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Cloning and characterization of ovine β2-microglobulin cDNAs
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Veterinary Immunology and Immunopathology 123 (2008) 360–365 www.elsevier.com/locate/vetimm
Short communication
Cloning and characterization of ovine b2-microglobulin cDNAs Changxin Wu a,b,*, Ian McConnell a, Barbara Blacklaws a a
Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK b Department of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China Received 9 August 2007; received in revised form 13 February 2008; accepted 27 February 2008
Abstract Beta-2-microglobulin (b2m) is the light chain of the major histocompatibility complex (MHC) class I cell surface heterodimer. b2m is well conserved across most species with few polymorphisms seen within species. The aims of this study were to clone and express ovine b2m and investigate if allelic variation of ovine b2m exists. Ovine b2m clones were isolated from five sheep of three breeds by reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that four ovine b2m sequences were obtained. Within breeds and individual animals there was evidence of allelic variation of ovine b2m. An expression system was established to express one of the alleles with an ovine MHC class I cDNA clone in human embryo kidney cells (HEK293) and quail cells (QT35). Transfection experiments showed that ovine b2m was expressed and directed the expression of ovine MHC class I heavy chain to the cell surface of the transfected cells. Both bovine and human b2m supported ovine MHC class I heavy chain cell surface expression. # 2008 Elsevier B.V. All rights reserved. Keywords: Sheep; Beta-2-microglobulin; Allele; MHC class I
1. Introduction Beta-2-microglobulin (b2m), an 11.8 kDa protein, has been found in a variety of physiological fluids as well as on the surfaces of all nucleated cells. On cells b2m is the non-covalently associated light chain of the major histocompatibility complex class I (MHC class I) glycoprotein (Grey et al., 1973). b2m plays an essential role in the structure and overall function of MHC class I by stabilizing classical MHC class I (Ia) with bound peptide (Van Kaer et al., 1992; Zijlstra et al., 1990). It is required for antigen recognition and peptide presentation to CD8+ T cells to initiate cell mediated
* Corresponding author at: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. Tel.: +44 1223 337648; fax: +44 1223 337610. E-mail address: [email protected] (C. Wu). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.02.017
cytotoxicity (Vitiello et al., 1990). b2m also interacts with many non-classical MHC class I molecules (Ib) such as HLA-E, -F, -G, and HFE in human and CD1, the neonatal Fc receptor, and H2-Q supporting their functions (Rodgers and Cook, 2005). Ia and Ib with malformed b2m are not stable on the cell surface, leading to reduced antigen presentation and increased susceptibility to invasion by intracellular pathogens (Ferrone and Marincola, 1995). Genes coding for b2m have been identified and characterized in many different animals, including mammals, birds, fish and prototherians. It is located on a different chromosome to that of MHC class I heavy chain genes (Goodfellow et al., 1976). The threedimensional protein structure of b2m has also been published (Becker and Reeke, 1985). In contrast to the highly polymorphic MHC class I heavy chain molecules, b2m molecules are either invariant or minimally polymorphic within species. In this paper the cloning,
C. Wu et al. / Veterinary Immunology and Immunopathology 123 (2008) 360–365
sequencing and expression of ovine b2m cDNA were carried out to show the clones obtained were functional and that there was limited polymorphism in the ovine b2m gene. 2. Materials and methods
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high fidelity DNA polymerase Pwo (Promega, Southampton, UK) according to the manufacturer’s instructions. PCR was hot started by holding the mixture at 80 8C after the 2 min 95 8C denaturation step then adding the DNA polymerase. This was followed by a 30 cycle PCR: 94 8C for 30 s, 42 8C for 30 s and 72 8C for 30 s, with a final extension step of 72 8C for 10 min.
2.1. Animals 2.4. Cloning and sequencing ovine b2m cDNAs Sheep used in this study were a Soay cross sheep (sheep 308), two Finnish Landrace Dorset crosses (sheep 1237A and B1125) and two Friesland sheep (sheep J74 and J99). All were more than 1 year old and kept in the animal facilities of the Dept of Veterinary Medicine, University of Cambridge, UK and they were used in accordance with procedures laid out in the Animals (Scientific Procedures) Act 1986 of the United Kingdom. 2.2. Primers Two sets of primers were used to isolate ovine b2m cDNAs. These were designed using the 50 and 30 untranslated consensus sequences present in mouse (NM_009735), human (NM_004048), rat (NM_ 012512), pig (AF452448), horse (X69083) and bovine (X69084) b2m cDNAs. ovB2mF1: 50 -C GCT GCA AGG ATG GC-30 (site: 10 to +5); ovB2mF2: 50 -GC AAG CTT AGG ATG GCT CGC TTC GTG GCC-30 (site: 3 to +18); ovB2mR1: 50 -TCT CGA TGG TGC TGC-30 (site: 372–358); ovB2mR2: 50 -GA GGT ACC TGC TTA CAG GTC TCG ATC CCA C-30 (site: 361– 340). All nucleotide positions in primers were taken from the bovine b2m cDNA sequence, X69084 (Ellis et al., 1993), start and stop codons are in bold and restriction enzyme sites (HindIII and KpnI) are underlined. Primers for PCR screening and sequencing insert positive plasmids were T3 and T7. 2.3. Amplification of ovine b2m cDNAs Total RNA was extracted from 1 107 density gradient purified sheep peripheral blood mononuclear cells (PBMC) using the Qiagen RNeasy Minikit (Qiagen, Crawley, UK). First strand cDNA was made using SuperscriptII (Invitrogen, Paisley, UK) in accordance with the manufacturer’s instructions. Amplification of ovine b2m cDNAs was carried out using two different sets of primers (ovB2mF1 + ovB2mR1 and ovB2mF2 + ovB2mR2). The PCR was carried out in a final volume of 50 ml containing 12–20 ng first strand cDNA, and 1 unit of
Ovine b2m cDNA PCR products were subcloned into the SmaI site of pBluescriptSKII+ and insert positive clones were screened by PCR using one vector specific primer (e.g. T3 or T7) and one b2m cDNA specific primer using Taq DNA polymerase (Invitrogen) with a cycling programme: 95 8C for 4 min, then 95 8C for 30 s, 42 8C for 30 s and 72 8C for 30 s, for 25 cycles. Plasmid DNA of insert positive clones was prepared using the Plasmid DNA Miniprep kit (Qiagen) and then sequenced by Sequence Laboratories (Gottingen GmbH, Germany). 2.5. Sequence data analysis Nucleotide sequences were assembled, edited and aligned using software CHROMAS (Version 1.45, Conor McCarthy, School of Health Science, Griffith University, Southport, Queensland, Australia). Multiple alignments of nucleotide and amino acid sequences were performed using CLUSTAL W. The GenBank accession numbers of the 4 sequences of ovine b2m cDNA obtained in this study are EF489533 (sheep1237A/B1125 called ovB2m.1), EF489534 (sheep 308 called ovB2m.2), EF489535 (sheepJ74/J99 called ovB2m.3) and EF489536 (sheepJ77/99 called ovB2m.4). The GenBank accession numbers of cDNA sequences of human and animal b2m used for analyses are as follows: NM_009735 (mouse), NM_012512 (rat), NM_004048 (human), M30683 (chimpanzee), X69084 (cattle), and M84767 (chicken). 2.6. Expression of ovine b2m The strategy to determine whether ovine b2m was expressed functionally was to co-express a b2m clone (ovB2m.1) with a full-length ovine MHC class I heavy chain clone from the same animal (B1125 c25, a clone recently generated in our laboratory, GenBank accession number: EF489540). To do this the two ovine clones were expressed in pcDNA3.1 (Invitrogen) separated by the IRES fragment from polio (pBluescriptSKII+ with the polio IRES (internal ribosome entry site) fragment was
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C. Wu et al. / Veterinary Immunology and Immunopathology 123 (2008) 360–365
kindly provided by Dr. L. Tiley, Dept. of Veterinary Medicine, University of Cambridge). The order of the inserts was ovine b2m, IRES fragment and ovine MHC class I heavy chain: the inserts were placed in the HindIII to KpnI, KpnI to EcoRI, and XhoI to XbaI sites of pcDNA3.1, respectively. The plasmids generated were pcDNA3.1ovB2m containing ovine b2m cDNA only, pcDNA3.1ovMHCIc25 containing ovine MHC class I heavy chain cDNA only, and pcDNAovB2m-IRESovMHCIc25 containing both ovine b2m and MHC class I heavy chain cDNAs. Confluent HEK293 cells (European Collection of Cell Cultures, ECACC No.: 85120602) and QT35 cells (ECACC No.: 93120832) were harvested and seeded in 6 well plates at 1.5 106 cells/well in Dulbecco’s modified Eagle’s medium (DMEM: HEK293 cells) or Glasgow minimal essential medium (GMEM: QT35 cells) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units penicillin per ml and 100 mg streptomycin per ml, and with 10% tryptose phosphate broth in GMEM. Cells were cultured at 37 8C, 5% CO2 overnight then monolayers were gently washed with serum free DMEM or GMEM twice, and transfections were carried out with lipofectin (Invitrogen) or fugene6 (Roche Diagnostics, Lewes, UK) as per the manufacturer’s instructions. With transient transfectants (QT35 cells), after 48 h the medium was replaced with FCS free medium and cultured for a further 72 h before flow cytometry. Stable cell lines (HEK293 cells) were generated by replacing medium with 800 mg/ml G418 (Melford Chemicals, Ipswich, UK) at 48 h post transfection and maintaining this for the next 3 weeks. Cell lines were then maintained in medium with G418 at 200–300 mg/ml. 2.7. Analysis of ovine MHC class I cell surface expression Cell surface expression of ovine MHC class I was detected by flow cytometry on a FACSCaliber Flow
Cytometer using CellQuest software (Becton Dickinson, Oxford, version 3.3). Cells were stained by indirect immunofluorescence with anti-ovine MHC class I specific monoclonal antibodies (mAb) VPM19 (Hopkins and Dutia, 1990) and 41–17 (Serotech, Oxon, UK) (5 mg/ml). Non-specific staining was controlled for with normal mouse serum (1/500) and primary antibodies were detected with 1/100 diluted FITC conjugated rabbit anti-mouse IgG (DakoCytomation, Ely, UK). 3. Results and discussion 3.1. Cloning and sequencing ovine b2m cDNAs RT-PCR with a high fidelity DNA polymerase was used to generate cDNA clones of b2m from five sheep of three breeds. The PCRs gave a single band of about 0.36 kb with both primer pairs used (data not shown). When the internal sequences of the PCR products from B1125 were shown to be the same from the different primer pairs (data not shown), the shorter primer pair (F1 and R1) was used for subsequent PCRs from sheep 1237A, 308, J74 and J99 to minimise the length of primer overlapping the open reading frame. Four unique b2m sequences were found. Each sequence was derived from at least 2 clones from 2 independent PCRs, either from different sheep or from independent PCRs from the same sheep. The size of the ovine b2m open reading frame was 357 bp coding for 119 amino acids in all sheep (Fig. 1). This is the same size as cattle and similar to other mammalian and chicken proteins which are 119 or 120 amino acids long (Fig. 1). Clones from two individual sheep (sheep 1237A and B1125, both Finnish Landrace Dorset crosses) were 100% identical (shown by ovB2m.1 in Fig. 1). Clones from sheep 308 (Soay cross) gave one sequence (shown by ovB2m.2 in Fig. 1). Clones from both sheep J74 and J99 (Friesland sheep) gave two sequences (shown by ovB2m.3 and ovB2m.4 in Fig. 1) indicating that both sheep J74 and J99 were
Fig. 1. Amino acid alignment of ovine b2m sequences compared to other vertebrate species. The predicted contact residues with the a1 (1), a2 (2) and a3 (3) domains of human HLA-A2 are noted below the alignment (Bjorkman et al., 1987). Residues the same as consensus are shown as (-), gaps in the sequence are shown as (.).
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heterozygotes with two b2m alleles expressed in both sheep. Allelic b2m is not common in animals and man, and heterozygotes are rarely reported. Since the allelic forms of mouse b2m were found, allelic variants of b2m have been reported in other animals including wild mice, cattle, owl monkey, chicken and fish (Magor et al., 2004). However, polymorphisms and multiple gene copies for b2m genes are common in some fish species, e.g. carp, barbs, and Siberian sturgeon (Magor et al., 2004). Indeed it has been reported that a single rainbow trout transcribes at least ten distinct b2m cDNAs and a b2m locus may encode three polymorphic genes (Magor et al., 2004). When all four ovine sequences were compared they were between 97.8% and 99.4% identical at the nucleotide level and 96.6% and 99.2% identical at the amino acid level. This level of identity is expected between alleles in a species although one of 5 mouse alleles varies more widely in comparison to the other mouse alleles (Hermel et al., 1993). OvB2m.3 was the same as the consensus sequence of the 4 aligned ovine sequences, and compared with this clone there were three coding changes located at positions 3, 4, and 5 in the signal peptide, and two non-coding changes at position 11 in the signal peptide and 98 in the mature b2m protein in ovB2m.4; 2 nucleotide differences with one coding change compared with ovB2m.2; and 2 nucleotide differences in one codon causing one amino acid change compared with ovB2m.2 (Fig. 1). The predicted amino acid sequences were 95.0%, 75.6%, 67.2% and 47.9% identical to bovine, human, mouse and chicken b2m, respectively. Partial goat b2m amino acid sequence has been published (31 amino acids of mature b2m protein (Groves et al., 1985)), and comparison of these residues between sheep and goat indicated they were 100% identical. The high level of homology with cattle and goat sequence was expected as these species are very closely related. In deed, rat (66.3% homology with sheep) and mouse b2m are 83.2% homologous, and human and chimpanzee (X69084) are 100% homologous. The 22 points of interaction between b2m and heavy chain have been determined from X-ray crystallographic studies of HLA-A2, H2Kb, and H2Db (Bjorkman et al., 1987). Comparison of the predicted ovine peptide with the bovine peptide (Fig. 1) indicated that the 22 amino acid residues predicted to interact with MHC class I heavy chain were highly conserved: specifically, 20 residues were completely conserved, 1 residue showed a conservative substitution (I–V) and one position encoded a charge change (K–E). Both variant positions putatively contact the a3 domain (Fig. 1). None of the
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allelic changes between the 4 ovine sequences affected any of these residues suggesting the changes may not affect structure and function of the variants. Research has indicated that mutated b2m containing structural changes (Trymbulak and Zeff, 1997) causes MHC class I structural changes (Schultz et al., 1998), and the loss of epitope presentation (Van Kaer et al., 1992; Zijlstra et al., 1990) resulting in increased susceptibility to invasion by intracellular pathogens (Ferrone and Marincola, 1995). However, an engineered variant of human b2m with a single serine (S) to valine (V) substitution at residue 55 improves its ability to bind to cell surface HLA-A1, A2 and A3 molecules, facilitates exogenous peptide loading (50–80-fold), and so promotes recognition by peptide-specific T cells (Shields et al., 1998). Mechanisms leading to diversity of b2m alleles are not understood at present. Pressure from pathogens has been suggested as an explanation, but this has been shown not to be the primary cause of the observed diversity in rainbow trout (Shum et al., 1996). 3.2. Expression of ovine b2m with ovine MHC class I heavy chain Several different transfection methods were used to try to express the ovine b2m clones in Daudi cells, a human cell line with a mutation in the b2m start codon and so no expression of b2m protein. This would have allowed ovine b2m expression to be analysed by, e.g. Western blot or flow cytometry using anti-human monoclonal antibodies known to cross-react with ovine b2m. However, we were unable to transfect these cells efficiently. We therefore designed an expression system to determine if expressed ovine b2m was functional using the fact that cell surface expression of the heavy chain of MHC class I molecules requires concomitant expression of b2m (Seong et al., 1988). We used ovB2m.1 (from sheep B1125) with a full-length ovine MHC class I heavy chain cDNA (c25) derived from the same sheep, using an IRES to allow polycistronic mRNA translation. A quail cell line, QT35, was shown not to express surface ovine MHC class I heavy chain in the absence of FCS and expressed ovine b2m (Fig. 2B), thus suggesting that either QT35 cells do not express b2m or quail b2m is unable to support ovine MHC class I expression at the cell surface. FCS is known to contain soluble bovine b2m which may rescue the surface expression of MHC class I heavy chain (Shields et al., 1998), as was indeed shown in Fig. 2E where FCS was added to cells. In the absence of FCS, ovine b2m rescued the surface expression of ovine MHC class I
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Fig. 2. Expression of ovine MHC class I on the surface of transfected cells. QT35 transient transfectants and HEK293 stable transfectants were cultured in FCS free medium and then were screened by flow cytometry for surface expression of ovine MHC class I antigen. Panels are, QT35 cells transfected with: (A) pcDNA3.1; (B) pcDNA3.1ovMHCIc25; (C) pcDNA3.1-ovb2m and pcDNA3.1ovMHCIc25; (D) pcDNA3.1-ovb2m-IRESovMHCIc25; (E) pcDNA3.1ovMHCIc25 cultured in medium with FCS; (F) sheep skin cells; HEK293 cells transfected with: (G) pcDNA3.1; (H) pcDNA3.1ovMHCIc25; and (I) pcDNA3.1-ovb2m-IRES-ovMHCIc25.
heavy chain on the quail cells, when the protein was expressed from pcDNA3.1 either in a polycistronic construct (Fig. 2D) or individual plasmids (Fig. 2C). Stable HEK293 transfectants were also analysed in the absence of FCS. Here the endogenous human b2m was able to support the surface expression of ovine MHC class I heavy chain and this expression was not increased by the addition of ovine b2m in the expression construct (Fig. 2H and I). The homology between human and mouse b2m is 70%, and research has shown that mouse b2m does not support efficient surface expression of human MHC class I heavy chain; it allows surface expression of HLA class I heavy chains at only 20–30% of levels observed for heavy chains assembled with human b2m. However, human b2m can allow mouse MHC class I expression and binds with higher affinity to mouse MHC class I heavy chains than mouse b2m (Pedersen et al., 1995). Of the 22 putative amino acid residues contacting MHC class I heavy chain, chicken
b2m has 13, human 17 and cattle 20 residues conserved with ovine b2m (Fig. 1). Therefore, avian b2m would be predicted to have the lowest or no affinity for ovine MHC class I heavy chain molecules. So it is not surprising that both human and bovine b2m supported ovine MHC class I heavy chain cell surface expression but avian b2m might not as homology between sheep and chicken b2m is very low (see above). In conclusion, we have cloned 4 allelic sequences of ovine b2m from different breeds of sheep and have obtained direct evidence of the existence of allelic variants of ovine b2m. Ovine, bovine and human b2m supported expression of ovine MHC class I molecules on the cell surface. Acknowledgements Changxin Wu was supported by the National Natural Science Foundation of China, the Henry Lester Trust,
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Great Britain-China Education Trust, and the Raymond and Beverly Sackler Trust. We thank Dr. Cyril Barbezange and Dr. Sangeeta Budhia for helpful discussions and Lisa Haring, Paul Tonks and Angela Wheatley for their technical support. References Becker, J.W., Reeke Jr., G.N., 1985. Three-dimensional structure of beta 2-microglobulin. Proc. Natl. Acad. Sci. U.S.A. 82, 4225– 4229. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., Wiley, D.C., 1987. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329, 506–512. Ellis, S.A., Braem, K.A., Payne, L.K., 1993. Nucleotide sequence of cattle beta 2-microglobulin cDNA. Immunogenetics 38, 310. Ferrone, S., Marincola, F.M., 1995. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol. Today 16, 487–494. Goodfellow, P.N., Barnstable, C.J., Bodmer, W.F., Snary, D., Crumpton, M.J., 1976. Expression of HLA system antigens on placenta. Transplantation 22, 595–603. Grey, H.M., Kubo, R.T., Colon, S.M., Poulik, M.D., Cresswell, P., Springer, T., Turner, M., Strominger, J.L., 1973. The small subunit of HL-A antigens is beta 2-microglobulin. J. Exp. Med. 138, 1608–1612. Groves, M.L., Greenberg, R., Farrell Jr., H.M., 1985. Isolation and properties of goat beta 2-microglobulin. Comp. Biochem. Physiol. B 81, 621–627. Hermel, E., Robinson, P.J., She, J.X., Lindahl, K.F., 1993. Sequence divergence of B2m alleles of wild Mus musculus and Mus spretus implies positive selection. Immunogenetics 38, 106–116. Hopkins, J., Dutia, B.M., 1990. Monoclonal antibodies to the sheep analogues of human CD45 (leucocyte common antigen), MHC class I and CD5. Differential expression after lymphocyte activation in vivo. Vet. Immunol. Immunopathol. 24, 331– 346.
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Magor, K.E., Shum, B.P., Parham, P., 2004. The beta 2-microglobulin locus of rainbow trout (Oncorhynchus mykiss) contains three polymorphic genes. J. Immunol. 172, 3635–3643. Pedersen, L.O., Stryhn, A., Holter, T.L., Etzerodt, M., Gerwien, J., Nissen, M.H., Thogersen, H.C., Buus, S., 1995. The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse beta 2m. Eur. J. Immunol. 25, 1609–1616. Rodgers, J.R., Cook, R.G., 2005. MHC class Ib molecules bridge innate and acquired immunity. Nat. Rev. Immunol. 5, 459–471. Schultz, C.S., Rodriguez, R.A., Chew, E.A., Dimaano, C., Li, F.M., Santos, M.D., Nieto, M.C., 1998. Effects of beta2-microglobulin mutations on the alpha-1 helical region of H2-Ld. Immunogenetics 48, 273–282. Seong, R.H., Clayberger, C.A., Krensky, A.M., Parnes, J.R., 1988. Rescue of Daudi cell HLA expression by transfection of the mouse beta 2-microglobulin gene. J. Exp. Med. 167, 288–299. Shields, M.J., Kubota, R., Hodgson, W., Jacobson, S., Biddison, W.E., Ribaudo, R.K., 1998. The effect of human beta2-microglobulin on major histocompatibility complex I peptide loading and the engineering of a high affinity variant. Implications for peptidebased vaccines. J. Biol. Chem. 273, 28010–28018. Shum, B.P., Azumi, K., Zhang, S., Kehrer, S.R., Raison, R.L., Detrich, H.W., Parham, P., 1996. Unexpected beta2-microglobulin sequence diversity in individual rainbow trout. Proc. Natl. Acad. Sci. U.S.A. 93, 2779–2784. Trymbulak, W.P., Zeff, R.A., 1997. Expression of HLA class I molecules assembled with structural variants of human beta2microglobulin. Immunogenetics 46, 418–426. Van Kaer, L., Ashton-Rickardt, P.G., Ploegh, H.L., Tonegawa, S., 1992. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4–8+ T cells. Cell 71, 1205–1214. Vitiello, A., Potter, T.A., Sherman, L.A., 1990. The role of beta 2microglobulin in peptide binding by class I molecules. Science 250, 1423–1426. Zijlstra, M., Bix, M., Simister, N.E., Loring, J.M., Raulet, D.H., Jaenisch, R., 1990. Beta 2-microglobulin deficient mice lack CD48+ cytolytic T cells. Nature 344, 742–746.
Veterinary Immunology and Immunopathology 123 (2008) 360–365 www.elsevier.com/locate/vetimm
Short communication
Cloning and characterization of ovine b2-microglobulin cDNAs Changxin Wu a,b,*, Ian McConnell a, Barbara Blacklaws a a
Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK b Department of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China Received 9 August 2007; received in revised form 13 February 2008; accepted 27 February 2008
Abstract Beta-2-microglobulin (b2m) is the light chain of the major histocompatibility complex (MHC) class I cell surface heterodimer. b2m is well conserved across most species with few polymorphisms seen within species. The aims of this study were to clone and express ovine b2m and investigate if allelic variation of ovine b2m exists. Ovine b2m clones were isolated from five sheep of three breeds by reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that four ovine b2m sequences were obtained. Within breeds and individual animals there was evidence of allelic variation of ovine b2m. An expression system was established to express one of the alleles with an ovine MHC class I cDNA clone in human embryo kidney cells (HEK293) and quail cells (QT35). Transfection experiments showed that ovine b2m was expressed and directed the expression of ovine MHC class I heavy chain to the cell surface of the transfected cells. Both bovine and human b2m supported ovine MHC class I heavy chain cell surface expression. # 2008 Elsevier B.V. All rights reserved. Keywords: Sheep; Beta-2-microglobulin; Allele; MHC class I
1. Introduction Beta-2-microglobulin (b2m), an 11.8 kDa protein, has been found in a variety of physiological fluids as well as on the surfaces of all nucleated cells. On cells b2m is the non-covalently associated light chain of the major histocompatibility complex class I (MHC class I) glycoprotein (Grey et al., 1973). b2m plays an essential role in the structure and overall function of MHC class I by stabilizing classical MHC class I (Ia) with bound peptide (Van Kaer et al., 1992; Zijlstra et al., 1990). It is required for antigen recognition and peptide presentation to CD8+ T cells to initiate cell mediated
* Corresponding author at: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. Tel.: +44 1223 337648; fax: +44 1223 337610. E-mail address: [email protected] (C. Wu). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.02.017
cytotoxicity (Vitiello et al., 1990). b2m also interacts with many non-classical MHC class I molecules (Ib) such as HLA-E, -F, -G, and HFE in human and CD1, the neonatal Fc receptor, and H2-Q supporting their functions (Rodgers and Cook, 2005). Ia and Ib with malformed b2m are not stable on the cell surface, leading to reduced antigen presentation and increased susceptibility to invasion by intracellular pathogens (Ferrone and Marincola, 1995). Genes coding for b2m have been identified and characterized in many different animals, including mammals, birds, fish and prototherians. It is located on a different chromosome to that of MHC class I heavy chain genes (Goodfellow et al., 1976). The threedimensional protein structure of b2m has also been published (Becker and Reeke, 1985). In contrast to the highly polymorphic MHC class I heavy chain molecules, b2m molecules are either invariant or minimally polymorphic within species. In this paper the cloning,
C. Wu et al. / Veterinary Immunology and Immunopathology 123 (2008) 360–365
sequencing and expression of ovine b2m cDNA were carried out to show the clones obtained were functional and that there was limited polymorphism in the ovine b2m gene. 2. Materials and methods
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high fidelity DNA polymerase Pwo (Promega, Southampton, UK) according to the manufacturer’s instructions. PCR was hot started by holding the mixture at 80 8C after the 2 min 95 8C denaturation step then adding the DNA polymerase. This was followed by a 30 cycle PCR: 94 8C for 30 s, 42 8C for 30 s and 72 8C for 30 s, with a final extension step of 72 8C for 10 min.
2.1. Animals 2.4. Cloning and sequencing ovine b2m cDNAs Sheep used in this study were a Soay cross sheep (sheep 308), two Finnish Landrace Dorset crosses (sheep 1237A and B1125) and two Friesland sheep (sheep J74 and J99). All were more than 1 year old and kept in the animal facilities of the Dept of Veterinary Medicine, University of Cambridge, UK and they were used in accordance with procedures laid out in the Animals (Scientific Procedures) Act 1986 of the United Kingdom. 2.2. Primers Two sets of primers were used to isolate ovine b2m cDNAs. These were designed using the 50 and 30 untranslated consensus sequences present in mouse (NM_009735), human (NM_004048), rat (NM_ 012512), pig (AF452448), horse (X69083) and bovine (X69084) b2m cDNAs. ovB2mF1: 50 -C GCT GCA AGG ATG GC-30 (site: 10 to +5); ovB2mF2: 50 -GC AAG CTT AGG ATG GCT CGC TTC GTG GCC-30 (site: 3 to +18); ovB2mR1: 50 -TCT CGA TGG TGC TGC-30 (site: 372–358); ovB2mR2: 50 -GA GGT ACC TGC TTA CAG GTC TCG ATC CCA C-30 (site: 361– 340). All nucleotide positions in primers were taken from the bovine b2m cDNA sequence, X69084 (Ellis et al., 1993), start and stop codons are in bold and restriction enzyme sites (HindIII and KpnI) are underlined. Primers for PCR screening and sequencing insert positive plasmids were T3 and T7. 2.3. Amplification of ovine b2m cDNAs Total RNA was extracted from 1 107 density gradient purified sheep peripheral blood mononuclear cells (PBMC) using the Qiagen RNeasy Minikit (Qiagen, Crawley, UK). First strand cDNA was made using SuperscriptII (Invitrogen, Paisley, UK) in accordance with the manufacturer’s instructions. Amplification of ovine b2m cDNAs was carried out using two different sets of primers (ovB2mF1 + ovB2mR1 and ovB2mF2 + ovB2mR2). The PCR was carried out in a final volume of 50 ml containing 12–20 ng first strand cDNA, and 1 unit of
Ovine b2m cDNA PCR products were subcloned into the SmaI site of pBluescriptSKII+ and insert positive clones were screened by PCR using one vector specific primer (e.g. T3 or T7) and one b2m cDNA specific primer using Taq DNA polymerase (Invitrogen) with a cycling programme: 95 8C for 4 min, then 95 8C for 30 s, 42 8C for 30 s and 72 8C for 30 s, for 25 cycles. Plasmid DNA of insert positive clones was prepared using the Plasmid DNA Miniprep kit (Qiagen) and then sequenced by Sequence Laboratories (Gottingen GmbH, Germany). 2.5. Sequence data analysis Nucleotide sequences were assembled, edited and aligned using software CHROMAS (Version 1.45, Conor McCarthy, School of Health Science, Griffith University, Southport, Queensland, Australia). Multiple alignments of nucleotide and amino acid sequences were performed using CLUSTAL W. The GenBank accession numbers of the 4 sequences of ovine b2m cDNA obtained in this study are EF489533 (sheep1237A/B1125 called ovB2m.1), EF489534 (sheep 308 called ovB2m.2), EF489535 (sheepJ74/J99 called ovB2m.3) and EF489536 (sheepJ77/99 called ovB2m.4). The GenBank accession numbers of cDNA sequences of human and animal b2m used for analyses are as follows: NM_009735 (mouse), NM_012512 (rat), NM_004048 (human), M30683 (chimpanzee), X69084 (cattle), and M84767 (chicken). 2.6. Expression of ovine b2m The strategy to determine whether ovine b2m was expressed functionally was to co-express a b2m clone (ovB2m.1) with a full-length ovine MHC class I heavy chain clone from the same animal (B1125 c25, a clone recently generated in our laboratory, GenBank accession number: EF489540). To do this the two ovine clones were expressed in pcDNA3.1 (Invitrogen) separated by the IRES fragment from polio (pBluescriptSKII+ with the polio IRES (internal ribosome entry site) fragment was
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kindly provided by Dr. L. Tiley, Dept. of Veterinary Medicine, University of Cambridge). The order of the inserts was ovine b2m, IRES fragment and ovine MHC class I heavy chain: the inserts were placed in the HindIII to KpnI, KpnI to EcoRI, and XhoI to XbaI sites of pcDNA3.1, respectively. The plasmids generated were pcDNA3.1ovB2m containing ovine b2m cDNA only, pcDNA3.1ovMHCIc25 containing ovine MHC class I heavy chain cDNA only, and pcDNAovB2m-IRESovMHCIc25 containing both ovine b2m and MHC class I heavy chain cDNAs. Confluent HEK293 cells (European Collection of Cell Cultures, ECACC No.: 85120602) and QT35 cells (ECACC No.: 93120832) were harvested and seeded in 6 well plates at 1.5 106 cells/well in Dulbecco’s modified Eagle’s medium (DMEM: HEK293 cells) or Glasgow minimal essential medium (GMEM: QT35 cells) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units penicillin per ml and 100 mg streptomycin per ml, and with 10% tryptose phosphate broth in GMEM. Cells were cultured at 37 8C, 5% CO2 overnight then monolayers were gently washed with serum free DMEM or GMEM twice, and transfections were carried out with lipofectin (Invitrogen) or fugene6 (Roche Diagnostics, Lewes, UK) as per the manufacturer’s instructions. With transient transfectants (QT35 cells), after 48 h the medium was replaced with FCS free medium and cultured for a further 72 h before flow cytometry. Stable cell lines (HEK293 cells) were generated by replacing medium with 800 mg/ml G418 (Melford Chemicals, Ipswich, UK) at 48 h post transfection and maintaining this for the next 3 weeks. Cell lines were then maintained in medium with G418 at 200–300 mg/ml. 2.7. Analysis of ovine MHC class I cell surface expression Cell surface expression of ovine MHC class I was detected by flow cytometry on a FACSCaliber Flow
Cytometer using CellQuest software (Becton Dickinson, Oxford, version 3.3). Cells were stained by indirect immunofluorescence with anti-ovine MHC class I specific monoclonal antibodies (mAb) VPM19 (Hopkins and Dutia, 1990) and 41–17 (Serotech, Oxon, UK) (5 mg/ml). Non-specific staining was controlled for with normal mouse serum (1/500) and primary antibodies were detected with 1/100 diluted FITC conjugated rabbit anti-mouse IgG (DakoCytomation, Ely, UK). 3. Results and discussion 3.1. Cloning and sequencing ovine b2m cDNAs RT-PCR with a high fidelity DNA polymerase was used to generate cDNA clones of b2m from five sheep of three breeds. The PCRs gave a single band of about 0.36 kb with both primer pairs used (data not shown). When the internal sequences of the PCR products from B1125 were shown to be the same from the different primer pairs (data not shown), the shorter primer pair (F1 and R1) was used for subsequent PCRs from sheep 1237A, 308, J74 and J99 to minimise the length of primer overlapping the open reading frame. Four unique b2m sequences were found. Each sequence was derived from at least 2 clones from 2 independent PCRs, either from different sheep or from independent PCRs from the same sheep. The size of the ovine b2m open reading frame was 357 bp coding for 119 amino acids in all sheep (Fig. 1). This is the same size as cattle and similar to other mammalian and chicken proteins which are 119 or 120 amino acids long (Fig. 1). Clones from two individual sheep (sheep 1237A and B1125, both Finnish Landrace Dorset crosses) were 100% identical (shown by ovB2m.1 in Fig. 1). Clones from sheep 308 (Soay cross) gave one sequence (shown by ovB2m.2 in Fig. 1). Clones from both sheep J74 and J99 (Friesland sheep) gave two sequences (shown by ovB2m.3 and ovB2m.4 in Fig. 1) indicating that both sheep J74 and J99 were
Fig. 1. Amino acid alignment of ovine b2m sequences compared to other vertebrate species. The predicted contact residues with the a1 (1), a2 (2) and a3 (3) domains of human HLA-A2 are noted below the alignment (Bjorkman et al., 1987). Residues the same as consensus are shown as (-), gaps in the sequence are shown as (.).
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heterozygotes with two b2m alleles expressed in both sheep. Allelic b2m is not common in animals and man, and heterozygotes are rarely reported. Since the allelic forms of mouse b2m were found, allelic variants of b2m have been reported in other animals including wild mice, cattle, owl monkey, chicken and fish (Magor et al., 2004). However, polymorphisms and multiple gene copies for b2m genes are common in some fish species, e.g. carp, barbs, and Siberian sturgeon (Magor et al., 2004). Indeed it has been reported that a single rainbow trout transcribes at least ten distinct b2m cDNAs and a b2m locus may encode three polymorphic genes (Magor et al., 2004). When all four ovine sequences were compared they were between 97.8% and 99.4% identical at the nucleotide level and 96.6% and 99.2% identical at the amino acid level. This level of identity is expected between alleles in a species although one of 5 mouse alleles varies more widely in comparison to the other mouse alleles (Hermel et al., 1993). OvB2m.3 was the same as the consensus sequence of the 4 aligned ovine sequences, and compared with this clone there were three coding changes located at positions 3, 4, and 5 in the signal peptide, and two non-coding changes at position 11 in the signal peptide and 98 in the mature b2m protein in ovB2m.4; 2 nucleotide differences with one coding change compared with ovB2m.2; and 2 nucleotide differences in one codon causing one amino acid change compared with ovB2m.2 (Fig. 1). The predicted amino acid sequences were 95.0%, 75.6%, 67.2% and 47.9% identical to bovine, human, mouse and chicken b2m, respectively. Partial goat b2m amino acid sequence has been published (31 amino acids of mature b2m protein (Groves et al., 1985)), and comparison of these residues between sheep and goat indicated they were 100% identical. The high level of homology with cattle and goat sequence was expected as these species are very closely related. In deed, rat (66.3% homology with sheep) and mouse b2m are 83.2% homologous, and human and chimpanzee (X69084) are 100% homologous. The 22 points of interaction between b2m and heavy chain have been determined from X-ray crystallographic studies of HLA-A2, H2Kb, and H2Db (Bjorkman et al., 1987). Comparison of the predicted ovine peptide with the bovine peptide (Fig. 1) indicated that the 22 amino acid residues predicted to interact with MHC class I heavy chain were highly conserved: specifically, 20 residues were completely conserved, 1 residue showed a conservative substitution (I–V) and one position encoded a charge change (K–E). Both variant positions putatively contact the a3 domain (Fig. 1). None of the
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allelic changes between the 4 ovine sequences affected any of these residues suggesting the changes may not affect structure and function of the variants. Research has indicated that mutated b2m containing structural changes (Trymbulak and Zeff, 1997) causes MHC class I structural changes (Schultz et al., 1998), and the loss of epitope presentation (Van Kaer et al., 1992; Zijlstra et al., 1990) resulting in increased susceptibility to invasion by intracellular pathogens (Ferrone and Marincola, 1995). However, an engineered variant of human b2m with a single serine (S) to valine (V) substitution at residue 55 improves its ability to bind to cell surface HLA-A1, A2 and A3 molecules, facilitates exogenous peptide loading (50–80-fold), and so promotes recognition by peptide-specific T cells (Shields et al., 1998). Mechanisms leading to diversity of b2m alleles are not understood at present. Pressure from pathogens has been suggested as an explanation, but this has been shown not to be the primary cause of the observed diversity in rainbow trout (Shum et al., 1996). 3.2. Expression of ovine b2m with ovine MHC class I heavy chain Several different transfection methods were used to try to express the ovine b2m clones in Daudi cells, a human cell line with a mutation in the b2m start codon and so no expression of b2m protein. This would have allowed ovine b2m expression to be analysed by, e.g. Western blot or flow cytometry using anti-human monoclonal antibodies known to cross-react with ovine b2m. However, we were unable to transfect these cells efficiently. We therefore designed an expression system to determine if expressed ovine b2m was functional using the fact that cell surface expression of the heavy chain of MHC class I molecules requires concomitant expression of b2m (Seong et al., 1988). We used ovB2m.1 (from sheep B1125) with a full-length ovine MHC class I heavy chain cDNA (c25) derived from the same sheep, using an IRES to allow polycistronic mRNA translation. A quail cell line, QT35, was shown not to express surface ovine MHC class I heavy chain in the absence of FCS and expressed ovine b2m (Fig. 2B), thus suggesting that either QT35 cells do not express b2m or quail b2m is unable to support ovine MHC class I expression at the cell surface. FCS is known to contain soluble bovine b2m which may rescue the surface expression of MHC class I heavy chain (Shields et al., 1998), as was indeed shown in Fig. 2E where FCS was added to cells. In the absence of FCS, ovine b2m rescued the surface expression of ovine MHC class I
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Fig. 2. Expression of ovine MHC class I on the surface of transfected cells. QT35 transient transfectants and HEK293 stable transfectants were cultured in FCS free medium and then were screened by flow cytometry for surface expression of ovine MHC class I antigen. Panels are, QT35 cells transfected with: (A) pcDNA3.1; (B) pcDNA3.1ovMHCIc25; (C) pcDNA3.1-ovb2m and pcDNA3.1ovMHCIc25; (D) pcDNA3.1-ovb2m-IRESovMHCIc25; (E) pcDNA3.1ovMHCIc25 cultured in medium with FCS; (F) sheep skin cells; HEK293 cells transfected with: (G) pcDNA3.1; (H) pcDNA3.1ovMHCIc25; and (I) pcDNA3.1-ovb2m-IRES-ovMHCIc25.
heavy chain on the quail cells, when the protein was expressed from pcDNA3.1 either in a polycistronic construct (Fig. 2D) or individual plasmids (Fig. 2C). Stable HEK293 transfectants were also analysed in the absence of FCS. Here the endogenous human b2m was able to support the surface expression of ovine MHC class I heavy chain and this expression was not increased by the addition of ovine b2m in the expression construct (Fig. 2H and I). The homology between human and mouse b2m is 70%, and research has shown that mouse b2m does not support efficient surface expression of human MHC class I heavy chain; it allows surface expression of HLA class I heavy chains at only 20–30% of levels observed for heavy chains assembled with human b2m. However, human b2m can allow mouse MHC class I expression and binds with higher affinity to mouse MHC class I heavy chains than mouse b2m (Pedersen et al., 1995). Of the 22 putative amino acid residues contacting MHC class I heavy chain, chicken
b2m has 13, human 17 and cattle 20 residues conserved with ovine b2m (Fig. 1). Therefore, avian b2m would be predicted to have the lowest or no affinity for ovine MHC class I heavy chain molecules. So it is not surprising that both human and bovine b2m supported ovine MHC class I heavy chain cell surface expression but avian b2m might not as homology between sheep and chicken b2m is very low (see above). In conclusion, we have cloned 4 allelic sequences of ovine b2m from different breeds of sheep and have obtained direct evidence of the existence of allelic variants of ovine b2m. Ovine, bovine and human b2m supported expression of ovine MHC class I molecules on the cell surface. Acknowledgements Changxin Wu was supported by the National Natural Science Foundation of China, the Henry Lester Trust,
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