart DNA polymerase (Qiagen) under the conditions described above.
∗01
∗03
∗02
∗02 ∗03
∗01 ∗03
∗01 ∗02
1
2
3
4
5
6
Fig. 1. PCR-single-strand conformational polymorphism of the caprine IGHA gene. Representative samples of three unique SSCP banding patterns corresponding to three allelic sequences *01, *02 and *03 are shown. PCR products were amplified from genomic DNA (lanes 1, 3–6) and a plasmid cloned IGHA fragment (lane 2).
After the addition of an A-overhang to the bluntended PCR products using an A-Addition Kit (Qiagen), amplimers were then cloned into the pDrive Cloning vector (Qiagen), and transformed into competent Escherichia coli cells (One ShotTM INVaF’, Invitrogen), following the manufacturers’ instructions. For each transformation, six or twelve insert positive clones, for ‘‘single’’ or ‘‘double’’ SSCP patterns, respectively, were selected and incubated overnight in Terrific broth (Invitrogen) at 37 1C in a shaking rotary incubator (225 rpm). Plasmids were recovered from bacterial cells by boiling for 10 min in 0.8% (vol/vol) Triton X-100 solution and following centrifugation at 12,000 g, 1 ml of the supernatant was used as a template for PCR amplification using Taq DNA polymerase (Qiagen). Amplimers from these clones and the corresponding genomic DNA were run adjacent to each other on SSCP gels for comparison of the SSCP patterns, and only those clones for which the SSCP patterns matched those of the corresponding genomic DNA were selected for subsequent DNA sequencing. Plasmids from the selected clones were extracted using a QIApreps Spin Miniprep kit (Qiagen), and were then sequenced in both directions using M13 forward and reverse primers at the Waikato DNA Sequencing Facility, the University of Waikato, New Zealand. Identical sequences obtained from at
ARTICLE IN PRESS H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
least three clones from different goats, or independent PCR amplifications from the same goat, were subjected to further sequence analysis. Sequence analysis using DNAMANTM (Version 5.2.10, Lynnon BioSoft, Vaudreuil, Canada), revealed that these unique SSCP patterns represented three different nucleotide sequences, two sequences of 316 bp and a sequence of 310 bp. All of these sequences were novel, but shared high homology to the IGHA sequences from a variety of species, with the highest homology being to sheep sequences (GenBank accession no. AY956424–AY956426; [9]). There were either one or two unique sequences identified in individual goats, and this suggests that the sequences isolated in this study represent different alleles of the caprine IGHA gene. The sequences from both breeds were identical for each of the alleles. These alleles were named followed the rules proposed by the international ImMunoGeneTics information systems (IMGT) ([13]; http:// imgt.cines.fr/) and sequences were deposited into the NCBI GenBank with the accession no. DQ118110–DQ118112. The three caprine IGHA nucleotide sequences were predicted to encode three different IGHA molecules, two with a longer hinge and one with a shorter hinge. The two ‘‘long-hinged’’ IGHA molecules (*01 and *02) had an identical hinge of nine residues and only differed at a single residue at the carboxyl-end of the CH1 region. The ‘‘shorthinged’’ IGHA molecule (*03) however, not only
743
was two amino acids shorter, but also possessed a different nucleotide and amino acid sequence in the hinge region (Fig. 2). The considerable differences between the ‘‘long-hinged’’ and ‘‘short-hinged’’ sequences raise a question of whether these sequences represent two loci of the IGHA gene as the case in humans and some primates [3]. However, as no more than two sequences were identified in any single goat, it is unlikely that these sequences are from more than one locus. This suggests a single IGHA gene in goats, consistent with the results reported in sheep and other species [3,9]. Two of the caprine sequences, caprine-IGHA*01 and caprine-IGHA*02, shared close homology to ovine sequences (ovine-IGHA*02 and ovineIGHA*03). This is consistent with the close relatedness of these species as evidenced by the similarity of alleles of their MHC genes [14,15]. However, the remaining caprine sequence (caprine-IGHA*03) had its 50 end similar or identical to ovine and caprine sequences while its 30 end was identical to the bovine sequence (GenBank accession no. AF109167; [16]) in the hinge-coding region. This suggests that either these similar sequences have arisen independently by convergent evolution, or that the IGHA hinge region sequences pre-date the divergence of goats, sheep and cattle. Sequence alignment of the caprine IGHA amino acid sequences using DNAMANTM revealed that variation was located at the carboxyl-end of CH1 and in the hinge region (Fig. 3). This pattern of
Fig. 2. Nucleotide sequences of the three caprine IGHA alleles. Nucleotides in exons are indicated in upper-case and those in the intron are in lower-case. Nucleotides identical to the top sequence are presented by dashes and dots have been introduced to improve the alignment.
ARTICLE IN PRESS 744
H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
Fig. 3. Alignment of the predicted amino acid sequences of the ovine, caprine and bovine IGHA gene. Amino acids are presented in oneletter code. A dash represents a sequence identical to the top, and dots have been introduced to improve the alignment. The most polymorphic region is shaded and the hinge region is boxed. The sequences of ovine-IGHA*01, *02, *03 and bovine-IGHA*01 are from the GenBank with accession no. AY956424, AY956425, AY956426 and AF109167, respectively.
polymorphism is similar to that reported in sheep [9]. As the carboxyl-end of CH1 and the hinge region are mostly affected, the possible consequences of this variation in the IGHA gene could be twofold. Firstly, the impact of length variation on hinge structure has been demonstrated with crystallization analysis showing that the long hinge of human IgA1 has an extended structure while the short hinge of IgA2 is compact [10]. The short hinge of caprine IGHA contained only seven amino acid residues, which is the shortest hinge reported in ruminants. Sequence variation may also affect the structure of the hinge and hence the hinge flexibility. An interesting characteristic of variation in the caprine IGHA gene was variation in the number and position of cysteine residues in the polymorphic region. If some of the cysteines form disulfide bonds, they may alter the flexibility of the hinge. IgA with a more flexible hinge may bind more effectively to antigens. Secondly, it has been shown that a single amino acid mutation in the hinge region can change the susceptibility of human IgA1 to streptococcal IgA1 protease [17]. Variation in hinge sequence may create or destroy amino acid motifs sensitive to cleavage by bacterial or parasitic proteases. Overall the consequences of caprine IGHA polymorphism await detailed study of the proteins encoded by these alleles. We thank W.-K. Lin and Y.-S. Lin for technical assistance.
[2] [3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] Flanagan JG, Lefranc MP, Rabbitts TH. Mechanisms of divergence and convergence of the human immunoglobulin
[14]
alpha 1 and alpha 2 constant region gene sequences. Cell 1984;36:681–8. Lefranc MP, Lefranc G, editors. The immunoglobulin factsbook. London, UK: Academic Press; 2001. Woof JM, Kerr MA. IgA function—variations on a theme. Immunology 2004;113:175–7. Spieker-Polet H, Yam PC, Knight KL. Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues. J Immunol 1993;150:5457–65. Phillips-Quagliata JM. Mouse IgA allotypes have major differences in their hinge regions. Immunogenetics 2002;53: 1033–8. Scinicariello F, Attanasio R. Intraspecies heterogeneity of immunoglobulin alpha-chain constant region genes in rhesus macaques. Immunology 2001;103:441–8. Brown WR, Kacskovics I, Amendt BA, Blackmore NB, Rothschild M, Shinde R, et al. The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C alpha intron. J Immunol 1995;154: 3836–42. Peters IR, Helps CR, Calvert EL, Hall EJ, Day MJ. Identification of four allelic variants of the dog IGHA gene. Immunogenetics 2004;56:254–60. Zhou H, Hickford JGH, Fang Q. Polymorphism of the IGHA gene in sheep. Immunogenetics 2005;57: 453–7. Furtado PB, Whitty PW, Robertson A, Eaton JT, Almogren A, Kerr MA, et al. Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1. J Mol Biol 2004;338:921–41. Senior BW, Woof JM. Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases. Infect Immun 2005;73:1515–22. Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80–3. Lefranc MP, Giudicelli V, Kaas Q, Duprat E, JabadoMichaloud J, Scaviner D, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2005;33:D593–7. Hickford JGH, Zhou H, Slow S, Fang Q. Diversity of the ovine DQA2 gene. J Anim Sci 2004;82:1553–63.
ARTICLE IN PRESS H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745 [15] Zhou H, Hickford JGH, Fang Q. Polymorphism of the DQA2 gene in goats. J Anim Sci 2005;83:963–8. [16] Brown WR, Rabbani H, Butler JE, Hammarstrom L. Characterization of the bovine C alpha gene. Immunology 1997;91:1–6.
745
[17] Batten MR, Senior BW, Kilian M, Woof JM. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases. Infect Immun 2003;71:1462–9.
Developmental & Comparative Immunology
Developmental and Comparative Immunology 30 (2006) 741–745
www.elsevier.com/locate/devcompimm
Short communication
Identification of allelic polymorphism in the caprine IGHA gene Huitong Zhou, Jon G.H. Hickford, Qian Fang Cell Biology Group, Agriculture and Life Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand Received 14 July 2005; received in revised form 10 October 2005; accepted 30 October 2005 Available online 22 November 2005
Abstract Variation in the immunoglobulin heavy alpha chain (IGHA) constant region has been reported in a number of species. In this study, the IGHA gene was investigated in goats using PCR-single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences were identified from 111 Boer and Angora goats. Either one or two sequences were detected in individual goats, and all the sequences shared high homology to the published ovine and bovine IGHA sequences. These sequences were predicted to encode three amino acid sequences, two with a longer hinge region and one with a shorter hinge region. The variation reported here may affect the structure of the hinge and hence the function of IgA. r 2005 Elsevier Ltd. All rights reserved. Keywords: Goat IgA; IGHA gene; Hinge; Polymorphism
Immunoglobulin A (IgA) is the major antibody class present in the mucosal secretions of most mammals and is also found at measurable concentrations in the serum of many species. On mucosal surfaces, IgA may represent a key first line of defense by binding to invading pathogens; whereas in the serum, IgA can mediate the elimination of pathogens that have breached the mucosal surface and thus function as a second line of defense. IgA appears to be genetically heterogeneous, and genetic variation in the constant region of the heavy chain of IgA (IGHA) has been documented in a number of species. Humans, chimpanzees, gorillas and gibbons have two IGHA genes which differ mainly in the hinge-coding region [1,2], giving rise to two IgA subtypes, IgA1 and IgA2 [3]. Corresponding author. Tel.: +64 3 325 2811; fax: +64 3 325 3851. E-mail address: [email protected] (J.G.H. Hickford).
Rabbits have 13 IGHA genes that differ mainly in the hinge-coding region, of which 11 appear to be expressed [3,4]. In all other species examined, there is only a single IGHA gene, but a number of species including mouse [5], macaque [6], pig [7], dog [8] and sheep [9] have variation in the constant region nucleotide sequence and length of the hinge-coding region. Variation in the hinge region may have an impact on the flexibility of IgA and antigen binding [10], and may affect the sensitivity of IgA to cleavage by bacterial or parasitic proteases [11]. In parasitized ruminants, IgA is probably the most intensively investigated immunoglobulin, but investigations have been focused on the quantity of IgA and little attention has been paid to the IgA heterogeneity. In this study, we describe the search for IGHA polymorphism in goats using PCR-single-strand conformational polymorphism (SSCP) analysis.
0145-305X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.10.012
ARTICLE IN PRESS 742
H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
Twenty-three Boer goats and 88 Angora goats were investigated. Genomic DNA was isolated from blood samples collected on FTAs cards (Whatman, Middlesex, UK) following the manufacturer’s protocol. A fragment of the caprine IGHA gene containing the entire hinge-coding region was amplified using PCR primers designed for the ovine IGHA gene [9]. These primers were IGHA-up (50 CCAAAGCCAGCAAGACCGT-30 ) and IGHA-dn (50 -ACTCAGGAGCAGATCCTCGA-30 ). Primers were synthesized by Proligo, Boulder, CO, USA. Amplification was performed in a 20-ml reaction containing the genomic DNA on one 1.2-mm punch of FTA paper, 0.25 mM of each primer, 150 mM dNTPs (Eppendorf, Hamburg, Germany), 2.5 mM Mg2+, 0.5 U Taq DNA polymerase (Qiagen, Hilden, Germany) and 1 reaction buffer supplied. Amplification was carried out in an iCycler (BioRad Laboratories, Hercules, CA, USA), and the thermal profile consisted of denaturation at 94 1C for 2 min, followed by 32 cycles of 94 1C for 30 s, 60 1C for 30 s and 72 1C for 30 s, with a final extension step at 72 1C for 5 min. PCR amplimers of two different lengths were visualized by electrophoresis in 1% Seakems LE agarose (BioWhittaker) gels using 1 TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA), containing 200 ng ml1 ethidium bromide. To screen for polymorphism in the caprine IGHA gene, all amplimers were subjected to SSCP analysis. A 0.7-ml aliquot of each amplimer was mixed with 7 ml of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol), and after denaturation at 95 1C for 5 min, samples were rapidly cooled on wet ice and then loaded on 16 cm 18 cm, 14.5% acrylamide:bisacrylamide (37.5:1) (Bio-Rad) gels containing 1% glycerol. Electrophoresis was performed using Protean II xi cells (Bio-Rad), at 410 V for 18 h at 17 1C in 0.5 TBE buffer, and gels were silverstained according to the method of Bassam et al. [12]. Under these conditions, three unique SSCP banding patterns could be detected (Fig. 1). One to two patterns were observed for each animal, which was consistent with there either being homozygous or heterozygous genotypes. To determine the nucleotide sequences corresponding to the different SSCP patterns, genomic DNA samples representative of different SSCP patterns were selected for amplification using the proofreading enzyme ProofStart DNA polymerase (Qiagen) under the conditions described above.
∗01
∗03
∗02
∗02 ∗03
∗01 ∗03
∗01 ∗02
1
2
3
4
5
6
Fig. 1. PCR-single-strand conformational polymorphism of the caprine IGHA gene. Representative samples of three unique SSCP banding patterns corresponding to three allelic sequences *01, *02 and *03 are shown. PCR products were amplified from genomic DNA (lanes 1, 3–6) and a plasmid cloned IGHA fragment (lane 2).
After the addition of an A-overhang to the bluntended PCR products using an A-Addition Kit (Qiagen), amplimers were then cloned into the pDrive Cloning vector (Qiagen), and transformed into competent Escherichia coli cells (One ShotTM INVaF’, Invitrogen), following the manufacturers’ instructions. For each transformation, six or twelve insert positive clones, for ‘‘single’’ or ‘‘double’’ SSCP patterns, respectively, were selected and incubated overnight in Terrific broth (Invitrogen) at 37 1C in a shaking rotary incubator (225 rpm). Plasmids were recovered from bacterial cells by boiling for 10 min in 0.8% (vol/vol) Triton X-100 solution and following centrifugation at 12,000 g, 1 ml of the supernatant was used as a template for PCR amplification using Taq DNA polymerase (Qiagen). Amplimers from these clones and the corresponding genomic DNA were run adjacent to each other on SSCP gels for comparison of the SSCP patterns, and only those clones for which the SSCP patterns matched those of the corresponding genomic DNA were selected for subsequent DNA sequencing. Plasmids from the selected clones were extracted using a QIApreps Spin Miniprep kit (Qiagen), and were then sequenced in both directions using M13 forward and reverse primers at the Waikato DNA Sequencing Facility, the University of Waikato, New Zealand. Identical sequences obtained from at
ARTICLE IN PRESS H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
least three clones from different goats, or independent PCR amplifications from the same goat, were subjected to further sequence analysis. Sequence analysis using DNAMANTM (Version 5.2.10, Lynnon BioSoft, Vaudreuil, Canada), revealed that these unique SSCP patterns represented three different nucleotide sequences, two sequences of 316 bp and a sequence of 310 bp. All of these sequences were novel, but shared high homology to the IGHA sequences from a variety of species, with the highest homology being to sheep sequences (GenBank accession no. AY956424–AY956426; [9]). There were either one or two unique sequences identified in individual goats, and this suggests that the sequences isolated in this study represent different alleles of the caprine IGHA gene. The sequences from both breeds were identical for each of the alleles. These alleles were named followed the rules proposed by the international ImMunoGeneTics information systems (IMGT) ([13]; http:// imgt.cines.fr/) and sequences were deposited into the NCBI GenBank with the accession no. DQ118110–DQ118112. The three caprine IGHA nucleotide sequences were predicted to encode three different IGHA molecules, two with a longer hinge and one with a shorter hinge. The two ‘‘long-hinged’’ IGHA molecules (*01 and *02) had an identical hinge of nine residues and only differed at a single residue at the carboxyl-end of the CH1 region. The ‘‘shorthinged’’ IGHA molecule (*03) however, not only
743
was two amino acids shorter, but also possessed a different nucleotide and amino acid sequence in the hinge region (Fig. 2). The considerable differences between the ‘‘long-hinged’’ and ‘‘short-hinged’’ sequences raise a question of whether these sequences represent two loci of the IGHA gene as the case in humans and some primates [3]. However, as no more than two sequences were identified in any single goat, it is unlikely that these sequences are from more than one locus. This suggests a single IGHA gene in goats, consistent with the results reported in sheep and other species [3,9]. Two of the caprine sequences, caprine-IGHA*01 and caprine-IGHA*02, shared close homology to ovine sequences (ovine-IGHA*02 and ovineIGHA*03). This is consistent with the close relatedness of these species as evidenced by the similarity of alleles of their MHC genes [14,15]. However, the remaining caprine sequence (caprine-IGHA*03) had its 50 end similar or identical to ovine and caprine sequences while its 30 end was identical to the bovine sequence (GenBank accession no. AF109167; [16]) in the hinge-coding region. This suggests that either these similar sequences have arisen independently by convergent evolution, or that the IGHA hinge region sequences pre-date the divergence of goats, sheep and cattle. Sequence alignment of the caprine IGHA amino acid sequences using DNAMANTM revealed that variation was located at the carboxyl-end of CH1 and in the hinge region (Fig. 3). This pattern of
Fig. 2. Nucleotide sequences of the three caprine IGHA alleles. Nucleotides in exons are indicated in upper-case and those in the intron are in lower-case. Nucleotides identical to the top sequence are presented by dashes and dots have been introduced to improve the alignment.
ARTICLE IN PRESS 744
H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745
Fig. 3. Alignment of the predicted amino acid sequences of the ovine, caprine and bovine IGHA gene. Amino acids are presented in oneletter code. A dash represents a sequence identical to the top, and dots have been introduced to improve the alignment. The most polymorphic region is shaded and the hinge region is boxed. The sequences of ovine-IGHA*01, *02, *03 and bovine-IGHA*01 are from the GenBank with accession no. AY956424, AY956425, AY956426 and AF109167, respectively.
polymorphism is similar to that reported in sheep [9]. As the carboxyl-end of CH1 and the hinge region are mostly affected, the possible consequences of this variation in the IGHA gene could be twofold. Firstly, the impact of length variation on hinge structure has been demonstrated with crystallization analysis showing that the long hinge of human IgA1 has an extended structure while the short hinge of IgA2 is compact [10]. The short hinge of caprine IGHA contained only seven amino acid residues, which is the shortest hinge reported in ruminants. Sequence variation may also affect the structure of the hinge and hence the hinge flexibility. An interesting characteristic of variation in the caprine IGHA gene was variation in the number and position of cysteine residues in the polymorphic region. If some of the cysteines form disulfide bonds, they may alter the flexibility of the hinge. IgA with a more flexible hinge may bind more effectively to antigens. Secondly, it has been shown that a single amino acid mutation in the hinge region can change the susceptibility of human IgA1 to streptococcal IgA1 protease [17]. Variation in hinge sequence may create or destroy amino acid motifs sensitive to cleavage by bacterial or parasitic proteases. Overall the consequences of caprine IGHA polymorphism await detailed study of the proteins encoded by these alleles. We thank W.-K. Lin and Y.-S. Lin for technical assistance.
[2] [3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [1] Flanagan JG, Lefranc MP, Rabbitts TH. Mechanisms of divergence and convergence of the human immunoglobulin
[14]
alpha 1 and alpha 2 constant region gene sequences. Cell 1984;36:681–8. Lefranc MP, Lefranc G, editors. The immunoglobulin factsbook. London, UK: Academic Press; 2001. Woof JM, Kerr MA. IgA function—variations on a theme. Immunology 2004;113:175–7. Spieker-Polet H, Yam PC, Knight KL. Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues. J Immunol 1993;150:5457–65. Phillips-Quagliata JM. Mouse IgA allotypes have major differences in their hinge regions. Immunogenetics 2002;53: 1033–8. Scinicariello F, Attanasio R. Intraspecies heterogeneity of immunoglobulin alpha-chain constant region genes in rhesus macaques. Immunology 2001;103:441–8. Brown WR, Kacskovics I, Amendt BA, Blackmore NB, Rothschild M, Shinde R, et al. The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C alpha intron. J Immunol 1995;154: 3836–42. Peters IR, Helps CR, Calvert EL, Hall EJ, Day MJ. Identification of four allelic variants of the dog IGHA gene. Immunogenetics 2004;56:254–60. Zhou H, Hickford JGH, Fang Q. Polymorphism of the IGHA gene in sheep. Immunogenetics 2005;57: 453–7. Furtado PB, Whitty PW, Robertson A, Eaton JT, Almogren A, Kerr MA, et al. Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1. J Mol Biol 2004;338:921–41. Senior BW, Woof JM. Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases. Infect Immun 2005;73:1515–22. Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80–3. Lefranc MP, Giudicelli V, Kaas Q, Duprat E, JabadoMichaloud J, Scaviner D, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2005;33:D593–7. Hickford JGH, Zhou H, Slow S, Fang Q. Diversity of the ovine DQA2 gene. J Anim Sci 2004;82:1553–63.
ARTICLE IN PRESS H. Zhou et al. / Developmental and Comparative Immunology 30 (2006) 741–745 [15] Zhou H, Hickford JGH, Fang Q. Polymorphism of the DQA2 gene in goats. J Anim Sci 2005;83:963–8. [16] Brown WR, Rabbani H, Butler JE, Hammarstrom L. Characterization of the bovine C alpha gene. Immunology 1997;91:1–6.
745
[17] Batten MR, Senior BW, Kilian M, Woof JM. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases. Infect Immun 2003;71:1462–9.