Owl monkey MHC-DRB exon 2 reveals high similarity with several HLA-DRB lineages

ORIGINAL PAPER

Owl monkey MHC-DRB exon 2 reveals high similaritywith several HLA-DRB lineages

Carlos F. Suárez & Manuel E. Patarroyo &

Esperanza Trujillo & Mónica Estupiñán &

Juan E. Baquero & Carlos Parra & Raúl Rodriguez

Received: 25 April 2005 /Accepted: 3 May 2006 / Published online: 22 June 2006# Springer-Verlag 2006

Abstract One hundred and ten novel MHC-DRB geneexon 2 nucleotide sequences were sequenced in 96monkeys from three owl monkey species (67 from Aotusnancymaae, 30 from Aotus nigriceps and 13 from Aotusvociferans). Owl monkeys, like humans, have high MHC-DRB allele polymorphism, revealing a striking similaritywith several human allele lineages in the peptide bindingregion and presenting major convergence with DRBlineages from several Catarrhini (humans, apes and OldWorld monkeys) rather than with others New Worldmonkeys (Platyrrhini). The parallelism between humanand Aotus MHC-DRB reveals additional similarities re-garding variability pattern, selection pressure and physico-chemical constraints in amino acid replacements. Theseobservations concerning previous findings of similaritybetween the Aotus immune system molecules and theirhuman counterparts affirm this specie’s usefulness as anexcellent animal model in biomedical research.

Keywords Animal models . NewWorld monkeys .

Molecular evolution .Molecular convergence .MHC class IImolecules .MHC-DRB

AbbreviationsPBR peptide binding regionTSP trans-specific polymorphismOWM old world monkeyNWM new world monkeyMYA million years ago

Introduction

The major histocompatibility complex (MHC) is a multi-gene family belonging to the immunoglobulin super-family,encoding receptor glycoproteins expressed on cell mem-brane. MHC proteins play a central role in recognising selfand non-self by presenting foreign peptides to be recog-nised by T cells, therefore constituting key pieces indefence against foreign substances.

Two large groups of MHC molecules, which can befunctionally and structurally distinguished. MHC class Imolecules have a peptide binding region (PBR) consisting oftwo domains (α1 and α2) encoded by a single gene andexpressed on all types of nucleated cells presentingintracellular peptides to CD8+T lymphocytes. MHC classII encoded molecules have a PBR encoded by two genes(α and β chains) expressed on antigen-presenting cells(APC), such as monocytes, B lymphocytes and macrophagesto CD4+ T lymphocytes (Edwards and Hedirck 1998).

MHC alleles from several non-human primate specieshave been studied, i.e. apes such as chimpanzees (Kenter etal. 1992; Mayer et al. 1992), Old World monkeys (OWM)such as Macaca (Zhu et al. 1991; Doxiadis et al. 2000,2001, Otting et al. 2000) and Cercopithecus (Rosal et al.1998), New World monkeys (NWM) such as cotton-toptamarins (Gyllensten et al. 1994; Kriener et al. 2001),

Immunogenetics (2006) 58:542–558DOI 10.1007/s00251-006-0127-0

Experiments carried out in this work complied with currentColombian Ministry of Health law and regulations governing animalcare and handling.

C. F. Suárez :M. E. Patarroyo (*) : E. Trujillo :M. Estupiñán :J. E. Baquero :C. Parra : R. RodriguezFundación Instituto de Inmunmología de Colombia (FIDIC),Carrera 50 No. 26-00,Bogotá, Colombiae-mail: [email protected]

M. E. PatarroyoUniversidad Nacional de Colombia,Bogotá, Colombia

common marmosets (Wu et al. 2000; Antunes et al. 1998),owl monkeys (Niño-Vasquez et al. 2000) and lemurs (Go etal. 2002). MHC molecules present common characteristicsin all these groups (such as polymorphism and diversifyingselection) tied to functional and structural restrictions.Some lineages are shared amongst species, but a trueorthologous relationship has only been found within simiansuborders such as Catarrhini (humans, apes and OWM) andPlatyrrhini, NWM and within Prosimians (lemurs andrelatives; see O’hUigin 1995; Kriener et al. 2000a,b,2001; Go et al. 2002; Trtkova et al. 1995). This contrastswith other MHC-II regions (DP, DQ, DO and DM)exhibiting great conservation amongst primates (thesebeing genuinely orthologous) and having similar chromo-some organisation (Satta et al. 1996).

Several New World monkey species are currently beingemployed in biomedical research, Aotus monkeys being animportant representative. They can predictably develophuman malaria, particularly Plasmodium falciparum (Collins1994; Gysin 1988; Rodriguez et al. 1990) and Plasmodiumvivax asexual/blood stage infections (Pico de Coaña et al.2003). Previous work concerning MHC-DRB has revealedthe presence of at least six different allele lineages (Niño-Vasquez et al. 2000). None of these has exhibited sequenceshaving stop codons or any kind of sequence alteration(insertions or deletions) at exon 2 level, but they haveexhibited great similarity with several HLA-DRB lineages.

Ninety-six Aotus monkeys (71 Aotus nancymaae, 15Aotus nigriceps and 10 Aotus vociferans) were sequencedin this work to establish whether they possessed allelesdifferent to those reported so far. Human and Aotus monkeyMHC-DRB characteristics were then compared. Analysiswas focused on variability patterns within the MHC-DRBβ-domain (exon 2 encoded). A phylogenetic approach wastherefore used for defining the main groups of sequences inthe Aotus species considered here and the variability soanalysed, focusing on the number and type of changesinvolved in the protein sequences. The extent and nature ofselection in these Aotus sequences by comparing them withHLA-DRB and MHC-DRB molecule sequences from otherprimates. Emphasis was placed on those residues involvedin PBR conformation for further analysis of this model’ssuitability for evaluating vaccine candidates for human use.

Materials and methods

Animals

The owl monkeys analysed in this study were caught in theAmazon area close to Leticia (Colombia) and selected atrandom to ensure the presence of a representative repertoireof different alleles. Mononuclear cells were isolated by

density gradient separation of peripheral blood obtained byvenous puncture (Garraud et al. 1994). The blood came from96 healthy monkeys from three different Aotus species: 71 A.nancymaae (29 male, 42 female), 15 A. nigriceps (six male,nine female) and 10 A. vociferans (six male, four female).

RNA extraction, polymerase chain reaction, DNA cloningand sequencing

Total cellular RNA was isolated from peripheral bloodmononuclear cells using the TRIzol one-step procedure(Invitrogen Life Technologies, CA, USA). Moloney MurineLeukemia Virus (Promega, Madison, WI, USA) was used forcDNA synthesis, according to the Manufacturer’s instructions.

cDNA samples were amplified by polymerase chainreaction (PCR) in 100-μl reaction mixtures containing5 mM MgCl2, 10 mM of each of the four dNTPs, 0.2 μMof each primer, 1× PCR buffer and 6 U of taq polymerase(Promega). A Perkin Elmer 9700 thermocycler was used forthe amplifications, using the following profile: 28 cycles at96°C, 58°C and 72°C each for 1 min, followed by 1 cycleof 72°C for 5 min. Sense (5′-GGTGCTGAGCTCCCGACTGGC-3′) and anti-sense (5′-AGGTTGTGGTGCTGCAGGGG-3′) MHC-DRB primers were used for amplify-ing exon 2, as previously described (Niño-Vasquez et al.2000). PCR products were purified with Wizard PCR prepspurification kit (Promega) and cloned into pGEM-T Easyvector (Promega). An ABI PRISM 310 genetic analyser(Perkin Elmer) was used for double-strand plasmid DNAsequencing; ABI PRISM 310 sequencing analysis 3.3 andMT Navigator 1.0.2.b3 software were used for analysingthe data. The reported alleles represented at least twoidentical clones obtained by screening 5 to 22 randomlychosen colonies from the same animal.

Sequence nomenclature

The sequences obtained were reported to the Immuno-genetics’ database at EBI http://www.ebi.ac.uk) and werenamed here according to the rules for standardised MHCnomenclature according to Klein et al. (1990).

Sequence analysis

A. nancymaae (Aona), A. nigriceps (Aoni) and A. vocifer-ans (Aovo) MHC-DRB sequences were analysed (Gen-Bank accession numbers AY563180–AY563263,DQ162624–DQ162736, respectively), together with a rep-resentative sample of simian sequences, i.e. Hominidae(human and apes), Cercopithecidae (OWM) and Platyrrhini(NWM) from the GenBank database for which completeexon 2 MHC-DRB data is available. Human sequences wereobtained from the Anthony Nolan institute (Marsh 1999,

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http://www.ebi.ac.uk/imgt/hla/); only non-identical nucleotidesequences were used. A sample of DP and DQ sequences wasused for providing out-groups (one from each primatespecies). A total of 820 sequences (including Aotus MHC-DRB) were used. The simian species considered were(Ceae) Cercopithecus aethiops, (Maar) Macaca arctoides,(Mafa)Macaca fascicularis, (Mamu)Macaca mulatta, (Mane)Macaca nemestrina, (Masi) Macaca silenus, and (Paca) Papiocynocephalus anubis for OWM; (Aotr) Aotus trivirgatus,(Aona) A. nancymaae, (Aoni) A. nigriceps, (Aovo) A.vociferans, (Aoaz) Aotus azarae, (Camo) Callicebus moloch,(Caja) Callithrix jacchus, (Ceap) Cebus apella, (Saoe)Saguinus oedipus, (Sasc) Saimiri sciureus, (Pipi) Pitheciapithecia, (Capy) Callithrix pygmaea, and (Atbe) Atelesbelzebuth for NWM; and (HLA) Homo sapiens, (Gogo) Gorillagorilla and (Patr) Pan troglodytes for human and apes.

Clustal X software (Thompson et al. 1994) was used foraligning sequences; 267/89 nucleotide/amino acid positionswere analysed altogether. Phylogenetic trees were con-structed with MEGA-2.1 software (Kumar et al. 2001),using minimum-evolution (ME) and neighbour-joining (NJ)distance methods (Rzhetsky and Nei 1993). Geneticdistances were estimated by using P distance, Jukes–Cantor(Jukes 1969) and Kimura 2-parameter (Kimura 1980)methods for nucleotide sequences, and gamma and Poissoncorrected distances for amino acid sequences. Bootstrapanalysis (Hillis 1993) and internal branch test (Sitnikova1996; with 1,000 replicates for each) were used forassigning confidence levels to branch nodes.

MEGA 2.1 software was used for calculating meandifferences between and within groups (nucleotide andamino acids). Standard errors were assessed by the boot-strap method with 1,000 replicates.

MEGA 2.1 software was used for calculating synonymousand non-synonymous substitutions and associated variancerates (assessed by the bootstrap method with 1,000 repli-cates) by Nei–Gojobori’s method (Nei and Gojobori 1986).

Mismatch distribution analysis (Go et al. 2002; Figueroaet al. 2000; Di Rienzo and Wilson 1991) detectedconvergence in Aotus species MHC-DRB. Gene conversionwas estimated using the method proposed by Sawyer(1989) using GENECONV software (Sawyer 1999); thisperforms statistical procedures for aligning possible geneconversion events and provides multiple-comparison cor-rected P values for each instance.

Each position’s variation for a given set of amino-acidaligned human and Aotus MHC-DRB pockets was repre-sented by a one-letter code in its respective position withinthe sequence. Conservative and non-conservative substitu-tion could thus be determined for each position wherevariation in an amino acid symbol’s colour indicated non-conservative changes; maintaining the colour representedconservative changes based on PAM 250 substitution

matrix parameter groups (DENQH, SAT, KR, FYW, LIVM,C, G and P; see Dayhoff et al. 1978).

Results and discussion

Isolated sequences

The MHC-DRB exon 2 from 96 Aotus monkeys (71 A.nancymaae, 10 A. vociferans and 15 A. nigriceps) wasamplified by cDNA PCR. Seven hundred ninety-sevenclones were sequenced, and 67 different sequences wereobserved for A. nancymaae, 30 for A. nigriceps and 13sequences for A. vociferans. Three identical sequences werefound in previous a work on A. nancymaae (Aona-DRB3*0612, Aona-DRB*W1802 and Aona-DRB*W1302;Niño-Vasquez et al. 2000).

The Aotus MHC-DRB sequences reported in this paperwere divided into 12 lineages according to the Immuno-genetics’ database nomenclature; MHC-DRB1*03 wassubdivided into three groups of sequences (GA, GB, GC)and MHC-DRB3*06 into two groups (GA, GB) havingclearly distinctive characteristics. The most frequentlyfound alleles in the 96 owl monkeys analysed here camewithin the MHC-DRB*W13 group (23%), followed byDRB1*03 (18%), DRB*W47 (15%), DRB3*06 (12%),DRB*W29 (11%), DRB*W43 (5%), DRB*W18 (5%),DRB*W38 (3%), DRB*W42 (3%), DRB*W44 (2%),DRB*W46 (2%) and DRB*W45 (1%). The most variablegroups were DRB*W43 (mean 9.9±1.7 nt), DRB*W18(mean 6.4±1.5) and DRB*W13 (mean 5.9±1.2 nt).

Figure 1 shows the MHC-DRB exon 2 deduced proteinalignment, from groups of Aotus and some alleles from theirhuman counterparts’ lineages. The sequences were groupedinto nine blocks based on the most representative AotusDRB* lineages found (see names in the left-hand column).Each block was based on Aotus amino-acid sequencesimilarity with the closest HLA-DRB* homologue (HLA-DRB1*-0403: -0422; -0301; -0701; -0801; -1001; -1130-150101 and HLA-DRB3*-0201) and are highlighted inyellow. Amino acids shown in colour in the figure indicatethe residues forming a particular pocket (Kaufman et al.

�Fig. 1 Aligning Aotus MHC-DRB deduced amino acid sequencesfrom exon 2. One hundred ten amino acid sequences were obtainedafter sequencing 797 recombinant colonies randomly screened from96 Aotus (71 Aona, 10 Aovo and 15 Aoni). Sequences previouslyreported by Niño-Vasquez et al. 2000 (21) are preceded by a dot (∙).The sequence names are provided in the left-handcolumn. Each blockwas based on Aotus amino acid sequence similarity with the closestHLA-DRB* homologue. Dashes correspond to amino-acid identity.Colouredpositions denote amino acid constituted pockets at the PBR:fuchsia P1, blue P4, ochre P6, grey P7, green P9. Secondary structuralfeatures are shown at the bottom of the figure: α1 and α2 alphahelices, β1 to β3 beta sheets, T1 to T5 turns, RC random coil

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HLA-DRβ 1*0301 R F L E Y S T S E C H F F N G T E R V R Y L D R Y F H N Q E E N V R F D S D V G E F R A V T E L G R P D A E Y W N S Q K D L L E Q K R G R V D N Y C R H N Y G V V E S F T V Q R R• Aona DRB1*0303 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - Q - - - - - - - - - - - F - - - - - - - -• Aona DRB1*0304 - - - F Q T - - - - - - - - - - - - - - F - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -• Aona DRB1*0305 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -• Aona DRB1*0307 - - - F Q T - - - - Y - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - R - - - - - - - - - - - - - - - G - - - - - - - -• Aona DRB1*0309 - - - F Q T - - - - Y - - - - - - - - - F - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - R - - - Q - - - - - - - - - - - G - - - - - - - -• Aona DRB1*0311 - - - F Q T - - - - - - - - - - - - - - F - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - - - - - - - - - -• Aona DRB1*0312 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - - - - - - - - - -• Aona DRB1*0316 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - K - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aona DRB1*031701 T - - V Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aona DRB1*031702 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aona DRB1*0318 - - - F Q T - - - - - - - - - - K - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aona DRB1*0319 - - - F Q T - - - - - - - - - - - - - - H - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aoni DRB1*0303 - - - F Q T - - - - Y - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - R - - - - - - - - - - - - - - - G - - - - - - - -Aoni DRB1*0304 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - K - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aoni DRB1*0306 - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aoni DRB1*0307 - - - F Q T - - - - - - - - - - - - - - H - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aovo DRB1*0301 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - R - - - Q - - - - - - - - - - - G - - - - - - - -Aovo DRB1*0302 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - K - Y - - - A - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aovo DRB1*0303 - - - F Q T - - - - - - - - - - - - - - - - H - - - Y - - K - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -Aovo DRB1*0304 - - - F Q T - - - - - - - - - - - - - - - - - - - - Y - - K - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - Y V - - - - - - - - - - - - - - - - - G - - - - - - - -

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HLA DRB1*150101 R F L W Q P K R E C H F F N G T E R V R F L D R Y F Y N Q E E S V R F D S D V G E F R A V T E L G R P D A E Y W N S Q K D I L E Q A R A A V D T Y C R H N Y G V V E S F T V Q R R• Aona DRB*W2901 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -Aona DRB*W2904 - - - E E T - S - - - - L - - - - - - - L - Q - - - - S - - - Y A - - - - - - - - - - - - - - P - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -Aona DRB*W2905 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -Aona DRB*W2906 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - V - - - - - - - -Aona DRB*W2907 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - - - - - A - - - -Aona DRB*W2908 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - - - - - - - - - -Aona DRB*W2909 - - - E E T - S - - - - - T - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - V - G - - - - - -Aoni DRB*W2901 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - - - - - - - - - -Aoni DRB*W2903 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - C - - - - - - - V - - - - - - - -Aoni DRB*W290402 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - C - - - - - - - V - - - - - - - -Aoni DRB*W2902 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y - - - V C - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - C - - - - - - - V - - - - - - - -Aovo DRB*W2901 - - - E E T - S - - - - L - - - - - - - L - Q - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - C - - - - - - - V - - - - - - - -• Aona DRB3*0608 - - - E L V - H - - - - - - - - - - - - Y - D - - - - - - K - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -• Aona DRB3*0609 - - - E L V - H - - - - - - - - - - - - Y - D G - I H - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - V - - - - - - - -• Aona DRB3*0610 - - - E L V - H - - - - - - - - - - - - Y - D - - - - - - K - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -• Aona DRB1*0316 - - - F E T T S - - - - - - - - - - - - Y - D - - - - - - - - Y A - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - - - - - - - F - - - - - - - -Aona DRB*W4201 - - - E E V - D - - - - L - - - - - - - F - E - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - K - - - - - V - - - - E - - -Aoni DRB*W4201 - - - E E V - D - - - - L - - - - - - - F - E - - - - - - - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - L - E R - - Y L - - - - - - - - K - - - - - V - - - - - - - -HLA DRB1*160101 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - F - - D R - - - - - - - - - - - - - - G - - - - - - - -

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- - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F I - E T - - - - - - - - - - - - - - - - - - - - - - -Aona DRB*W1307 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - L - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aona DRB*W1308 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - E - - - F - - L - - F M - E T - - - - - - - - - - - - - - A - - - - - - - -Aona DRB*W1309 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - E - - - F - - L - - F M - E T - - - M - - - - - - - - - - A - - - - - - - -Aona DRB*W1310 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aona DRB*W1311 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - E - - - F - - L - - F M - E T - - - - - - - - K - - - - - - - - - - - - - -Aona DRB*W1312 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - F - - - - - - - - - Y - - - - - - - - R T - - - F - - L - - F M - E T - - - - - - - - K - - - - - - - - - - - - - -Aona DRB*W1313 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - P - - - - - - - E - - - F - - L - - F M - E T - - - - - - - - - - - - - - A - - - - - - - -Aoni DRB*W1301 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F I - E T - - - - - - - - - - - - - - - - - - - - - - -Aoni DRB*W1302 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - H F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aoni DRB*W1303 - - - E - F - P - - - L - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aoni DRB*W1304 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aoni DRB*W1305 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - E - - - F - - L - - F M - E T - - - M - - - - - - - - - - A - - - - - - - -Aoni DRB*W1306 - - S E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - R - -Aoni DRB*W1307 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - G - - - - - - - -Aoni DRB*W1308 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - D - - - - - - - - - - W -Aoni DRB*W1309 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - - - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aovo DRB*W130102 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - F - - - - - - - - - Y - - - - - - - - R T - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aovo DRB*W130101 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - F - - - - - - - - - Y - - - - - - - - R T - - - F - - L - - F M - E T - - - - - - - - K - - - - - - - - - - - - - -Aovo DRB*W1302 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - F - - - - - - - - - Y - - - - - - - - R T - - - F - - L - - F M - E T - - - - - S F - K - - - - - - - - - - - - - -Aovo DRB*W1303 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - - - - -Aovo DRB*W1304 - - - E - F - P - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - Y - - - - - - - - - - - - - F - - L - - F M - E T - - - - - - - - - - - - - - - - - - - A - - -HLA DRB1*160101 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - - - - - - - - - - - F - - D R - - - - - - - - - - - - - - G - - - - - - - -

HLA DRB1*1130 R F L E L L K S E C H F F N G T E R V R F L D R Y F Y N Q E E Y V R F D S D V G E F R A V T E L G R P D E E Y W N S Q K D F L E D R R A A V D T Y C R H N Y G V G E S F T V Q R RAona DRB*W4401 - - - - Q A - - - - Y - - - - - - - - - Y - - - - - - - - K - - - - - - - - - - - L - - - - - - - - R T A Q - - - - - - - - - - - H - - - - - - - - K - - - - - - - - - - - - - -Aoni DRB*W4401 - - - - Q A - - - - Y - - - - - - - - - Y - - - - - - - - K - - - - - - - - - - - L - - - - - - - - R T A Q - - - - - - - - - - - H - - - - - - - - K - K - - - - - - - - - - - -SECONDARY STRUCTURE T

5 β 1 T1 β 2 T2 α 1 α 2 RC β 3 T3 4 T4α

Immunogenetics (2006) 58:542–558 545

1984, Stern et al. 1994): fuchsia for P1, blue for P4, ochrefor P6, grey for P7 and green for P9. PBR sequence secondarystructural features are show at the bottom of the figure.

Slightly less variability was observed in Aotus MHC-DRB beta sheets cf alpha helices. The most polymorphicpositions in the Aotus MHC-DRB locus β sheet were: 9,10, 11, 13, 26, 28, 30, 31, 33 and 37. As these are highlypolymorphic residues in humans, this shows that bothgroups co-varied in these positions. Comparative analysisof alpha helices revealed that the alpha 2 helix had a greaterdegree of polymorphism and a greater number of conservedsubstitutions in each Aotus MHC-DRB lineage. The mostpolymorphic positions in the alpha chain were: 57, 67, 68,70, 73 and 74; they were also highly polymorphic inhumans. Positions 63–68 were more variable in AotusMHC-DRB than in HLA-DRB. The fragment encoding therandom coil structure between residues 86 and 94 showedsimilar polymorphism in Aotus MHC-DRB to that observedfor human lineages.

Some Aotus MHC-DRB sequences were identical at thenucleotide level amongst the species analysed. Only thesequence corresponding to A. nancymaae DRB*W1301was shared by the three species. Identity was only sharedby two species in another ten cases; these numbers are fartoo small to be indicative of evolutionary relationshipamongst the species being studied because of the differencein sample size. However, they do suggest a closerelationship between them. This indicates long-lastingmaintenance of allelic variants, until the ancient origin ofthis species (8 million years ago; see Setoguchi andRosenberger 1987). The Aotus DRB*W43 lineage is worthnoting, as it mainly consisted of A. nigriceps sequences.Given that this species has been less sampled compared toA. nancymaae (even with our sampling limitations), thisgroup suggested that there could be preferential represen-tation for some lineages according to a particular Aotusspecies.

Number of MHC-DRB sequences expressed in Aotus

A maximum of four sequences was observed per monkeyfor A. nancymaae, four for A. nigriceps and three for A.vociferans. The differences between different species couldhave been due to the varying number of monkeys beingsampled for each species and the random method used forchoosing the clones to be sequenced. This number agreedwith the number of MHC-DRB genes expressed in A.trivirgatus (5), determined by denaturing gradient gelelectrophoresis (Middleton et al. 2004). Duplication eventswere detected in several lineages (expression of the samelineage several times in one specimen) such as DRB1*03and DRB*W13 for all species studied, DRB*W29 for A.nancymaae and A. nigriceps, DRB*W43 for A. nigriceps

and DRB*W18, DRB*W47, DRB*W38 and DRB3*06 forA. nancymaae. This issue is still being clarified for furtherstudies.

Evolutionary analysis of Aotus and humanswithin the context of primate MHC-DRB exon 2

Figure 2 shows minimum evolution analysis with Kimura 2parameter distance, including a selection of 170 OTUs,Operational Taxonomic Units (267 nucleotide positions)from 820 primate sequences considered. A sample of allPlatyrrhini MHC-DRB sequences, an MHC-DRB sequencesample from M. mulatta and humans as Catarrhini members,and a representative sample of Aotus MHC-DRB sequencesreported in this paper were used, and some sequences ofMHC-DPB1 and MHC-DQB1 were taken as out-groups.

The tree shows that the groups made up of primateDRB sequences tended to become associated according todescribed allele lineage, but such association was seldomsupported by a statistically solid reliability value, inde-pendently of the type of sequence from which thetopology was calculated (nucleotides or amino acids).This observation is common to other studies using theMHC-DRB exon 2 (Kriener et al. 2000a; Antunes et al.1998; Wu et al. 2000; Go et al. 2002; Figueroa et al. 2000;Middleton et al. 2004), explained by the large number ofvariable positions, the enormous structural restriction cfselective pressure, molecular convergence, and the recom-bination phenomena to which this sector was submitted(Ohta 1991; Kriener et al. 2001; Hughes and Yeager 1998;Yeager and Hughes 1999; Klein et al. 1998; Kriener et al.2000a,b; O’hUigin 1995).

Seventeen groups (shown in bold in Fig. 2) of sequenceswere recovered from topologies generated just from thegenus’ sequences with the complete MHC-DRB exon 2sequence, whether by distance methods (ME, NJ) orparsimony, and independently of the type of sequence used(DNA or proteins). Some groups of Aotus sequences didnot have statistical support, even though most werewell-supported (Fig. 2). These groups were also

�Fig. 2 Minimum evolution tree for primate MHC-DRB exon 2sequences. The tree was constructed based on Kimura 2 parameterscalculated from aligning 267 nt nucleotide from primate MHC-DRBexon 2 sequences. Bootstrap values were calculated based on 1,000replicates, which only showed values higher than 60% (>70% wassignificant). Values shown in bold indicate values that were alsosignificant for internal branch test (IBT; >95%); circles denote valuesthat were only significant for IBT. We have included Catarrhini[Hominoidea (humans shown by boldgreylines) and Cercopithecoidea(OWM, Macaca mulatta) in grey] and Platyrrhini sequences (NWM)including Aotus (blackboldlines) and other NWM genera (black). DPBand DQB sequences from several primate genera were used as out-groups. The scalebar represents 0.05 substitutions per site. See“Materials and methods” section for species’ abbreviations

546 Immunogenetics (2006) 58:542–558

Aona-DRB1*0307Aoni-DRB1*0303

Aotr-DRB1*0301Aona-DRB1*0319

Aoni-DRB1*0306Aotr-DRB1*0303Aoni-DRB1*0304

Aovo-DRB1*0303Aona-DRB1*0303

Aona-DRB1*0312Aona-DRB1*0309Aovo-DRB1*0301

Aotus MHC-DRB1*03 GA

Aona-DRB1*0301Aona-DRB1*0302


Aotus MHC-DRB1*03 GB


Aoni-DRB1*0305Aotus MHC-DRB1*03 GC

Aona-DRB3*0601Aotr-DRB3*0602

Aona-DRB3*0613Aotr-DRB




Aoaz-DRB3*0601Aoni-DRB3*0601


HLA-DRB1*030101HLA-DRB1*030201

Mamu-DRB1*0315Mamu-DRB1*0316

Catarrihini MHC-DRB1*03

Mamu-DRB*W2002HLA-DRB3*010101HLA-DRB3*0201

HLA-DRB3*030101Catarrihini MHC-DRB3

Saoe-DRB3*0508Saoe-DRB3*0510

Pipi-DRB3*0501Camo-DRB3*0501

Atbe-DRB3*0501

Platyrrhini MHC-DRB3*05

Camo-DRB3*0701Aotr-DRB*W1801

Aona-DRB*W1802Aona-DRB*W1801

Aoni-DRB*W1801Aotus MHC-DRB*W18

Aona-DRB*W2901Aoni-DRB*W2901


Aovo-DRB*W2901Aotus MHC-DRB*W29

Aona-DRB*W4201Aoni-DRB*W4201 Aotus MHC-DRB*W42Aona-DRB3*0609


Aotus MHC-DRB3*06 GBCamo-DRB3*0601

Aona-DRB*W4701Aona-DRB*W4702Aona-DRB*W4706

Aona-DRB*W4709Aotus MHC-DRB*W47Aotus MHC-DRB*W45Aona-DRB*W4501Aotus MHC-DRB*W46Aona-DRB*W4601

Ceap-DRB*W1301Ceap-DRB*W1302 Ceap-DRB*W13

Aoni-DRB*W4301Aoni-DRB*W4304

Aovo-DRB*W4301Aoni-DRB*W4303

Aoni-DRB*W4305

Aotus MHC-DRB*W43

Aotus MHC-DRB*W41Aona-DRB*W4101Aona-DRB*W4401

Aoni-DRB*W4401 Aotus MHC-DRB*W44Ceap-DRB*W1303



Aovo-DRB*W1302Aona-DRB*W1310


Aoni-DRB*W1303Aovo-DRB*W1304

Aoni-DRB*W1308Aona-DRB*W1306Aona-DRB*W1307

Aotus MHC-DRB*W13

HLA-DRB5*010101HLA-DRB5*0103 HLA-DRB5

Mamu-DRB*W2507Mamu-DRB*W502

Mamu-DRB19HLA-DRB1*150101

HLA-DRB1*150102 HLA-DRB1*15HLA-DRB1*160101




HLA-DRB1*080201 HLA-DRB1*08Mamu-DRB1*0317

Mamu-DRB1*0318 Mamu-DRB1*03HLA-DRB1*110101


HLA-DRB1*130201 HLA-DRB1*13Caja-DRB1*0301

Caja-DRB1*0302Capy-DRB1*0301

Saoe-DRB1*0301Camo-DRB1*0302

Camo-DRB1*0303

CallitrichidaeMHC-DRB1*03

Ceap-DRB*W1501Ceap-DRB*W1502 Camo-DRB1*03

Sasc-DRB*W1901Sasc-DRB*W1903 Sasc-DRB*W19

Caja-DRB11*0101Saoe-DRB11*0101Caja-DRB*W1611

Saoe-DRB11*0108Caja-DRB*W1601

Caja-DRB*W1605

CallitrichidaeMHC-DRB11/DRB*W16

Caja-DRB*W1201Capy-DRB*W1201

Ceap-DRB*W3201Pipi-DRB*W1201

Saoe-DRB*W1205

CallitrichidaeMHC-DRB*W12

Camo-DRB*W1401Camo-DRB*W1402

Sasc-DRB*W1401Sasc-DRB*W1404

PlatyrrihiniMHC-DRB*W14

Caja-DRB*W1606Caja-DRB*W1607 Caja-DRB*W16

Sasc-DRB*W1203Sasc-DRB*W1204 Sasc-DRB*W12

Caja-DRB*W1608Saoe-DRB*W2201

Saoe-DRB*W2209Sasc-DRB*W1201

PlatyrrihiniMHC-DRB*W22

Aona-DRB*W3801Aoaz-DRB*W3801

Aoni-DRB*W3801Atbe-DRB*W3801

Aotus MHC-DRB*W38

HLA-DRB1*0707HLA-DRB1*0705 HLA-DRB1*07

Mamu-DRB110HLA-DRB1*0406



HLA-DRB1*090102 HLA-DRB1*09Mamu-DRB51

Saoe-DRB5*0701Mamu-DRB5*0307

Mamu-DRB111HLA-DRB1*10HLA-DRB1*100101

Mamu-DRBw1Mamu-DRB41

HLA-DRB4*010101 Catarrihini MHC-DRB4HLA-DPB1*010101

Mamu-DPB1*15 MHC-DPB1Caja-DQB1*2302

HLA-DQB1*0201Mamu-DQB1*1703

MHC-DQB1

99

8499

9994

96

95

99

99

99

99

99

99

99

99

75

99

92

78

89

90

71

98

80

99

97

99

9299

99

64

60

99

98

87

99

97

96

98

99

99

73

99

96

99

83

87

90

77

82

97

65

71

75

88

92

92

76

63

86

61

91

64

70

98

85

62

98

0,05

Aotus MHC-DRB*W30

Immunogenetics (2006) 58:542–558 547

distinguished at both nucleotide and amino acid levelswhen only Aotus MHC-DRB sequences were analysed(data not shown).

No identical MHC-DRB alleles was observed betweenAotus and other primate species; cases of total identityamongst alleles from different primate species are rare(Otting et al. 2000), generally being restricted to speciesfrom the same genus and more rarely from the same family.This indicates that most alleles in primates originated inpost-speciation phenomena; even though a very few havehigh identity at genus level, this is rarely maintainedbeyond this taxonomic division.

Most NWM monkey sequences, irrespective of methodand type of sequence (NT or AA), were grouped into asingle, highly consistent group. This group includedpractically all Platyrrhini lineages (Pithecinae, Atelinae,Cebinae and Callitrichinae) but did not contain Aotus(classified as Cebidae, together with Saimiri and Cebus;Fig. 2). Aotus was seen to be a highly differentiated groupwithin the exon 2 context at nucleotide level, being mainlyrelated to Catarrhini lineages such as DRB3 (from allprimates including other NWM) and DRB1*07 (human)(Fig. 2). Some groups of Aotus MHC-DRB presented anassociation with HLA-DRB1*03 and HLA-DRB1*10 andwith HLA-DRB1*04, HLA-DRB1* 08-11-12-13 and HLA-DRB1*01 at the amino acid level, in addition to thosealready described (data not shown).

Aotus MHC-DRB variability within the context of primateMHC-DRB exon 2

Overall, Aotus DRB had the same variability as humans andapes in the MHC-DRB β-domain at both nucleotide andamino acid levels. It was notable how this variability wasless than that shown for other NWM and OWM. These twogroups of species (the former having lower variation) couldbe distinguished at the PBR, non-PBR and pocket levels(Table 1). Like all primates considered, Aotus had greatervariability (almost double) at protein sequence than nucleo-tide level in exon 2, implying active protein variantproduction (data not shown). Variability trends were verysimilar between nucleotides and amino acids for the threespecies considered. DRB exon 2 analyses showed greatermutation accumulation in the PBR than in the region notdirectly involved in peptide binding (non-PBR in Table 1).In fact, variation at the nucleotide level in the non-PBR wasequivalent to that of amino acids, indicating evolutionarydynamics different to those of the PBR (Table 1). P1 was themost conserved pocket in primates; P4 showed mediumvariation and P6, P7 and P9 showed high variability. AotusMHC-DRB followed a similar variation pattern to thatexhibited by HLA-DRB; however (bearing in mind thedifferent number of monkeys sampled for each species), the

least variable was A. vociferans, and A. nancymaae and A.nigriceps were the most variable (with equivalent values)(Table 1).

Recombination, population expansion and convergencein Aotus MHC-DRB exon 2

Mismatch distribution analysis showed that none of theAotus species analysed showed population expansion or totalrecombination at sequence or MHC-DRB secondary struc-ture level (in spite of evidence suggesting that recombinationphenomena are more frequent in alpha helices than betasheets; Go et al. 2002; see below). The frequencies shownwere clearly bimodal, disagreeing with a Poisson distribution(P<0.0001) in all cases; this was confirmed by theKolmogorov–Smirnov test (data not shown). Such distribu-tion arising from this analysis indicated the loss of allelelineages, thus producing islands of similarity detected aslocal maximums in the distribution of pairwise differencefrequencies amongst the sequences analysed. If sequencedivergence had not occurred due to a random process(bottlenecks, recombination phenomena, high rate of muta-tion provoking convergence phenomena, etc.), then histo-grams would have tended to be unimodal and would havefollowed Poisson distribution (data not shown) (Go et al.2002; Di Rienzo and Wilson 1991; Figueroa et al. 2000).This result indicated that Aotus MHC-DRB did not undergothe aforementioned processes during its evolution.

Additional analysis, following the method implementedin GENECONV software (Sawyer 1989, 1999) showed thatAotus MHC-DRB has suffered 96 significant globalrecombination events (<0.05 P values; Table 2) and 4,756significant pairwise recombination events (<0.05 P values;data not shown) involving all groups of sequences and largeexon 2 segments in each one. Gene conversion was relatedto the search for these genes’ diversity as an adaptiveresponse, increasing MHC-DRB global polymorphism asdescribed for other vertebrate species (Ohta 1991; Yeagerand Hughes 1999). It is noticeable that its very infrequentconversion events in positions 6–13, considered diagnosticfor determining allele lineage (Bergstrom et al. 1999) (e.g.the 9EYSTS13 motif), was thus characteristic of HLA-DRB1*03 (Fig. 1). This criterion was generally compliedwith in Aotus MHC-DRB lineages.

Natural selection in Aotus MHC-DRB exon 2

Two methods were used for analysing the type of selectionto which Aotus MHC-DRB has been submitted withinprimates. The first consisted of the Nei–Gojobori test fordetermining the significance of disequilibrium (if therewere any) between synonymous (dS) and non-synonymous(dN) substitutions. The second was a graphical representa-

548 Immunogenetics (2006) 58:542–558

Tab

le1

Average

aminoacid

identityin

prim

ateMHC-D

RB

DRBsequ

ences

All

PBR

Pockets

NoPBR

Pocket1

Pocket4

Pocket6

Pocket7

Pocket9

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Aminoacids

+DRB

81.71

2.34

67.18

4.23

66.97

4.50

93.57

1.31

85.88

8.63

63.92

8.85

48.22

6.11

50.64

4.78

58.71

5.14

APE-G

ogo-DRB

83.51

3.53

69.48

5.58

66.06

5.09

94.98

1.82

76.25

8.50

61.23

6.77

41.67

7.33

61.56

7.67

56.57

11.29

APE-Patr-DRB

82.96

3.26

68.23

5.13

64.88

3.84

94.98

1.82

86.50

10.25

62.23

6.69

45.11

8.00

49.44

6.33

48.43

6.00

APE-H

LA-D

RB

86.81

2.47

74.93

4.83

73.69

4.03

96.51

1.22

91.75

7.25

71.69

5.62

55.67

5.44

62.11

6.33

66.00

9.14

NWM-A

ona-DRB

84.06

2.62

71.00

4.48

73.25

4.34

94.71

1.59

87.25

10.38

66.31

6.85

59.89

6.44

56.22

6.11

74.71

4.86

NWM-A

oni-DRB

83.71

2.84

71.23

4.63

72.78

4.59

93.90

1.84

88.75

7.13

64.15

7.54

57.00

7.67

51.67

5.22

71.14

7.71

NWM-A

otr-DRB

85.39

2.87

74.50

4.50

75.94

6.00

94.29

2.39

86.25

10.25

70.00

6.85

61.11

7.22

74.4

5.33

85.71

9.14

NWM-A

ovo-DRB

84.82

3.03

70.83

4.95

72.00

5.25

96.22

1.65

89.63

8.13

64.23

7.38

59.22

8.67

53.89

6.78

67.00

6.14

NWM-Caja-DRB

84.11

2.63

70.13

4.83

70.31

4.00

95.53

2.10

86.25

10.38

67.15

6.69

60.22

5.22

50.00

5.56

60.00

11.57

NWM-Cam

o-DRB

75.53

2.88

63.48

4.55

64.44

3.78

85.37

3.29

75.13

14.75

66.08

5.92

51.67

7.89

50.00

7.89

56.14

14.29

NWM-Ceap-DRB

77.45

2.69

65.18

4.03

65.41

4.34

87.49

2.73

76.63

9.50

60.54

7.77

57.78

10.67

42.22

5.33

49.57

7.29

NWM-Saoe-DRB

78.91

2.51

67.53

5.85

67.25

5.44

88.18

2.45

97.75

11.63

61.38

8.54

49.44

10.33

36.44

3.89

41.86

6.71

NWM-Sasc-DRB

75.98

3.19

63.13

6.10

61.81

4.97

86.45

2.57

79.38

9.50

65.15

8.62

43.00

12.33

34.33

5.78

33.29

6.14

OWM-Ceae-DRB

81.67

3.34

66.13

5.23

66.03

5.25

94.37

1.71

85.88

7.25

55.92

5.54

51.33

8.78

43.89

7.56

56.14

8.29

OWM-M

af-D

RB

80.70

2.85

65.95

5.55

64.91

4.31

92.73

2.10

83.00

8.50

62.92

7.23

49.11

6.56

45.44

3.89

51.43

4.86

OWM-M

amu-DRB

81.57

2.99

66.50

5.30

65.56

3.94

93.88

1.76

84.63

9.25

63.15

6.15

50.89

6.44

47.44

5.11

50.00

7.14

OWM-M

ane-DRB

82.90

2.57

68.65

5.65

67.56

4.19

94.53

2.12

84.75

10.00

64.31

7.08

46.56

6.89

47.89

5.67

60.71

5.29

OWM-M

asi-DRB

80.62

3.70

64.65

6.68

61.94

5.22

93.65

2.24

89.25

9.00

58.00

8.00

43.22

9.11

37.67

4.78

40.86

4.29

OWM-Paca-DRB

79.39

3.25

62.93

5.65

62.84

5.28

92.84

2.18

86.75

8.25

56.62

7.15

41.33

7.11

41.56

3.56

55.00

6.71

The

averagewas

obtained

from

allpo

ssible

pairwisecomparisons

ofexon

2anddedu

cedMHC-D

RBproteinsequ

enceswith

ineach

species.Stand

arderrorwas

calculated

bybo

otstrapsampling

with

1,00

0replicates

Immunogenetics (2006) 58:542–558 549

tion of dN cf dS, allowing the intensity of the selectiveprocess to which different DRB exon 2 sectors have beensubmitted to be appreciated; the more the divergence fromcentral tendency (the line of neutrality) in a determinedgroup of sequences, the greater the pressure to which it hasbeen submitted. Presence in the upper left-hand sector of aspecie marker indicated positive or diversifying pressure,and presence in the lower left-hand sector indicated negative

Table 2 Gene conversion events

Sequences Begin(aln)

End(aln)

Length

Aoni_DRB1*0304–Aona_DRB3*0608 39 190 152Aovo_DRB1*0304–Aona_DRB3*0608 39 190 152Aoni_DRB1*W1308–Aona_DRB1*0309 47 181 135Aona_DRB1*0311–Aona_DRB3*0614 112 279 168Aona_DRB*W1303–Aona_DRB1*0309 47 170 124Aona_DRB*W1304–Aona_DRB1*0309 47 181 135Aona_DRB*W1306–Aona_DRB1*0309 47 181 135Aoni_DRB*W1301–Aona_DRB1*0309 47 181 135Aovo_DRB*W1304–Aona_DRB1*0309 47 181 135Aoni_DRB*W1302–Aona_DRB1*0309 47 177 131Aona_DRB*W1310–Aona_DRB1*0309 47 190 144Aoni_DRB*W1306–Aona_DRB1*0309 47 190 144Aona_DRB*W1307–Aona_DRB1*0309 47 180 134Aoni_DRB*W1303–Aona_DRB1*0309 50 181 132Aona_DRB*W1311–Aona_DRB1*0309 47 170 124Aoni_DRB*W1308–Aona_DRB1*0304 43 181 139Aona_DRB1*0303–Aona_DRB3*0616 112 279 168Aona_DRB1*0311–Aona_DRB3*0615 112 279 168Aona_DRB1*0312–Aona_DRB3*0614 112 279 168Aona_DRB*W1302–Aona_DRB1*0309 47 181 135Aovo_DRB*W1303–Aona_DRB1*0309 47 181 135Aona_DRB*W1303–Aona_DRB1*0304 43 170 128Aoni_DRB*W1309–Aona_DRB1*0309 47 190 144Aona_DRB*W1309–Aona_DRB1*0309 47 170 124Aoni_DRB*W1305–Aona_DRB1*0309 47 170 124Aovo_DRB1*0303–Aona_DRB3*0608 83 190 108Aona_DRB*W1304–Aona_DRB1*0304 43 181 139Aona_DRB*W1306–Aona_DRB1*0304 43 181 139Aoni_DRB*W1301–Aona_DRB1*0304 43 181 139Aovo_DRB*W1304–Aona_DRB1*0304 43 181 139Aoni_DRB*W1302–Aona_DRB1*0304 43 177 135Aona_DRB*W1311–Aona_DRB1*0304 43 170 128Aona_DRB*W1307–Aona_DRB1*0304 43 180 138Aoni_DRB*W1307–Aona_DRB1*0309 47 181 135Aona_DRB1*0303–Aona_DRB3*0617 112 279 168Aona_DRB1*0312–Aona_DRB3*0615 112 279 168Aona_DRB*W1310–Aona_DRB1*0304 43 190 148Aoni_DRB*W1306–Aona_DRB1*0304 43 190 148Aoni_DRB*W1303–Aona_DRB1*0304 50 181 132Aona_DRB*W1308–Aona_DRB1*0309 47 170 124Aona_DRB*W1302–Aona_DRB1*0304 43 181 139Aovo_DRB*W1303–Aona_DRB1*0304 43 181 139Aoni_DRB*W1302–Aona_DRB1*0302 43 166 124Aoni_DRB*W1302–Aona_DRB1*0326 43 166 124Aoni_DRB*W1308–Aona_DRB1*0302 43 166 124Aoni_DRB*W1308–Aona_DRB1*0326 43 166 124Aona_DRB*W4711–Aona_DRB1*0311 39 166 128Aona_DRB*W2906–Aona_DRB3*0609 95 279 185Aona_DRB*W1309–Aona_DRB1*0304 43 170 128Aoni_DRB*W1305–Aona_DRB1*0304 43 170 128Aona_DRB1*0304–Aona_DRB3*0614 112 256 145Aona_DRB1*0305–Aona_DRB3*0614 112 256 145Aona_DRB1*0318–Aona_DRB3*0614 112 256 145Aona_DRB1*0319–Aona_DRB3*0614 112 256 145Aoni_DRB1*0307–Aona_DRB3*0614 112 256 145

Table 2 (continued)

Sequences Begin(aln)

End(aln)

Length

Aotr_DRB1*0301–Aona_DRB3*0614 112 256 145Aovo_DRB1*0303–Aona_DRB3*0614 112 256 145Aona_DRB*W2902–Aona_DRB*W2905 16 190 175Aona_DRB*W2907–Aona_DRB1*0316 94 255 162Aona_DRB*W2907–Aona_DRB3*0609 95 256 162Aona_DRB1*0303–Aona_DRB3*0601 112 282 171Aoni_DRB*W1309–Aona_DRB1*0304 43 190 148Aona_DRB*W2909–Aona_DRB3*0609 95 261 167Aona_DRB1*0311–Aona_DRB3*0623 104 279 176Aovo_DRB1*0303–Aona_DRB3*0605 104 279 176Aovo_DRB1*0303–Aona_DRB3*0606 104 279 176Aona_DRB*W1303–Aona_DRB1*0302 43 166 124Aona_DRB*W1304–Aona_DRB1*0302 43 166 124Aona_DRB*W1304–Aona_DRB1*0326 43 166 124Aona_DRB*W1306–Aona_DRB1*0302 43 166 124Aona_DRB*W1306–Aona_DRB1*0326 43 166 124Aona_DRB*W1307–Aona_DRB1*0302 43 166 124Aona_DRB*W1307–Aona_DRB1*0326 43 166 124Aoni_DRB*W1301–Aona_DRB1*0302 43 166 124Aoni_DRB*W1301–Aona_DRB1*0326 43 166 124Aoni_DRB*W1302–Aona_DRB1*0320 43 166 124Aoni_DRB*W1302–Aotr_DRB*W1303 43 166 124Aoni_DRB*W1308–Aona_DRB1*0320 43 166 124Aoni_DRB*W1308–Aotr_DRB*W1303 43 166 124Aovo_DRB*W1304–Aona_DRB1*0302 43 166 124Aovo_DRB*W1304–Aona_DRB1*0326 43 166 124Aona_DRB*W4101–Aona_DRB1*0305 89 181 93Aona_DRB*W4101–Aona_DRB1*0318 89 181 93Aona_DRB*W4101–Aotr_DRB1*0301 89 181 93Aona_DRB*W4101–Aotr_DRB1*0302 89 181 93Aona_DRB1*0318–Aona_DRB3*0605 78 279 202Aona_DRB1*0319–Aona_DRB3*0605 78 279 202Aoni_DRB1*0307–Aona_DRB3*0605 78 279 202Aotr_DRB1*0301–Aona_DRB3*0605 78 279 202Aoni_DRB*W1307–Aona_DRB1*0304 43 181 139Aona_DRB*W1308–Aona_DRB1*0304 43 170 128Aona_DRB1*0308–Aona_DRB3*0622 47 166 120Aona_DRB*W2902–Aona_DRB3*0614 141 256 116Aona_DRB*W2905–Aona_DRB1*0316 94 279 186Aona_DRB*W1807–Aona_DRB*W3001 102 168 67Aotr_DRB*W1301–Aona_DRB*4701 47 173 127

Global inner significant fragments (Sim P values <0.05)were calculated with GENECONV software. The table shows thebeginning, end and length of the fragments

550 Immunogenetics (2006) 58:542–558

or conservative pressure. The same graph shows whether thegroup of sequences have (or did not have) a significantselection value according to Nei–Gojobori test (Nei andGojobori 1986).

The type of selection between different Aotus MHC-DRB PBR sectors was similar to HLA-DRB. Figure 3presents the result of the above analysis. All speciesshowed a slight tendency to accumulate more non-synon-

AllGogo

Patr

HLA

Aovo

Camo

CeapSaoe

Sasc

CeaeMane

Masi

AonaAoni

Aotr

Caja

Mafa

Mamu

Paca

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

HLA

Aoni

Aovo

Camo Ceap

SaoeSasc

MafaMasi

PacaAll

GogoPatr

AonaAotrCaja

CeaeMamuMane

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18

AllGogo

Patr

HLA

AonaAoni

Aotr

Aovo

Camo

Ceap

SaoeSasc

Mafa

MamuMane

Masi

Paca

Caja

Ceae

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

AllGogoPatr

HLA

Aona

Aoni

Aotr

AovoCamo

Ceap

Ceae

Mane

Masi

Paca

Caja

SaoeSasc

Mafa

Mamu

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

All

Gogo

Patr

HLA

AonaAotr

Aovo

Camo

Ceap

Saoe

Sasc

Ceae

Mafa

Mamu

Mane

Masi

Paca

Aoni

Caja

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Gogo

Patr

HLAAona

Aotr

Aovo

Camo

CeapSaoe

Sasc

Ceae

Mafa

MamuMane

Paca

All

Aoni

Caja

Masi

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.05 0.10 0.15 0.20 0.25 0.30 0.35

AllGogo

Patr

HLAAona

Aoni

Aotr

Aovo

Camo

Ceap

Saoe

Sasc

Mane

Masi

PacaCajaCeae

MafaMamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

Gogo

HLA

Aoni

Aotr

Camo Ceap

Saoe

Sasc

Ceae

Mafa

Mane

Masi

Paca

AllPatr

Aona

Ao

vo

Caja

Mamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.00 0.10 0.20 0.30 0.40 0.50 0.60

Gogo

Aotr

Camo

Ceap

SaoeSasc

CeaeMafa

Mamu

MasiPaca

AllPatr

HLA

Aona

Aoni

Ao

vo Caja

Mane

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

dN

dS

whole exon 2 non-PBR PBR

Set of pocket residues

OWM NWM AotusHLA-Apes All DRB

Pocket 1 Pocket 4

Pocket 6 Pocket 7 Pocket 9

AllGogo

Patr

HLA

Aovo

Camo

CeapSaoe

Sasc

CeaeMane

Masi

AonaAoni

Aotr

Caja

Mafa

Mamu

Paca

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

HLA

Aoni

Aovo

Camo Ceap

SaoeSasc

MafaMasi

PacaAll

GogoPatr

AonaAotrCaja

CeaeMamuMane

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18

AllGogo

Patr

HLA

AonaAoni

Aotr

Aovo

Camo

Ceap

SaoeSasc

Mafa

MamuMane

Masi

Paca

Caja

Ceae

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

AllGogoPatr

HLA

Aona

Aoni

Aotr

AovoCamo

Ceap

Ceae

Mane

Masi

Paca

Caja

SaoeSasc

Mafa

Mamu

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

All

Gogo

Patr

HLA

AonaAotr

Aovo

Camo

Ceap

Saoe

Sasc

Ceae

Mafa

Mamu

Mane

Masi

Paca

Aoni

Caja

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Gogo

Patr

HLAAona

Aotr

Aovo

Camo

CeapSaoe

Sasc

Ceae

Mafa

MamuMane

Paca

All

Aoni

Caja

Masi

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.05 0.10 0.15 0.20 0.25 0.30 0.35

AllGogo

Patr

HLAAona

Aoni

Aotr

Aovo

Camo

Ceap

Saoe

Sasc

Mane

Masi

PacaCajaCeae

MafaMamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

Gogo

HLA

Aoni

Aotr

Camo Ceap

Saoe

Sasc

Ceae

Mafa

Mane

Masi

Paca

AllPatr

Aona

Ao

vo

Caja

Mamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.00 0.10 0.20 0.30 0.40 0.50 0.60

Gogo

Aotr

Camo

Ceap

SaoeSasc

CeaeMafa

Mamu

MasiPaca

AllPatr

HLA

Aona

Aoni

Ao

vo Caja

Mane

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

dN

dS




Pocket 1 Pocket 4


AllGogo

Patr

HLA

Aovo

Camo

CeapSaoe

Sasc

CeaeMane

Masi

AonaAoni

Aotr

Caja

Mafa

Mamu

Paca

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

HLA

Aoni

Aovo

Camo Ceap

SaoeSasc

MafaMasi

PacaAll

GogoPatr

AonaAotrCaja

CeaeMamuMane

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18

AllGogo

Patr

HLA

AonaAoni

Aotr

Aovo

Camo

Ceap

SaoeSasc

Mafa

MamuMane

Masi

Paca

Caja

Ceae

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

AllGogoPatr

HLA

Aona

Aoni

Aotr

AovoCamo

Ceap

Ceae

Mane

Masi

Paca

Caja

SaoeSasc

Mafa

Mamu

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

All

Gogo

Patr

HLA

AonaAotr

Aovo

Camo

Ceap

Saoe

Sasc

Ceae

Mafa

Mamu

Mane

Masi

Paca

Aoni

Caja

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Gogo

Patr

HLAAona

Aotr

Aovo

Camo

CeapSaoe

Sasc

Ceae

Mafa

MamuMane

Paca

All

Aoni

Caja

Masi

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.05 0.10 0.15 0.20 0.25 0.30 0.35

AllGogo

Patr

HLAAona

Aoni

Aotr

Aovo

Camo

Ceap

Saoe

Sasc

Mane

Masi

PacaCajaCeae

MafaMamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

Gogo

HLA

Aoni

Aotr

Camo Ceap

Saoe

Sasc

Ceae

Mafa

Mane

Masi

Paca

AllPatr

Aona

Ao

vo

Caja

Mamu

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.00 0.10 0.20 0.30 0.40 0.50 0.60

Gogo

Aotr

Camo

Ceap

SaoeSasc

CeaeMafa

Mamu

MasiPaca

AllPatr

HLA

Aona

Aoni

Ao

vo Caja

Mane

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

dN

dS




Pocket 1 Pocket 4


AB

C

D E F

G H I

Fig. 3 Primate MHC-DRB natural selection. The graphs for specificpockets show dN cf dS substitutions; the line represents the tendencytowards neutrality. The regions above and below the line of neutralitydenote positive selection pressure and negative selection pressure,respectively. Names are shown in bold and greatersymbolsizeindicates significant selection pressure as revealed by Nei–Gojobori

test. A rhombus denotes the set of all MHC-DRB studied. Afilledcircle denotes human and ape MHC-DRB. A filledsquaredenotes OWM MHC-DRB, a filledtriangle denotes NWM MHC-DRB and an emptytriangle denotes Aotus MHC-DRB sequences. Seethe “Materials and methods” section for species’ abbreviations

Immunogenetics (2006) 58:542–558 551

ymous (dN) than synonymous (dS) mutations for the wholeof exon 2 (Fig. 3a); however, this tendency was onlysignificant in the Nei–Gojobori test for some NWM species(Aoni, Caja, Sasc and Ceap, shown in bold). The closestgroup to HLA-DRB was Aotus MHC-DRB, showing thattotal variability tendency followed a similar pattern foraccumulating dN cf dS mutations.

The non-peptide binding region (non-PBR, Fig. 3b)consistently exhibited negative selective pressure andseparation between Catarrhini (OWM and humans andapes) and Platyrrhini (NWM), as the first accumulatedsynonymous cf non-synonymous mutations with signifi-cantly greater intensity, whilst NWM presented a patternclose to neutrality. Aotus MHC-DRB moved away from thistendency, and (even though not having significant negativepressure) it approached the tendency exhibited by HLA-DRB more than other NWM, once more confirming thesetwo species’ close similarity at this level.

PBR (Fig. 3c) and direct peptide binding sites (pockets)(Fig. 3d) had positive selective pressure, being significantfor most DRB exon 2 from the species analysed. AotusMHC-DRB and HLA-DRB presented similar values in bothcases, just like the whole exon.

Except for P1, primate DRB pockets (P1–P9, Figs. 3e–i)followed the trend observed in the PBR and the set ofpockets. P1 showed a tendency to have negative selectivepressure in most Catarrhini species including humans, whilstPlatyrrhini did not. Some species even exhibited significantpositive selective pressure, such as C. apella and A.nigriceps (Fig. 3e). It is notable that this highly specificlevel followed the same tendency seen when analysingvariability regarding low and high variability pockets andhigh and low variability species; a more diffuse associationwas observed between HLA-DRB and Aotus MHC-DRB.Even though HLA-DRB and Aotus MHC-DRB had a similaroverall pattern of diversity and dN cf dS, the way they accu-mulated changes at each pocket level varied more specifical-ly. Both Aotus and human species had the same tendenciesregarding selective pressures, except for P1; however, AotusMHC-DRB presented a pattern more like Catarrhini MHC-DRB, always being presented as an atypical NWM.

Some characteristics divided NWM (excluding Aotus)from OWM, humans and apes (such as greater variationand quasi-neutral non-PBR pattern and greater diversity inP1), pointing to specific specialisation between both orders.Except for this behaviour, selection tendencies in distinctprimate DRB exon 2 regions were generally similar.

Behaviour within each lineage was more conservativethan for the species’ set of DRB, each exhibiting its owncharacteristic stamp (data not shown). Such conservationcould have indicated functional specialisation within eachspecies; however, HLA-DRB and Aotus MHC-DRB line-ages shared characteristics a having common variation,

reflecting similar pressures and indicating a commonfunctional role.

Aotus showed a high degree of convergence with humanMHC-DRB at PBR

When only the PBR was considered and the same analysisperformed, it was then found that these relationships weregenerally maintained, but that more groups of Aotus MHC-DRB sequences converging with Catarrhini allele lineagesbecame clearly defined, being well above those presented inother Platyrrhini (data not shown). The relationshipbetween Aotus MHC-DRB and MHC-DRB allele lineagescharacteristic of humans and other Catarrhini became moreevident at the nucleotide and amino acid residue levels(being directly related to conforming pockets), presenting asignificant degree of identity amongst them and being veryclearly differentiated from MWM allele lineages. A tree forAotus and human sequences (Fig. 4) clearly showed theconvergences detected at this level between these twospecies’ DRB groups.

All Aotus MHC-DRB were grouped with human andother Catarrhini MHC-DRB sequences in exon 2 at thelevel of amino acids directly involved in peptide binding(35 residues). The following associations were found(Fig. 4), showing only associations with HLA-DRB:

1. HLA-DRB1* 08/11/12/13/14 converged with AotusMHC-DRB1*03 GB.

2. HLA-DRB1*1130 converged with Aotus MHC-DRB*W44.

3. HLA-DRB1*03 converged with Aotus MHC-DRB1*03GA.

4. HLA-DRB3 and HLA-DRB1*0422 converged withAotus MHC-DRB*W18 and DRB3*06 GA.

5. HLA-DRB1*04 and HLA-DRB1*1122 converged withAotus MHC-DRB*W45, W46 and W47.

6. HLA-DRB1*09 and HLA-DRB1*07 converged withAotus MHC-DRB*W30 and W38.

7. HLA-DRB4 and HLA-DRB1*10 converged with AotusMHC-DRB*W41 and W43.

8. HLA-DRB1*15 and DRB1*16 were related to AotusMHC-DRB*W29, W42, DRB6*03 GB, DRB1*03 GCand MHC-DRB*W13.

�Fig. 4 Minimum evolution tree for owl monkey–human MHC-DRBpocket positions. The tree was constructed using a P-distance matrixcalculated from aligning deduced amino acid sequences. Bootstrapvalues were calculated based on 1,000 replicates. Only values higherthan 60% are shown (>70% was significant); bold values indicatevalues that were also significant for internal branch test (IBT; >95%)and circles denote values only significant for IBT. Collapsed brancheswere denoted by triangles. Human and Aotus sequences are drawn ingrey and black, respectively. HLA DPB and DQB (grey) sequenceswere used as out-group. The scalebar represents 0.1 substitutions persite. See “Materials and methods” section for species’ abbreviations

552 Immunogenetics (2006) 58:542–558

HLA-DRB1*130101HLA-DRB1*1309HLA-DRB1*1304







HLA-DRB1*1409HLA-DRB1*1402HLA-DRB1*1406HLA-DRB1*1414HLA-DRB1*1423


HLA-DRB1*1404Aona-DRB1*0301






HLA-DRB1*08/11/12/13/14

HLA-DRB1*11HLA-DRB1*1130Aona-DRB*W4401

Aoni-DRB*W4401 Aotus MHC-DRB*W44Aona-DRB1*0305

Aovo-DRB1*0303Aona-DRB1*0307

Aotr-DRB1*0303Aona-DRB1*0304


Aona-DRB1*0309Aovo-DRB1*0301




HLA-DRB1*03



HLA-DRB3*0108

HLA-DRB3


Aona-DRB*W1802Aotr-DRB*W1801

Aotus MHC-DRB*W18

HLA-DRB1*0422Aona-DRB3*0620



Aoni-DRB3*0601Aona-DRB3*0604







HLA-DRB1*04

HLA-DRB1*0402HLA-DRB1*0412 HLA-DRB1*04

Aotus MHC-DRB*W45Aona-DRB*W4501Aotus MHC-DRB*W46Aona-DRB*W4601HLA-DRB1*11HLA-DRB1*1122




Aotus MHC-DRB*W47

HLA-DRB1*15HLA-DRB1*150101HLA-DRB1*16HLA-DRB1*160101


Aona-DRB*W2907Aovo-DRB*W2901

Aotus MHC-DRB*W29

Aona-DRB*W4201Aoni-DRB*W4201 Aotus MHC-DRB*W42Aona-DRB3*0608


Aotus MHC-DRB3*06 GB


Aoni-DRB1*0305Aotus MHC-DRB1*03 GC



Aovo-DRB*W1302Aona-DRB*W1310

Aoni-DRB*W1307Aona-DRB*W1307

Aoni-DRB*W1302Aovo-DRB*W1304Aona-DRB*W1304


Aotus MHC-DRB*W13

HLA-DRB1*10HLA-DRB1*100101HLA-DRB4HLA-DRB4*010101Aotus MHC-DRB*W41Aona-DRB*W4101



Aoni-DRB*W4302

Aotus MHC-DRB*W43


Aoni-DRB*W3801Aotus MHC-DRB*W38

HLA-DRB1*09HLA-DRB1*090102Aotus MHC-DRB*W30Aona-DRB*W3001



HLA-DPB1*010101HLA-DQB1*0201

8698

98

78

92

92

95

96

68

92

87

63

92

96

98

90

80

73

84

73

74

71

97

73

63

60

0,1

Immunogenetics (2006) 58:542–558 553

Aotus MHC-DRB thus became a case having a highdegree of convergence, until clear evidence of the indepen-dent origin of MHC-DRB from Catarrhini and Platyrrhiniwas presented (Kriener et al. 2000a,b, 2001). The homol-ogy presented with HLA-DRB and MHC DRB from otherCatarrhini was thus an analogy; this denotes similarimmune-functional restrictions for Aotus and humans,guiding the generation and evolution of an MHC-DRBrepertoire. On the other hand, Aotus MHC-DRB has a highdegree of differentiation regarding other NWM, indicatingindependent MHC-DRB evolution and, maybe, other func-tional pressures.

Further analysis at lineage level showed the high degreeof convergence between owl monkeys and humans inMHC-DRB. Table 3 presents values (weighted by PAM

250 matrix) for the homology between allele lineages fromthese two species. Mean homology value was greater than90% in all cases, and top homology limit exceeded 85% forall exon 2 protein deduced sequences. For the pocketspositions (the most variable residues in PBR), mean valuewas around 80% and top homology reached 100%.

Aotus MHC-DRB cf HLA-DRB1 pocket variability

Figure 5 shows pocket variability and how residues inter-acted with the TCR. A change of colour indicated that suchchange was considered to be non-conservative; if the colourwere maintained, then the change was considered to beconservative. The colours corresponded to groups deducedfrom PAM 250 (Dayhoff et al. 1978). This amino acid

Table 3 Mean and range homology between Aotus MHC-DRB and HLA–DRB in exon 2 deduced amino acid sequences and just pocket ositions,as assessed by PAM 250 matrix

Aotus Human Exon 2 (aa) Pockets

Mean % Min % max % mean % min % max %

MHC-DRB*W13 vs HLA-DRB1*15 92 89 94 81 77 85MHC-DRB*W13 vs HLA-DRB1*16 90 87 92 76 71 80MHC-DRB*W18 vs HLA-DRB1*0422 92 89 94 88 82 94MHC-DRB*W18 vs HLA-DRB3 91 86 93 84 74 91MHC-DRB*W29 vs HLA-DRB1*15 88 86 89 72 68 74MHC-DRB*W29 vs HLA-DRB1*16 87 85 89 71 68 74MHC-DRB*W30 vs HLA-DRB1*07 91 89 92 78 74 80MHC-DRB*W30 vs HLA-DRB1*09 88 – – 71 – –MHC-DRB*W38 vs HLA-DRB1*07 90 87 93 81 77 88MHC-DRB*W38 vs HLA-DRB1*09 88 87 93 78 74 80MHC-DRB*W41 vs HLA-DRB4 84 83 85 62 62 65MHC-DRB*W41 vs HLA-DRB*10 88 – – 77 – –MHC-DRB*W42 vs HLA-DRB*15 89 88 91 74 71 77MHC-DRB*W42 vs HLA-DRB*16 88 87 89 72 68 74MHC-DRB*W43 vs HLA-DRB4 83 78 86 66 62 71MHC-DRB*W43 vs HLA-DRB1*10 89 83 91 82 77 85MHC-DRB*W44 vs HLA-DRB1*1130 92 92 92 91 91 91MHC-DRB*W45 vs HLA-DRB1*1122 93 – – 88 – –MHC-DRB*W45 vs HLA-DRB1*04 94 92 96 89 82 94MHC-DRB*W46 vs HLA-DRB1*1122 92 – – 82 – –MHC-DRB*W46 vs HLA-DRB1*04 94 93 95 84 80 88MHC-DRB*W47 vs HLA-DRB1*1122 96 94 97 96 91 97MHC-DRB*W47 vs HLA-DRB1*04 97 93 100 94 88 100MHC-DRB1*03 GA vs HLA-DRB1*03 93 89 95 85 80 91MHC-DRB1*03 GB vs HLA-DRB1*08 92 88 94 85 82 88MHC-DRB1*03 GB vs HLA-DRB1*11 90 86 93 83 74 88MHC-DRB1*03 GB vs HLA-DRB1*12 84 83 86 72 68 74MHC-DRB1*03 GB vs HLA-DRB1*13 89 85 93 82 74 91MHC-DRB1*03 GB vs HLA-DRB1*14 89 85 93 81 74 88MHC-DRB1*03 GC vs HLA-DRB1*15 91 89 92 79 77 80MHC-DRB1*03 GC vs HLA-DRB1*16 88 87 89 73 71 74MHC-DRB3*06 GA vs HLA-DRB1*0422 95 93 97 91 88 94MHC-DRB3*06 GA vs HLA-DRB3 91 86 94 82 71 91MHC-DRB3*03 GB vs HLA-DRB1*15 87 86 88 71 68 74MHC-DRB3*03 GB vs HLA-DRB1*16 87 85 88 70 65 74

554 Immunogenetics (2006) 58:542–558

classification preserved and combined both physicochemicalattribute-defined groups (volume, charge, hydrophobicityand acid–base division) and genetic code constraints.

All Aotus MHC-DRB lineages were polymorphic.Regarding HLA-DRB, Aotus MHC-DRB usually presentedthe greatest polymorphism, showing more variation thanHLA-DRB in P1 and P4 (the least variable in primates),whereas HLA-DRB was more variable in P9 than Aotus

MHC-DRB. Pockets 6 and 7 (the most variable withinprimates) revealed a varied tendency between the twospecies; however, substitutions observed in Aotus cfhumans were mostly conservative.

Overall conservation between Aotus and human MHC-DRB(even in polymorphic positions) meant that the same aminoacids frequently co-varied in both species even though theproportion differed in each lineage (amino acid order in Fig. 5

81 Y Y828385 A A Q86 F V V V F G V F G R V F V V F G89 L S90 A91 E L14 V1526 N Y F H F Y L F F28 I H E Q Y D40 S F70 E D R Y D R V71 T R K A E K R E7273 A74 E E L Q Q S78 Y V Y C79 R9 Q K10 Q Q11 K A F V A T A R12 F13 Y D P28 I H E Q Y D29 S30 R Y L71 T R K A E K R E13 Y D P28 I H E Q Y D30 R Y L38 R V V A L V47 F F F Y61 F F L64 L L67 Y I L I I F I I F71 T R K A E K R E9 Q K V13 Y P D30 R Y L37 V S F V S F H N Y F G F L Y V S F N38 R V V A L V57 D S D A V Y S A S60 H H G K61 F F L60 H G K64 L L6670 E D R Y D R V77 N T N T81 Y85 A A Q

V

T

L

Y

Y

L Q

DY

DY

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RS

LE W

A

L QR I

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RS

R

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Fig. 5 Owl monkeys (Aotus) and Human (Homo) MHC-DRB lineagesequence diversity at pocket level (Pockets 1, 4, 6, 7 and 9) and atTCR contact residue level. The nature of amino acid change(conservative/non-conservative) was deduced using PAM 250 ma-trix-inferred groups: DENQH (green), SAT (blue), KR (orange), FYW

(grey), LIVM (purple), C (yellow), G (brown) and P (red). If colourwas maintained, a conservative change occurred; if not, then a non-conservative change took place. Relative residue abundance in thelineages was denoted by the order of amino acids for each positionconsidered

Immunogenetics (2006) 58:542–558 555

corresponded to their frequency when appearing in allelelineage). The positions generally presented slight changesrespecting property conservation; however, some positionshad high variability, especially in P6, P7 and P9, as thesegenerally shared residues or types of property in HLA-DRBand Aotus MHC-DRB. Positions interacting with the TCR alsoshowed a high degree of conservation. It is worth noting thatpolar amino acids predominated in the positions involved(Fig. 5). The foregoing implies that similarity between humansand Aotus reflects a discretely structured DRB variabilitypattern, suggesting that both species’ MHC-DRB could besubmitted to the same functional demands within this sector(the most variable in the PBR).

Each lineage can be analysed in detail using theinformation in Fig. 5. For example, the Aotus lineagesMHC-DRB*W45, *W46 and W*47 (taken as a group)converge with HLA-DRB1*04, showing great homology atpocket level, ranging from 80 to 100% and having 95%mean (Table 3). Figure 5 shows that variant position 86 inP1 presented G and V in both species, these residues’alternation being the tendency for other allele lineages inboth HLA-DRB and Aotus MHC-DRB. Other Aotus MHC-DRB lineages also showed F in this position.

These changes may be considered non-conservative byPAM 250, indicating major changes in physicochemical-genetic code attributes. V and G were separated by onenucleotide substitution in the second position at geneticcode level (V: GGN→G: GTN), and F was separated fromV by a change in first position (F: TTY→V: GTT, GTC).The interchange between G and F could only occur as V asan intermediary state; this change may easily have beenpresented by point mutation in positive selection stressedregions. These V and G changes shared similar surface areain folded proteins (V, 23.7 Å2 and G, 24.5 Å2) (Janin et al.1978) and the same charge. F presented a similar surfacearea as V and G (25.5 Å2) and the same polarity but sharedhigh hydrophobicity and non-polar nature with V. Themajor change between these three residues was presented interms of volume (G, 60 Å3; F, 189 Å3; V, 141.4 Å3;Goldsack and Chalifoux 1973).

This example indicates that a single point mutation mayexplain the emergence of non-conservative polymorphismin several pocket positions having variable physicochemicalproperty conservation in both HLA-DRB and Aotus MHC-DRB. For example, previously analysed lineage positions40 (F→L) and 70 (D→Y) in P4, 9 (E→K), 11 (V→F) and13 (H→P, H→D) in P6, 64 (Q→L) and 67 (L→F, I→F) inP7, 37 (Y→V, Y→S), 57 (D→Y) and 60 (Y→H) in P9 andin TCR binding position 70 (D→Y), 77 (T→N) and 91(V→E) may be explained by this process. In most cases,these non-conservative changes implied fewer sequencescompared to the predominant residue in the allele lineage.

This pattern explained most polymorphic non-conserva-tive changes in the remaining lineages shown in Fig. 5.Very distinct physicochemical properties were presented toa much lesser degree than other changes involving moresteps at the codon level. This fact, the high degree ofhomology and the co-variation exhibited could implysimilar Aotus MHC-DRB and HLA-DRB peptide presenta-tion ability, highlighting the similarity between these twospecies’ immune systems.

Concluding remarks

The hallmark of classic MHC in all vertebrates is theirenormous polymorphism as a response to the dynamics ofthe host–pathogen relationship. Such polymorphism isactively maintained by adaptive evolution in the form ofbalanced selection (Hughes and Yeager 1998). Mutationand recombination processes (cross-over, micro-recombi-nation and gene conversion) have contributed towardsmoulding MHC polymorphism (Ohta 1991; Yeager andHughes 1999). They could be trans-specific (TSP), as allelevariants increasing an individual’s suitability are maintainedbeyond the speciation process, leading to different speciesexhibiting common allele lineages (Klein et al. 1998).Natural selection could also have provoked a level ofmolecular PBR convergence amongst distantly relatedMHC alleles, indicating that the need for adapting MHCto peptide presentation may have optimum solutions at thesequence level (O’hUigin 1995). Both processes haveproduced similarity in divergent species’ PBR, clearly seenin the case of Catarrhini and Platyrrhini MHC-DRBevolution (Trtkova et al. 1995; Kriener et al. 2000a,b,2001). However, whilst similarity between genes caused byTSP denotes homology, similarity due to convergencedenotes functional analogy, as could be occurring inHLA-DRB cf Aotus MHC-DRB.

Aotus MHC-DRB was highly idiosyncratic (presentinghigh recombination rates) and preferentially presentedassociation with MHC-DRB from humans, apes andOWM rather than with NWM MHC-DRB. Aotus MHC-DRB extensively converged with HLA-DRB. This conver-gence with HLA-DRB happened at different levels; itoccurred in the amount of overall variability, types ofsubstitution found, similar natural selective pressures andcommon pocket amino acid property signatures at thesequence level. The functional implications of suchconvergence are evident. Aotus has obvious potentialamongst NWM as a promising animal model for thecomparative study of processes involving peptide presenta-tion with MHC-DRB. The overall evidence regarding bio-logical similarity between human and owl monkey immune

556 Immunogenetics (2006) 58:542–558

systems justifies its use as an excellent animal model forpharmacological and vaccine-development research.

Acknowledgements This research has been supported by theColombian President’s Office and the Colombian Ministry of SocialProtection. We would like to thank Jason Garry for patientlyreviewing the manuscript.

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Owl monkey MHC-DRB exon 2 reveals high similarity with several HLA-DRB lineages

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