| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Wisconsin Regional Primate Research Center and Departments of
Genetics and
Obstetrics and Gynecology, University of Wisconsin, Madison, WI 53715;
§
Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands;
¶
Department of Biology, Pennsylvania State University, University Park, PA 16802; and
||
Histocompatibility Laboratory, Division of Laboratory Medicine, Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53792
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Classical MHC class I glycoproteins are expressed in almost every cell type and play a central role in immune recognition of nonself by binding pathogen-derived peptides and presenting them to CD8-positive CTLs. The function of nonclassical MHC class I glycoproteins is just beginning to be understood. MHC class II glycoproteins are expressed largely on cells of the immune system and bind exogenously derived peptides and present them to CD4-positive T helper cells. MHC class II genes have been extensively studied in the rhesus monkey. These class II genes are very well conserved and are stable over very long evolutionary periods, such that allelic lineages are preserved even between humans and macaques (9, 14, 15). Methods for molecular typing of the MHC class II alleles in the macaque have now been developed and nearly 100 rhesus DRB alleles have been identified (16).
Although analysis of the MHC class II loci in the rhesus macaque has been relatively straightforward, the MHC class I loci of this species have been more difficult to define. We and others have identified orthologs of the human HLA-A, -B, -E, -F, and -G genes in the rhesus monkey (8, 17, 18, 19). The orthologs of HLA-G in the rhesus monkey are pseudogenes, but the recently identified nonclassical Mamu-AG (Macaca mulatta-AG) locus may have evolved to serve the function of HLA-G in this primate (17, 20, 21, 22). We have also shown that duplication of the rhesus MHC class I A and B loci has occurred, making the study of the MHC class I loci in this species exceptionally complex (our unpublished observations and Ref. 8). In particular, the presence of multiple MHC class I A and B loci makes the development of molecular technologies for MHC class I typing of this species even more challenging.
Therefore, definition of the MHC class I loci and alleles in the rhesus macaque is central to the development of this animal model for understanding human diseases. In this paper we describe a new MHC class I locus in rhesus macaques, Mamu-I (Macaca mulatta-I), which is likely the result of a recent duplication of a classical MHC class I B locus. These results have implications for the development of the rhesus macaque as an animal model for human diseases and organ transplantation and, in addition, for the elucidation of cellular immune responses in this species.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We used two different methods for isolation and identification of full-length Mamu-I cDNAs. The first was described previously (23). The second method was as follows. Total cellular RNA was extracted from 2–7 x 106 rhesus monkey lymphocytes using RNAzol (Tel-Test, Friendswood, TX). cDNA was synthesized from 0.1 to 1 µg of RNA in a 20-µl reaction containing 50 mM Tris (pH 8.3), 5 mM MgCl2, 1 mM each of dATP, dGTP, dCTP, and dTTP (Gene AMP, Perkin-Elmer, Foster City, CA), 0.5 µg random primers (Promega, Madison, WI), 50 U of SuperScript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD), and 20 U of RNase inhibitor (Gene AMP-Perkin-Elmer). cDNA was synthesized at room temperature for 10 min, 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min in a Perkin-Elmer Cetus 9600 thermocycler (Norwalk, CT). PCR was then conducted in a Perkin-Elmer Cetus 9600 using the previously described rhesus macaque B locus-specific primers, 5'MBS and 3'MBS (8), each at a final concentration of 0.25 mM. The PCR mixture contained 2 mM MgCl2, 50 mM Tris (pH 8.3), and 2.5 U Amplitaq DNA polymerase (Perkin-Elmer Cetus) in a final volume of 100 µl. The reactions were denatured initially for 2 min at 94°C before being subjected to 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1.5 min, and a single final extension at 72°C for 10 min.
Amplified, B locus-specific products were gel purified using QIAEX II suspension (Qiagen, Santa Clarita, CA) and were subcloned into the pCR2.1 vector using the TA cloning kit from Invitrogen (Carlsbad, CA). A total of 500 ng of plasmid DNA was sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, (Perkin-Elmer-Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. At least three copies of each cDNA were sequenced to ensure that clones did not contain PCR-generated artifacts. Full-length sequences were generated using previously described primers (8), assembled using the AutoAssembler DNA sequence assembly software (Perkin-Elmer-Applied Biosystems), and analyzed with MacVector sequence analysis software (Oxford Molecular Group, Campbell, CA).
Mamu-I sequence-specific PCR (PCR-SSP),4 RT-PCR-SSP, and direct sequencing of PCR products
Mamu-I-specific primers were designed based on a rhesus macaque MHC class I sequence database generated by ourselves and others (MMIF2, 5'-CTCCCACTCCTTGAGGTATTTCG-3'; MMIR, 5'-TGCAGCGTCTCCTTCCCC-3'). PCR-SSP was performed either on diluted 5'MBS/3'MBS PCR product or on genomic DNA isolated using the QIAmp Blood kit (Qiagen).
Mamu-I PCR-SSP on genomic DNA was performed in 26 µl of 1x PCR buffer F from the Invitrogen PCR Optimizer kit, 2 mM MgCl2, 2.5 mM of each of the four dNTPs (Invitrogen), 1.9 µM of each specific primer, and 0.4 µM each Mamu-DRB internal control primer (24) and 1.25 U of Amplitaq DNA polymerase (Perkin-Elmer, Foster City, CA). The reactions were denatured initially for 2 min at 96°C before being subjected to the following cycling conditions: 10 cycles of 96°C for 25 s, 70°C for 50 s, and 72°C for 45 s; 21 cycles of 96°C for 25 s, 65°C for 50 s, and 72°C for 45 s; and a final four cycles of 96°C for 25 s, 55°C for 1 min, and 72°C for 1 min 20 s. A total of 6 µl of the PCR mix was electrophoresed in 1% agarose, stained with ethidium bromide, and visualized under UV light. A total of 5 µl of PCR product from positive reactions was then incubated for 15 min at 37°C with 2 U shrimp alkaline phosphatase and 10 U exonuclease I to remove unincorporated dNTPs and residual single-stranded PCR primers, respectively (United States Biochemical, Cleveland, OH). This reaction mixture was then diluted with 7–18 µl of H2O and 3 µl were sequenced directly with the same primers used for PCR-SSP using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit as described above. The forward and reverse sequences were assembled using the AutoAssembler DNA sequence assembly software (Perkin-Elmer-Applied Biosystems). A second set of Mamu-I-specific primers was designed to amplify from a few samples that failed to amplify with the MMIF2/MMIR primer set (MMIF3, 5'-CTTGAGGTATTTCGGCACCA-3'; MMIR2, 5'-TGTTCTGAGCCGCCTCAC-3'). The same conditions were used for amplification with this set of primers.
For Mamu-I PCR-SSP on diluted PCR product, full-length, locus-specific PCR products amplified with the MBS primers were diluted 1:250 in 500 µl H2O and were subjected to a second round of nested, Mamu-I-specific PCR using buffer F from the Invitrogen PCR Optimizer kit. Mamu-I primers were used at a final concentration of 0.5 µM. The reactions were denatured initially for 2 min at 96°C before being subjected to the following cycling conditions: five cycles of 96°C for 25 s, 70°C for 50 s, and 72°C for 45 s; 21 cycles of 96°C for 25 s, 69°C for 50 s, and 72°C for 45 s; and a final four cycles of 96°C for 25 s, 60°C–65°C for 1 min, and 72°C for 1 min 20 s.
For Mamu-I-specific RT-PCR, 1 µg of total RNA was reverse transcribed to cDNA using an oligo(dT) primer and Maloney murine leukemia virus reverse transcriptase (Perkin-Elmer). The cDNA was amplified with MMIF2/MMIR primers for 36 cycles, with each cycle consisting of 94°C for 45 s, 66°C for 45 s, and 72°C for 2 min, in a reaction mixture containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each dNTP, and 1 U Amplitaq DNA polymerase (Perkin-Elmer). RT-PCR products were directly sequenced as described above.
Nomenclature of macaque MHC class I alleles
The DNA and cDNA sequences were compared with a database of macaque MHC class I sequences generated by ourselves and others. Unique sequences were named in the order isolated according to established guidelines (25). In the nomenclature of the MHC class I genes, alleles that differ by even a single, non-amino acid-changing nucleotide substitution (synonymous substitution) are given separate designations. For example, the alleles designated Mamu-I*01011, Mamu-I*01012, Mamu-I*01013, and Mamu-I*01014 are identical at the amino acid level but differ by one or more nucleotides. The alleles designated Mamu-I*01011, Mamu-I*02011, Mamu-I*04, Mamu-I*05, etc. differ by one or more amino acids.
Transfection of Mamu-I molecules into an MHC class I null cell line
Clones containing the consensus cDNA for full-length Mamu-I*02011 were subcloned into the pKG5 expression vector (a gift from Andrew McMichael, Oxford University, Oxford, U.K.). This vector was then electroporated into the 721.221 cell line, a cloned EBV-transformed B lymphoblastoid cell line (BLCL) (4) with homozygous deletions of the MHC class I loci (26). A total of 5 x 106 721.221 cells were transfected in a 1-cm electroporation cuvette with 5 µg of plasmid DNA. Electroporation was conducted at 1000 V and a capacitance of 600 µF. The cells were then put in RPMI 1640 culture medium supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 5% defined FBS (HyClone, Logan, UT), and 10% defined/supplemented bovine calf serum (HyClone) and were plated onto four 24-well plates. The cells were incubated for 4 days at 37°C. On day 5, the cells were placed under selection by adding 1 ml of culture medium containing G418 (Life Technologies) for a final concentration of 500 µg/ml. About 4 wk later, viable transfectants were tested for MHC class I surface expression by flow cytometry with W6/32, BB7.7, or BBM.1 mAbs (American Type Culture Collection, Manassas, VA). The transfectant with the highest level of MHC class I expression was selected to be grown up for one-dimensional isoelectric focusing (1-D IEF) analysis.
Flow cytometry
A total of 2 x 105–1 x 106 cells were washed twice in PBS with 2% FBS and 0.1% sodium azide, and 50 µl of supernatant from the BBM.1 hybridoma was added to each sample and incubated on ice for 30 min. After two additional washes, FITC-conjugated goat anti-mouse Abs (Dako, Carpinteria, CA) were added. After 30 min on ice, samples were washed twice, and 250 µl 2% paraformaldehyde was added. Sample data were acquired on a Becton Dickinson FACSCalibur instrument and analyzed using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Radiolabeling and electrophoretic analysis
MHC class I glycoproteins were translabeled with [35S]methionine and immunoprecipitated with W6/32 and BB7.7 mouse mAbs directed against human MHC class I proteins, as described previously (27).
Phylogenetic analysis
Gene trees were constructed using the neighbor-joining method (28). Genetic distances were estimated using the method of Jukes and Cantor (29). At sites in which the alignment indicated a gap, nucleotides at this position in all the sequences were deleted. The reliability of the tree topology was tested by the bootstrap method (30). One thousand replications was used for bootstrap analysis, the results of which are reported as the strength of support for a given branch topology. Calculations of the rates of synonymous and nonsynonymous substitutions were performed using the method of Nei and Gojobori (31), and variances were calculated using the method of Nei and Jin (32). Phylogenetic analyses were performed using MEGA software (33). Tests of the significances of these differences were performed using a two-tailed t test. A binomial test was used to determine the significance of differences in %GC content.
GenBank accession numbers
The sequences described in this manuscript have been deposited
with GenBank and were assigned accession numbers AF161860–AF161882.
Mamu-I*02011, Mamu-I*05, and Mamu-I*06
were previously submitted with the names and accession numbers
Mamu-B*0902 AF157403, Mamu-B*0903 AF157404, and
Mamu-B*0904 AF157405, respectively. The GenBank accession
numbers for all other sequences used in Figs. 2
, 3, and 4 are:
Gogo-A*0101 X60258, Gogo-B*0101 X60255,
Gogo-B*0102 X60693, Gogo-C*0201 X60251,
HLA-A*0101 M24043, HLA-A*0201 K02883,
HLA-A*1101 X13111, HLA-B*0702 M32317,
HLA-B*2702 X03664 and X03667, HLA-B*3506 M84381,
HLA-B*3802 L22028, HLA-C*1203 U06695,
HLA-C*1402 U06487, HLA-C*1601 M24097,
Hyla-A*01 U50089, Hyla-A*02 U50090,
Mamu-A*01 U50836, Mamu-A*02 U50837,
Mamu-A*03 U41379, Mamu-A*04 U41880,
Mamu-A*05 U41831, Mamu-A*06 U41834,
Mamu-A*07 U41832, Mamu-A*08 (34),
Mamu-A*12 AF157398, Mamu-A*1301 AF157399,
Mamu-B*01 U42837, Mamu-B*02 U41833,
Mamu-B*03 U41825, Mamu-B*04 U41826,
Mamu-B*06 U41828, Mamu-B*07 U41829,
Mamu-B*08 U41830, Mamu-B*12 (34),
Paan-A*01 U35624, Paan-A*02 U35625,
Papa-A*01 L39093, Papa-A*02 L39094,
Papa-B*02 U05578, Papa-B*03 U05575,
Papa-B*04 U05577, Patr-A*01 M24047,
Patr-B*02 X13116, Patr-B*10 U05582,
Popy-A*01 M30680, Popy-A*02 U50084,
Popy-B*01 U50086, Popy-B*02 U50087, and
Popy-B*03 U50088
|
|
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Alleles at this new locus are inherited in a Mendelian fashion
To investigate whether the Mamu-B*09 variants that we
had observed in our cohort of Shigella-infected rhesus
macaques were indeed the products of a distinct B-like
locus, we examined the inheritance of these alleles in a family of
rhesus macaques by PCR-SSP and by cloning and sequencing (Fig. 1). We isolated two Mamu-I
alleles in addition to other Mamu-B alleles in the offspring
of two adult macaques, and the alleles of this locus were segregated
and inherited in a Mendelian fashion, suggesting that they represented
alleles of a separate locus. Because MHC class I A–H loci
already exist in primates (8, 17, 18, 19, 35), we have called
this new locus Mamu-I in accordance with established
nomenclature guidelines (25). We were unable to detect an
I locus on the a haplotype (Fig. 1). Animal 79167 does not
appear to be homozygous at the I locus, as was shown by the
fact that we were also unable to detect a second Mamu-I
allele in animal 88090 which inherited the a haplotype. This could be
due to the existence of a rare haplotype that lacks an MHC class I
I locus. Alternatively, this could be due to the lack of
sensitivity of our screening methods. We are currently screening a cDNA
library from an additional offspring in this family that has inherited
the a haplotype to address this question (H. Horton, manuscript in
preparation).
|
We then wished to investigate whether this new locus was classical or nonclassical. Generally, the difference between these two categories of MHC molecules is the level of polymorphism and cell-surface expression (36). Nonclassical molecules tend to have limited polymorphism and low cell-surface expression in restricted tissues, whereas classical molecules are highly polymorphic and are readily detected on the surface of nearly all cell types.
PCR-SSP on DNA or diluted Mamu-B-specific PCR product from
60 rhesus macaques revealed that, with one exception, all animals were
positive for at least one, often two, Mamu-I alleles (data
not shown and Fig. 2
). In only one case
did a DNA sample not amplify when subjected to PCR with two different
sets of Mamu-I-specific primers. This could be due to the
presence of a variant of I that would not amplify with
either set of primers. Alternatively, this animal could have two
haplotypes that do not contain a Mamu-I locus.
Cloning and sequencing revealed that the Mamu-I cDNAs were
1098 nt in length from the start codon in exon 1 to the end of exon 8
(Fig. 2). Exon 8 is untranslated in Mamu-I molecules due to
a stop codon just before the end of exon 7, a characteristic of
classical MHC class I B molecules. We showed previously that
some human A and B locus-specific nucleotides
(8) were conserved in rhesus monkey class I cDNAs
(37). Macaque I molecules shared several
B locus-specific nucleotide substitutions with
Mamu-B alleles (Fig. 2, see nucleotides marked with *).
Additionally, there were two areas of similarity between I
alleles and Mamu-B alleles that suggested that interlocus
recombination may have occurred (Figs. 2 and 3, boxed areas). Based on a comparison of
I alleles and a database of over 50 rhesus A and
B alleles, we noted only four nucleotide substitutions that
were specific for Mamu-I alleles (Fig. 2, nucleotides marked
with •).
The predicted amino acid sequence of MHC class I I alleles
is 362 aa in length, including the leader peptide. Macaque I proteins
contained several amino acid residues that are conserved in all known
mammalian and avian classical alleles (Fig. 3 and Ref.
38). They also contained the conserved cysteine
residues at positions 101 and 164, and 203 and 259 that form the
structurally important disulfide bonds. Furthermore, the
N-linked glycosylation site at position 86 in the 
1
domain was also present. There were only two residues that were
specific to and conserved among all macaque I molecules, cysteine at
position 99 and glycine at position 137 (Fig. 3).
The Mamu-I locus is derived from a recent duplication of an MHC class I B locus
Sequence comparisons showed a high degree of similarity between Mamu-I alleles and Mamu-B alleles. Therefore, we wished to determine whether the Mamu-I locus was derived from the ancestor of a B locus and used gene tree analysis to address this question.
Gene tree analysis of full-length Mamu-I sequences revealed
that this locus is derived from a classical MHC class I B
locus as is shown by the significant clustering of the
Mamu-I alleles with other primate B alleles (Fig. 4). Because exons 4–8 contain the
majority of the locus-specific nucleotide substitutions, we conducted
gene tree analysis of this region. Gene trees constructed with exons
4–8 confirmed the clustering seen in the full-length trees (data not
shown). Mamu-I alleles did not consistently cluster with any
particular Mamu-B alleles in gene trees of exon 2 (data not
shown).
To investigate when this duplication event may have occurred, we
conducted PCR-SSP using two different sets of
Mamu-I-specific primers on DNA samples from several other
Cercopithecine and Hominoid species: MHC class I
I DNA amplified from 10 of 10 stump-tailed macaques
(Macaca arctoides) and 6 of 9 cynomolgus macaques
(Macaca fascicularis) (Figs. 1 and 2, Maar and
Mafa sequences). We were unable to amplify DNA using these
primers from one diana monkey (Cercopithecus diana), two
sooty mangabeys (Cercocebus torquatus), four baboons
(Papio anubis), one orangutan (Pongo pygmaeus),
and one gibbon (Hylobates lar). These data suggest that the
I locus is the result of a very recent duplication event
that occurred after macaques diverged from other
Cercopithecine genera. Furthermore, this also suggests that
Mamu-I is not orthologous to HLA-C. It is
possible that the absence of amplified bands is due to a sequence
difference that may exist between these species. However, it would be
imprudent to rule out the possibility that the I locus is
present in these species without doing extensive screening of genomic
libraries. Nonetheless, given its low variability, it is unlikely that
we would not have detected this locus using two different sets of
primers.
Directly sequenced PCR-SSP products and sequences of full-length clones
revealed the presence of at least 21 different Mamu-I
variants (Fig. 2). In some cases, mixed sequences were apparent in
PCR-SSP products, suggesting heterozygosity at this locus. In several
cases, new alleles were identified in these heterozygous mixes, but
exact sequence of these alleles could not be resolved without cloning.
Therefore, these sequences were not reported.
Nonclassical MHC class I alleles and pseudogenes have a lower %GC
content at the third codon positions (38). Thus, the
longer the time between divergence of a locus from its classical
ancestor, the lower the %GC content at the third codon position.
Analysis of the %GC content of the third position of codons in the
peptide binding region (PBR) and the codons of the remainder of exons 2
and 3 not involved in Ag recognition further suggested a recent origin
of the I locus (Table I).
Table I shows that the %GC content of the third codon position of
I alleles is more characteristic of classical MHC class I
loci, suggesting a recent divergence from a classical MHC class I
locus.
|
Classical MHC class I mRNA transcripts are readily found in nearly
all tissues. Conversely, one characteristic of some nonclassical MHC
class I genes is their restricted tissue expression. Like the
nonclassical gene, HLA-G, mRNA transcripts from the recently
identified nonclassical Mamu-AG locus were detected at high
levels in placenta and amniotic membranes but at lower levels in other
tissues such as kidney, heart, and spleen. Mamu-AG mRNA
transcripts were absent from peripheral blood lymphocytes and lymph
nodes (39). We used RT-PCR with Mamu-I-specific
primers to investigate the expression pattern of Mamu-I mRNA
transcripts. Fig. 5 shows that
Mamu-I mRNA transcripts were found in PBL, placenta, thymus,
lung, spleen, heart, kidney, liver, and small intestine. Directly
sequenced PCR products from PBL1 and PBL2 confirmed that the reaction
was specific for Mamu-I. Thus, unlike the restricted
distribution of the nonclassical Mamu-AG mRNA,
Mamu-I mRNA transcripts are found in most tissues.
|
To ascertain whether Mamu-I protein is expressed, we used the
BBM.1 mAb to stain the MHC class I null cell line, 721.221, transfected
with Mamu-I*02011. We used BBM.1, which recognizes
ß2-microglobulin, to decrease allele to allele
differences in Ab binding. Flow cytometry histograms showed a shift in
fluorescence in .221-Mamu-I*02011 cells compared with .221
cells (Fig. 6). The level of staining
that we observed for .221-Mamu-I*02011 was lower than that
observed for other Mamu transfectant cell lines (Fig. 6).
Whether this low surface expression is unique to the 721.221
transfectants or is also found on macaque lymphocytes remains to be
shown. We also demonstrated using 1-D IEF analysis that the
Mamu-I*02011 protein produced in the 721.221 transfectants has the same
isoelectric point as a protein produced in the BLCL of the animal that
is positive for the particular allele (data not shown). Furthermore, we
also translated Mamu-I*07 in vitro and this product had the
same isoelectric point as a protein produced in BLCL from the animal,
88090, from which this allele was isolated (data not shown). Taken
together, these results suggest that the Mamu-I protein is produced. We
have not shown cell-surface expression directly. However, given the
close relationship of Mamu-I to other Mamu-B
glycoproteins and based on sequence analysis, there are no striking
differences that lead us to conclude that these molecules are not
transported to the cell surface.
|
One of the hallmarks of the MHC is the high polymorphism and variability of its loci. More than 149 alleles of HLA-B have now been sequenced (40), and in the 41 sequences that we analyzed for this report, there are between 1 and 49 aa differences over their entire length. The classical loci are much more polymorphic and diverse than nonclassical loci, which could be a result of their different functions in the immune system.
The macaque I locus is extremely limited in its variability
compared with other classical and nonclassical loci (Figs. 2, 3, and 7 and Table II). There are 16 variable sites in exon
2, and 14 variable sites in exon 3 of I alleles from the
three species of macaques. However, pairwise comparisons between the
macaque I alleles show that the nucleotide variablility is
very low, with numbers of nucleotide differences ranging from only 0–9
in exons 2 and 3. In this same region, HLA-A and
-B alleles differ by 1–51 and 0–37 nt, respectively (Fig. 7). Mamu-B alleles differ by 7–76 nt over this region.
However, note that this number may be artificially high due to the fact
that Mamu-B alleles probably derive from at least three
different loci (H. Horton, manuscript in preparation, and Ref.
8). Pairwise comparisons of the predicted glycoprotein
sequences revealed differences ranging only from 0 to 6 compared with
0–28 and 1–32 in HLA-A and -B, respectively, in
the 1 and 2 domains. The predicted protein sequences of
Mamu-B molecules differ by 2–46 aa in this same region.
Again, this number may be misleading in that the comparison includes
Mamu-B alleles from different loci.
|
|
and 3).
Moreover, Mafa-I*1011 and Maar-I*01 are identical
over the entire region of exons 2 and 3 that we sequenced. This was not
due to cross-contamination of samples because there were differences in
intron 2 (data not shown). However, these two alleles may differ in
other exons not included in the region sequenced here. Although several
groups have now identified identical alleles between species
(41, 42, 43), the lack of variability of I alleles
between species is striking. The I locus has evolved under purifying selection
Classical MHC class I loci differ in their evolution from
nonclassical loci. Positive selection acts on the PBR of classical loci
to select for nonsynonymous changes. This is reflected by a higher rate
of nonsynonymous substitution (dN)
(amino acid-altering substitutions) compared with the rate of
synonymous substitution (dS)
(non-amino acid-altering substitutions) in PBR codons of exons 2 and 3.
The remaining codons in exons 2 and 3 and codons in exon 4 evolve under
purifying selection which is characterized by a higher
dS than
dN (44). By contrast,
there is no evidence for positive selection acting on the PBR of
nonclassical loci (44, 45). To determine whether
Mamu-I has evolved in a manner similar to classical or
nonclassical loci, we compared the dS
and dN in the PBR of macaque
I alleles and found that evidence for positive selection in
the PBR is absent (Table III). In fact,
not only is dN not higher than
dS in PBR codons, but
dS is slightly higher than
dN at these sites, though the
difference is not statistically significant. However, in the remaining
codons of exons 2 and 3, dS is
significantly higher than dN,
indicating that the I locus has evolved under purifying
selection. Thus, Mamu-I has evolved in a manner more
characteristic of a nonclassical locus.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The I locus is similar to other MHC class I classical loci in its high polymorphism, wide tissue expression, and high %GC content at the third position of PBR codons. However, it is similar to the nonclassical Mamu-AG and HLA-E in its lack of positive selection in the PBR. Unlike the macaque I locus, the E locus is very old and so the purifying selection that has acted on it is evident from a significantly higher dS than dN in the PBR (46). The function, or lack thereof, of nonclassical MHC class I (class Ib) molecules has been greatly debated. It has been hypothesized that these genes are largely nonfunctional and are pseudogenes (38, 47). It is now known that HLA-E binds MHC class I-derived leader peptides and is the ligand for the CD94-NKG2A, B, and C receptors on NK cells. Recognition of HLA-E/leader peptide can either inhibit or trigger lysis by the NK cell, depending on the receptors involved (48, 49, 50). The function of HLA-G is still unknown but it is expressed in extravillous trophoblasts and is also an NK-inhibitory ligand. Thus, it is thought to play a role in maternal-fetal tolerance by protecting trophoblasts from NK cell killing (51, 52, 53, 54, 55, 56). The recent discovery of a new nonclassical locus in the rhesus monkey, Mamu-AG, that has similarities to HLA-G yet evolved independently further supports a functional role for these molecules. Thus, nonclassical loci appear to evolve from the duplication of classical loci and take on specific functions. Mamu-I may be at an intermediate step in this process, retaining some of its classical characteristics.
A striking feature of the MHC class I I locus is that it is
highly polymorphic, and yet the numerous alleles of this locus
demonstrate low variability. We isolated at least 21 Mamu-I
alleles from 60 rhesus monkeys, yet the alleles differed from each
other by only 0–9 nt. This lack of sequence variability is lower even
than that observed in nonclassical loci such as Mamu-AG and
HLA-E (Fig. 2 and Table II). Many more alleles of the
Mamu-I locus have been isolated than of any other
nonclassical locus. At the protein level, sequence diversity is even
lower with many of the I alleles being identical at the
amino acid level. The lack of sequence variation is most easily
explained by the recent origin of this locus. This locus appears to be
present in macaques but not in other Cercopithecine genera,
suggesting that it is only 5–7 million years old (57, 58). However, it should be noted that without extensive
screening of genomic libraries, we cannot say with certainty that this
locus is absent in other Cercopithecine genera.
The MHC is of great interest not only because of the important immunologic function of the glycoproteins that it encodes but also for its unique evolutionary characteristics. Evolution of the MHC class I region is characterized by duplication and differential expansion of loci (38, 59, 60). In New World primates, the classical MHC class I Ags are more similar to the nonclassical HLA-G molecule than they are to any other human MHC class I molecule (61). Thus, in these primates, the ancestral G locus duplicated and its products functioned as the classical MHC class I molecule, presenting peptides to CTL in tamarins, whereas in humans the HLA-G molecule appears to have evolved to serve a much more specialized function. In some cases, after duplication, a locus may accumulate deleterious mutations and degenerate into a pseudogene. For example, HLA-H originated as a duplication of the A locus but is now a pseudogene (62). Therefore, the fate of a locus after duplication depends on the selective forces acting on it. Based on the analysis of synonymous and nonsynonymous substitutions, evidence for positive selection in the PBR of Mamu-I is absent. One interpretation of these data is that the PBR of Mamu-I has evolved under neutral selection since its divergence from the B locus. However, an alternative hypothesis is that the PBR of Mamu-I has undergone purifying selection and, due to the short time since its divergence, dS is not signficantly higher than dN in this region.
The rhesus monkey has proved to be one of the most important animal models for the study of AIDS and transplantation (1, 2, 3, 4, 5, 6). The recent development of the MHC tetramer technology has led to new insight regarding T cell responses to HIV in humans and SIV in the rhesus macaque (63, 64). This technology has also led to the elucidation of the ligand for the nonclassical MHC class I molecule HLA-E (48). However, to utilize this new tetramer technology to further understand immune responses in the rhesus monkey, knowledge of the MHC class I molecules expressed by this species is critical. It is now becoming increasingly clear that the MHC class I locus of the rhesus macaque is much more complex than its human counterpart. Orthologs of the human HLA-A, -B, -E, -F, and -G loci have now been indentified in the rhesus monkey (8, 17, 18, 19). However, there is also evidence to suggest that the Mamu-A and Mamu-B loci have duplicated at least once (8). Furthermore, the nonclassical Mamu-AG locus is likely the result of a duplication of an A locus (45). In this paper we have described a new MHC class I locus in the rhesus macaque, Mamu-I, that shares features with both classical and nonclassical loci. These results have implications for the further development of the rhesus macaque animal model and for understanding cellular immune responses in this species.
Nucleotide sequence of macaque I alleles. Nucleotide sequences of rhesus, cynomolgus, and stump-tailed macaque I alleles were aligned to a macaque I consensus sequence. Mamu-A and -B sequences were included for comparison. Note that many alleles differ by non-amino acid-changing nucleotide substitutions (see Materials and Methods for further explanation of Mamu-I nomenclature). *, Locus-specific nucleotides shared between Mamu-B and Mamu-I cDNAs; •, nucleotides specific to and conserved in all Mamu-I alleles. Possible regions of interlocus recombination are boxed.
Continued
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109.
3 Address correspondence and reprint requests to Dr. David I. Watkins, Wisconsin Regional Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715. E-mail address:
4 Abbreviations used in this paper: PCR-SSP, sequence-specific PCR; BLCL, B lymphoblastoid cell line; PBR, peptide binding region; dN, rate of nonsynonymous substitution; dS, rate of synonymous substitution; 1-D IEF, one-dimensional isoelectric focusing.
Received for publication June 25, 1999. Accepted for publication November 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. R. Manuel, W. W. Yeh, M. S. Seaman, K. Furr, M. A. Lifton, S. L. Hulot, P. Autissier, and N. L. Letvin Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus J. Virol., October 1, 2009; 83(19): 10028 - 10035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Q. DeGottardi, A. Specht, B. Metcalf, A. Kaur, F. Kirchhoff, and D. T. Evans Selective Downregulation of Rhesus Macaque and Sooty Mangabey Major Histocompatibility Complex Class I Molecules by Nef Alleles of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2 J. Virol., March 15, 2008; 82(6): 3139 - 3146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Loffredo, J. Sidney, S. Piaskowski, A. Szymanski, J. Furlott, R. Rudersdorf, J. Reed, B. Peters, H. D. Hickman-Miller, W. Bardet, et al. The High Frequency Indian Rhesus Macaque MHC Class I Molecule, Mamu-B*01, Does Not Appear to Be Involved in CD8+ T Lymphocyte Responses to SIVmac239 J. Immunol., November 1, 2005; 175(9): 5986 - 5997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Hickman-Miller, W. Bardet, A. Gilb, A. D. Luis, K. W. Jackson, D. I. Watkins, and W. H. Hildebrand Rhesus Macaque MHC Class I Molecules Present HLA-B-Like Peptides J. Immunol., July 1, 2005; 175(1): 367 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Otting, C. M. C. Heijmans, R. C. Noort, N. G. de Groot, G. G. M. Doxiadis, J. J. van Rood, D. I. Watkins, and R. E. Bontrop Unparalleled complexity of the MHC class I region in rhesus macaques PNAS, February 1, 2005; 102(5): 1626 - 1631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Loffredo, J. Sidney, C. Wojewoda, E. Dodds, M. R. Reynolds, G. Napoe, B. R. Mothe, D. H. O'Connor, N. A. Wilson, D. I. Watkins, et al. Identification of Seventeen New Simian Immunodeficiency Virus-Derived CD8+ T Cell Epitopes Restricted by the High Frequency Molecule, Mamu-A*02, and Potential Escape from CTL Recognition J. Immunol., October 15, 2004; 173(8): 5064 - 5076. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Daza-Vamenta, G. Glusman, L. Rowen, B. Guthrie, and D. E. Geraghty Genetic Divergence of the Rhesus Macaque Major Histocompatibility Complex Genome Res., August 1, 2004; 14(8): 1501 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. P. Lafont, A. Buckler-White, R. Plishka, C. Buckler, and M. A. Martin Characterization of Pig-Tailed Macaque Classical MHC Class I Genes: Implications for MHC Evolution and Antigen Presentation in Macaques J. Immunol., July 15, 2003; 171(2): 875 - 885. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. U. Vogel, T. C. Friedrich, D. H. O'Connor, W. Rehrauer, E. J. Dodds, H. Hickman, W. Hildebrand, J. Sidney, A. Sette, A. Hughes, et al. Escape in One of Two Cytotoxic T-Lymphocyte Epitopes Bound by a High-Frequency Major Histocompatibility Complex Class I Molecule, Mamu-A*02: a Paradigm for Virus Evolution and Persistence? J. Virol., October 11, 2002; 76(22): 11623 - 11636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Muhl, M. Krawczak, P. ten Haaft, G. Hunsmann, and U. Sauermann MHC Class I Alleles Influence Set-Point Viral Load and Survival Time in Simian Immunodeficiency Virus-Infected Rhesus Monkeys J. Immunol., September 15, 2002; 169(6): 3438 - 3446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Ryan, R. L. Grendell, D. E. Geraghty, and T. G. Golos A Soluble Isoform of the Rhesus Monkey Nonclassical MHC Class I Molecule Mamu-AG Is Expressed in the Placenta and the Testis J. Immunol., July 15, 2002; 169(2): 673 - 683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Hershberger, R. Shyam, A. Miura, and N. L. Letvin Diversity of the Killer Cell Ig-Like Receptors of Rhesus Monkeys J. Immunol., April 1, 2001; 166(7): 4380 - 4390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Sidebottom, R. Kennedy, and W. H. Hildebrand Class I MHC Expression in the Yellow Baboon J. Immunol., March 15, 2001; 166(6): 3983 - 3993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Polakos, D. Drane, J. Cox, P. Ng, M. J. Selby, D. Chien, D. T. O'Hagan, M. Houghton, and X. Paliard Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine J. Immunol., March 1, 2001; 166(5): 3589 - 3598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Vigne, D. Hornung, M. D. Mueller, and R. N. Taylor Purification and Characterization of an Immunomodulatory Endometrial Protein, Glycodelin J. Biol. Chem., May 11, 2001; 276(20): 17101 - 17105. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
