Immune responsive role of MHC class II DQA1 gene in livestock

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1 LIVESTOCK RESEARCH INTERNATIONAL Journal homepage: Immune responsive role of MHC class II DQA1 gene in livestock REVIEW ARTICLE RK Vandre 1*, GR Gowane 2, AK Sharma 3 and SS Tomar 4 1# College of Veterinary science and Animal husbandry, Rewa (MP) , India 2 Central Sheep and Wool Research Institute, Avikanagar, via-jaipur (Rajasthan),India 3 Temperate Animal Husbandry Division, Indian Veterinary Research Institute, Mukteshwar (UK) , India. 4 College of Veterinary science and Animal husbandry, Jabalpur (MP) , India. *Corresponding Author: R.K. Vandre rajvetvan@gmail.com Received: 23/02/2014 Revised: 13/03/2014 Accepted: 15/03/2014 Abstract Major histocompatibility complex (MHC) molecules play a major role in immunological defense against pathogens. MHC is a group of genes on a single chromosome that codes the MHC antigens. MHC genes are inherited as a group (haplotypes), one from each parent, thus MHC genes are codominant in each individual. MHC molecules that function in the recognition event, which is termed antigen presentation, are polymorphic (genetically diverse) glycoprotein s found on cell membranes. The MHC participates in the development of both humoral and cell mediated immune responses. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes. The MHC system in cattle is known as the bovine leukocyte antigen (BoLA). A major rearrangement within the class II region has led to the division of the BoLA region into two distinct sub-regions such as class IIa and class IIb, on chromosome 23. The class IIa sub-region contains the functionally expressed DR and DQ genes. These gene products (the DR and DQ molecules) represent the main class II restriction elements for CD4+ T-helper cells. Cattle MHC DQ region comprises five DQA loci and five DQB loci, with exon 2 of the DQA1, DQA2, DQA3, DQB1, and DQB2 genes being highly polymorphic. The MHC Class II DQ genes encode a heterodimer consisting of α chain: DQA and β chain: DQB. Unlike the DR genes, both are polymorphic. Therefore several genes of BoLA which are not considered and has significance role should be investigated thoroughly for generating detailed knowledge regarding antigen presentation in livestock. Research on BoLA has yielded a few markers for disease, which can be used in the breeding programs; however more hard work on allele mining in this part of the livestock genome is required. Introduction Genetic variation in parasite and host and relative distribution across space and time is of great interest and serve as a basis for adaptive change. Spatial population structure can strongly influence the process of co-adaptation between parasite and host and the evolution of virulence (Biek and Real, 2010). The large number of potential host species, both wild and domestic, and their complex movement pattern clearly makes spatial inference particularly challenging in case of many infectious diseases. The increasing information on genetics of host and parasites and their interaction at molecular level can lead to insights into Keywords: BoLA, bovine, disease resistance, genetic variation, major histocompatibility complex, MHC. disease emergence and control. Plasticity in the host genome especially for the genes responsible for disease resistance gives an advantage to host against pathogens with respect to protection. Therefore, studying the variability in the host population for disease resistance genes is of utmost importance in practicing genetics of disease resistance. MHC is a large genomic region or gene family found in most vertebrates that encodes MHC molecules. MHC molecules play an important role in the immune system and autoimmunity. Proteins are continually synthesized and destroyed in the cell. These include normal proteins (self) and microbial pathogens (nonself). The MHC proteins act as "signposts" that Livestock Research International January-March, 2014 Vol 2 Issue 1 Pages 01-07

2 serve to alert the immune system if foreign material is present inside a cell. They achieve this by displaying fragmented pieces or antigens on the host cell's surface. The constitutive presentation of MHC: peptide on cell surfaces allows for pathogen surveillance by immune cells, usually a T cell or natural killer (NK) cell. If activating T or NK cell surface receptors recognize MHC: peptide through binding interactions, it can activate the immune cell and lead to the development of an immune response against the presented antigen. Because MHC genes must defend against a great diversity of microbes in the environment, the MHC molecules (coded for by the MHC genes) must be able to present a wide range of peptides. MHC genes achieve this through several mechanisms: (1) the MHC locus is polygenic, (2) MHC genes are highly polymorphic and numerous alleles have been described, and (3) several MHC genes are codominantly expressed. MHC complex is a group of genes on a single chromosome that codes the MHC antigens. Histocompatibility molecules of one individual act as antigens when introduced into a different individual. George Snell, Jean Dausset and Baruj Benacerraf received the Nobel Prize in 1980 for their contributions to the discovery and understanding of the MHC in mice and humans. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes. These antigens and their genes can be divided into three major classes: class I, class II and class III. It has been studied that two previously described cattle DQA loci are represented by the genomic clones W1 and A5 (Sigurdardóttir et al., 1991b; van der Poel et al., 1990). Because of sequence similarities between W1 and the sheep DQA1 genes on one hand and between A5 and sheep DQA2 on the other, the cattle loci were designated BoLA DQA1 and BoLA-DQA2, respectively (Ballingall et al., 1997; Fabb et al., 1993; Scott et al., 1991; Wright and Ballingall, 1994). Cattle haplotypes carry either a single DQA1 locus, or a single DQA1 locus together with a single DQA2 locus, or two DQA2 loci (Sigurdardóttir et al., 1991b). Recently, marked sequence differences between the alleles of the two DQA2 loci were found consistent with a third DQA locus, BoLA-DQA3 (Ballingall et al., 1997, 1998). Considerable diversity between two reported DQA3 allelic lineages suggested an additional BoLA-DQA4 locus (Ballingall et al., 1997, 1998). BoLA DQA1 exon II polymorphism studies in livestock The MHC Class II DQ genes encode a heterodimer consisting of α chain: DQA and β chain: DQB. Unlike the DR genes, both are polymorphic. In humans the DQ genes have been associated with autoimmune disease; however, no such associations have been noted in cattle. To date 51 DQA and 74 DQB alleles have been identified ( This region has a further layer of complexity unique to cattle, as in approximately half of the known haplotypes the DQ genes are duplicated (Andersson et al., 1988; Ballingall et al., 1997). The MHC class II molecules bind and present foreign peptides to CD4 + T lymphocytes, and extensive research has demonstrated a high level of variation concentrated in the peptide binding regions at several MHC loci across a wide range of species (Meyer et al., 2001). And when the genes are duplicated they are expressed (Xu et al., 1994). This has led to difficulties in accurately typing the alleles at the loci. Moreover it has meant that research on polymorphisms in the DQ genes has been limited, especially when compared to DRB3. Little is known about how BoLA DQ polymorphisms in the peptide binding cleft (PBC) influence the immune response to pathogens. BoLA DQA1 gene encode a mature protein of 233 amino acid (Ballingall et al., 1997) Davies et al. (1997) in cattle showed, that The DQ region consists of five DQA loci and five DQB loci, with exon 2 of the DQA1, DQA2, DQB1, and DQB2 genes being highly polymorphic. Ballingall et al. (1997) sequenced new PCR-RFLP patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families. Takeshima et al. (2007) showed that DQA1*0101 was the most common allele in British Frisian cattle (6 of 13, 46%), DQA1*0101 is mastitisassociated allele (Park et al., 2004). DQA1*10011 allele was the most frequent in Danish Black Pied (9 of 20 cattle, 45%). DQA1*0103 allele was most frequent in Danish Red (5 of 8 cattle, 63%), and DQA1*0301 allele was most frequent in Jersey (4 of 5 cattle, 80%). 2

3 Further, Takeshima et al. (2007) reported that alignments of nucleotide sequences and deduced amino acid sequences of BoLA-DQA1 showed 15 distinct alleles in term of nucleotide sequence and 13 distinct alleles in term of amino acid sequence. Miyasaka et al. (2011) worked on the diversity of bovine MHC class II DQA1 alleles in different herds of Japanese black and Holstein cattle in Japan, the distribution of frequencies for the BoLA DQA1 alleles are ranged per herd from 6 to 11. Donald et al. (2005) worked on the characterization of Bison MHC class IIa haplotypes, showed the Polymorphism of DRB3, DQA, and DQB genes. The 14 bison had 12 DRB3, 11 DQA, and 10 DQB alleles. Six of the DRB3 alleles were identical to alleles previously identified by Mikko et al. (1997); the other six alleles were new. The similarity of the DQA subtype configurations found in cattle, bison, and sheep is strong evidence for the trans-species evolution of MHC class II haplotypes in the family Bovidae (Klein et al., 1987). Brown et al. (1993a) worked on buffalo, showed that two groups of buffalo DQA1 and DQA2 alleles showed most of the residue changes at the PBS. Niranjan et al. (2009) worked on Two cdna sequences of water buffalo DQA1 (DQA*0101) and -DQA2 (DQA*2001). Niranjan et al. (2010) worked on the Allelic diversity at MHC class II DQ loci in Murrah buffalo, showed that DQA*01, DQA*02, DQA*04 and DQA*05 sequences were of DQA1 type and showed high homology with previously described buffalo DQA*0101 allele and DQA*03, DQA*06 and DQA*07 were of the DQA2 type, showing high homology with the DQA*2001 allele of buffalo. In study of Brown et al. (1993b), there were two pairs of ovine DQA1 alleles (*0101 and *0102; *0103 and *0104), which only had one AA difference between alleles within the pair. Ballingall et al. (1998) confirmed that the ovine MHC DQA1 is a highly polymorphic locus. The level of diversity detected for the ovine DQA1 locus appears to be lower than that for the human DQA1 (22 alleles; Marsh et al., 2002), but is similar with that for DQA1 in cattle. Snibson et al. (1998) showed that the nucleotide sequence of the exon 2 of the Ovine DQA1 and DQA2 genes with those of cattle has shown that the ovine DQA1 and DQA2 genes are more similar to their bovine counterparts than to each other. Hickord et al. (2011) worked on the ovine MHC-DQA2 polymorphism, showed that is the more common allele /haplotype such as *0101-*1401,*1201, *0602 are strongly associated with the presence of additional DQA2 allele or haplotypes. Long et al. (2004) observed that using rabbit leukocyte antigen (RLA), RLA-DQA gene had a very high polymorphism both in the DNA and AA sequences. Methodology for analysis of polymorphism of BoLA DQA1 exon II There are different methods which have been used previously in the polymorphism study of DQA gene that is polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP), PCR-single-strand conformation polymorphism (SSCP) and sequence based typing (SBT). Restriction fragment length polymorphism of the BoLA DQA1 exon II in cattle In molecular biology, restriction fragment length polymorphism, or RFLP (commonly pronounced rif-lip ), is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In addition to genetic fingerprinting, RFLP was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing. ( d/rflp.html) DQA and DQB RFLP patterns are simpler to interpret than DRB patterns. Furthermore, because some or all of the haplotypes with duplicated DQ genes express both sets of genes (Bissumbhar et al., 1994), DQ RFLP patterns probably reflect expressed polymorphism reasonably well. The number of DQA and DQB genes present in different haplotypes can be deduced from the number of fragments detected. Class IIa haplotypes with DQA*1, DQB*1, DQA*2, DQB*2, DQA*3, DQB*3, DQA*4, DQB*4 and DQA*14, DQB*14 have single DQA and DQB genes; haplotypes with DQA*13, DQB*13 have 2 DQA genes and 1 DQB gene; and the remaining haplotypes have duplicated DQA and DQB genes (Andersson and Rask, 1988; Sigurdardóttir et al., 1991b). Ballingall et al. (1997) did nucleotide sequence analysis of new PCR-RFLP 3

4 patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families. Single-strand conformation polymorphism (SSCP) analysis of the BoLA DQA1 exon II in cattle The ultimate character that can be used to distinguish species is variation in DNA sequence between homologous genes or regions. The distinguishing patterns obtained with PCR-SSCP are sequence dependant and utilize minor nucleotide differences across several hundred bases of sequence, but without recourse to sequencing. PCR-SSCP is a simple procedure where denatured PCR products are electrophoresed through a non-denaturing neutral polyacrylamide gel. The single strands adopt primary conformations that are dependent on their nucleotide sequence and this determines the rate at which they migrate through the gel matrix. Each PCR product with a different sequence therefore, will be theoretically represented by two bands corresponding to the two strands of the amplified molecule. SSCP has been shown to be able to detect single base changes in 99% of PCR products between 100 and 300 base pairs in length although this limit reduces to 89% with products between 300 and 450 base pairs (Hayashi, 1991; Hayashi and Yandell, 1993). However, the detection of minor sequence variation has been reported in molecules up to 775 nucleotides in length (Orti et al., 1997) although careful optimization of conditions is required. Optimization of the technique must be carried out empirically (Hayashi and Yandell, 1993; Orita et al., 1989) since it is not possible to predict the optimal conditions for band separation in advance (Spinardi et al., 1991), but once achieved; the reproducibility of profiles enables easy comparison between samples. Zhou and Hickford (2004) studied variation in the ovine DQA1 gene in a large number of sheep of different breeds using SSCP analysis, cloning, and sequencing. In their study 14 sequences identified, eight were identical to previously published ovine DQA1 sequences, whereas the remaining six sequences were unique, but shared close homology to previously published DQA1 sequences derived from sheep and cattle. Zhou and Hickford (2005) studied polymorphism of DQA2 gene in goat by the PCR- SSCP method, found 11 unique SSCP banding patterns of DQA2 gene. Cloning and sequencing for the identification of alleles The PCR-RFLP and PCR-SSCP methods give different pattern of BoLA DQA1 gene. The representative PCR samples of different pattern are use for cloning of DQA1 gene in vector and then after cloned product are used for sequencing. Niranjan et al. (2010) used cloning and sequencing for identification of allelic diversity at MHC class II DQ loci in Murrah buffalo. Microarray: The microarray is test for the presence of a nucleic acid sequence by hybridizing a probe bound to a matrix to the target sequence. Many different probes can be bound to the same matrix. Therefore, a single sample can be evaluated for many different target sequences simultaneously. Two type of microarray are Expression Arrays - test for mrna expressed in a tissue and Sequencing Arrays - test for nucleotide sequence in a fragment of DNA sequencing by hybridization.this is ideal for detection of single nucleotide polymorphisms (SNPs). Park et al. (2004) used microarray method for DQA gene, the DQA typing array was based on 47 sequences from the BoLA Nomenclature Web Site. This array was comprised of 8 series of exon 2 probes (15 for codons 9-16, 8 for codons 21-30, 11 for codons 32-39, 10 for codons 40-48, 19 for codons 49-58, 13 for codons 59-66, 21 for codons and 17 for codons 75-81) that define a minimum of 17 DQA haplotypes. Sequence based typing of DQA1 exon II in cattle Genotyping of BoLA is relatively complex because the genes within this family are extremely polymorphic. The genetic polymorphism of class II α and β genes occurs predominantly in exon 2 encoding the antigen binding site. BoLA-DQA typing has been achieved using restriction fragment length polymorphism (RFLP) analysis (Joosten et al., 1992) polymerase chain reaction (PCR)-RFLP (Ballingall et al., 1997) and sequencing of genomic DNA, cdnas, or cloned PCR products (van der Poel, 1990). Recently, Park et al. (2004) established a microarray-based class I, DRB3, and DQA typing methods. Nevertheless, these three typing methods were their ability to define all alleles including new alleles was limited. However, sequencing of cloned PCR product is too labour 4

5 intensive for applying in a more samples of cattle. More rapidly sequence-based typing (SBT), as developed for the BoLA DRB3 locus would improve the resolution and the accuracy of MHC allele typing in animals (Takeshima et al., 2001 and Miltiadou et al., 2003). The SBT method has the great advantage that it analyzes the complete sequence, including both conserved and polymorphic positions. The 3 -end of exon 2 of BoLA-DQA gene was conserved among DQA1 to DQA5 loci ( projects.roslin.ac.uk/bola/dqanuc.html), it is difficult to design PCR primers that are truly locus specific. Previously, three sets of primers have been constructed for amplification of sequences of BoLA-DQA1 exon 2 (Gelhaus et al., 1995). However, all these primers appeared to be unsuitable for PCR-SBT method in which a single amplification locus is used as a direct sequencing template because these primers can coamplify more than two DQA loci. Takeshima et al. (2007) at Japan, worked on the SBT of DQA1 exon 2 of cattle standardized an approach which uses two stages PCR for amplification of the desired sequence. Association of DQA1 polymorphism with immune response to various diseases in different animals MHC molecules play a major role in immunological defense against pathogens. These molecules loaded with peptides derived from invading pathogen are recognized by the immune system to produce a highly effective and specific response against foreign pathogens (Benacerraf, 1981). These genes have attracted much attention in farm animals due to the need of improved methods of disease control through the design of novel vaccines and selection of disease resistant animals (Gowane et al., 2013). Norimine and Brown (2005) identified that the product of the interhaplotype DQ molecule, DQA*2206/DQB*1301, as the MHC class II restriction element for MSP1a peptide B and the product of the intrahaplotype-matched allelic pair, DQA* 2206/DQB*1402, was functional in presenting MSP1a peptide F3-5. This indicates that the DQA*2206 allelic product plays a role in presenting at least two different CD4+ T-cell epitopes derived from MSP1a by pairing with DQB allelic products derived from the same and different haplotypes. Glass et al. (1991) showed that most of BoLA class II haplotypes which were examined, associated with FMDV- specific T-cell and antibody response, although few haplotypes were associated with low or non responsiveness even in presence of another haplotypes which conferred responsiveness. There was consistent qualitative difference between haplotypes, which suggested that different BoLA DR and DQ molecules bind different fragments of the 40-mer peptide with different affinities (Glass and Millar, 1994). According to Glass et al. (2000), the MHC of cattle encodes two distinct isotopes of class II molecules, DR and DQ. Unlike humans, cattle lack the DP locus and about half the common haplotypes express duplicated DQ genes. The number and frequency of DQA and DQB alleles means that most cattle are heterozygous. Norimine et al. (2004) worked on the cattle MHC class II, found that Babesia bovis small heat shock protein (HSP20) is recognized by CD4+ T lymphocytes from cattle that have recovered from infection and are immune to challenge. Schwaba et al. (2009) worked on association of DQA1 alleles with susceptibility to Neospora caninum and reproductive outcome in Quebec Holstein cattle and reported that no association was found between allele frequency distribution of DQA genes and infection with N. Caninum. Diaz et al. (2003) worked on the incidence rate of the Type 1 diabetes in human population, Santiago and reported that DQA1 and DQB1 gene related to the susceptibility of diabetes specially alleles DQA1 *0301, DQA1 *0501 and DQB1 *0201, DQB1 *0302 strongly associated with Type 1 diabetes. Conclusion In conclusion, it is clear that both MHC and non-mhc genes play a role in determining the responses of cattle to vaccination. Cattle have a complex set of BoLA class I and class II genes, with DQ-duplicated haplotypes giving rise to more expressed restriction elements than might be predicted from the number of genes present. In the future, both QTL studies and microarray experiments will be used to identify non-mhc genes, as well as to quantify the relative contributions of different genes that play a role in vaccine responsiveness. Ultimately, these studies should provide valuable information that will be essential for the development of more effective and safe vaccines for livestock world-wide study of the major histocompatibility complex assumes importance because of the critical role it plays in the immune system of the animal. The extensive structural polymorphism of class II molecules is responsible for the differences among individuals in immune response to infectious agents. This high degree of polymorphism 5

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