Hyperconservation of the N-Formyl Peptide Binding Site of M3: Evidence that M3 Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated This information is current as Molecular Pattern of September 27, 2021. C. Kuyler Doyle, Beckley K. Davis, Richard G. Cook, Robert R. Rich and John R. Rodgers J Immunol 2003; 171:836-844; ; doi: 10.4049/jimmunol.171.2.836 Downloaded from http://www.jimmunol.org/content/171/2/836

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hyperconservation of the N-Formyl Peptide Binding Site of M3: Evidence that M3 Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated Molecular Pattern1

C. Kuyler Doyle, Beckley K. Davis, Richard G. Cook, Robert R. Rich,2 and John R. Rodgers3

The mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochon- dria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR). To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera , Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and Peromyscus. We found that M3 is highly conserved, and the Downloaded from 10 residues coordinating the N-formyl group are almost invariant. The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50Ð65 million years. Consistent with this, M3 ␣1␣2 domains from Rattus norvegicus and Sigmodon hispidus and from the “null” allele H2-M3b specifically bound N-formyl peptides. The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap (“saltation”), perhaps involving intense selective pressure from bacterial pathogens. Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) http://www.jimmunol.org/ . Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns. Such MHC PRR mol- ecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation. The Journal of Immunology, 2003, 171: 836Ð844.

he mouse class I-b molecule H2-M3 preferentially binds domain (12). In contrast to the minimal oligomorphism of class I-b N-formyl peptides (1, 2), pathogen-associated molecular genes (6), class I-a genes are extremely polymorphic, allowing patterns (PAMP)4 (3) also recognized by neutrophil che- presentation of diverse intracellular Ags to T cells (13). Polymor-

T by guest on September 27, 2021 motactic receptors (4). Thus, M3 is a pattern-recognition receptor phism is pronounced especially in the Ag recognition site (ARS) (PRR). In this respect, M3 resembles CD1, which presents myco- and is thought to be generated through diversifying (positive) se- bacterial waxy lipids to T cells (5). M3 may be important for lection (13) evidenced by a high ratio of nonsynonymous to syn- protection against intracellular bacteria (6). Indeed, M3-restricted onymous substitutions in the ARS (14). CTL are protective in experimental infections by the intracellular The paucity of I-b orthologs shared among species of different pathogen Listeria monocytogenes (7–9). The laboratory of Fischer taxonomic orders led to the hypothesis that I-b genes are relatively Lindahl and colleagues (10) showed Norway rats have a gene young, formed by duplications of class I-a genes (15). Such du- nearly identical to H2-M3, suggesting that M3 has been conserved plicates are often redundant and may drift rapidly under neutral since the rat/mouse divergence ϳ14–40 million years ago (MYA). selection towards pseudogeny (15). Functional divergence of gene Like other class I-b genes, M3 is virtually monomorphic in Mus duplicates (16) probably requires positive selection (17). Phyloge- musculus (11). A minor allele, M3b, has been considered null be- netic analyses suggest that many mouse class I-b genes, such as cause it does not restrict lysis by known M3-specific CTL (11); Qa-2 (15) and H2-B1 (our unpublished observations) arose since null activity was mapped to a Leu95Gln substitution in the ␣2 the rat/mouse divergence from duplications of class I-a genes. Be- cause M3 has been unknown outside the murine genera Rattus and Baylor College of Medicine, Department of Immunology, Houston, TX 77030 Mus, it may also have evolved from murine or murid I-a genes. A contrasting model, similar to one proposed for H2-TL (18, 19), Received for publication December 23, 2002. Accepted for publication May 12, 2003. suggests that M3 arose before the mammalian radiation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance The unique ligand specificity of M3 makes it especially inter- with 18 U.S.C. Section 1734 solely to indicate this fact. esting as a model to study how MHC specificities evolve, presum- 1 This work was supported by National Institutes of Heatlh RO1 Grants AI30036 (to ably in response to pathogen or other immune pressure. The mech- R. R. R. and R. G. C.) AI18882 (to R. R. R. and J. R. R.), and RO1 AI17897 (to anism of N-formyl specificity in M3 is well-studied (11). The R. G. C. and J. R. R.). crystal structure of M3a (20) indicated 10 amino acids coordinated 2 Current address: Emory University School of Medicine, Atlanta, GA 30322. N-formyl specificity, with a key contribution from histidine in po- 3 Address correspondence and reprint requests to Dr. John R. Rodgers, Department of 9 Immunology Room M929, Baylor College of Medicine, One Baylor Plaza, Houston, sition 9 (His ). Five of these residues are rarely found in other class TX 77030. E-mail address: [email protected] I molecules. However, our unpublished studies in which we trans- b 4 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; planted these residues between M3 and H2-K suggested other ARS, Ag recognition site; NARS, non-ARS; MY, million years; MYA, MY ago; dN, residues are required to achieve N-formyl specificity. Moreover, rate of nonsynonymous substitution; dS, rate of synonymous substitution; indel, in- sertion and/or deletion; NJ, neighbor-joining; PRR, pattern recognition receptor; UP- the differences between M3 and murine class I-a molecules are not GMA, unweighted pair-group method with arithmetic means; cytb, cytochrome b. concentrated in the ARS but are spread throughout the ␣1 and ␣2

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 837 domains. This raises the question of how M3 evolved from a class GTG GGA GTT (reverse); exon 3 primers were 5Ј-AAG CTT GAT CCA I-a gene. Identification of M3 orthologs from related species AAC CTG GCA GAT (forward) and 5Ј-CTC GAG CCT AAG GTT GAG should identify transitional forms, or “missing links,” between mu- GGA TTT (reverse). PCR using these primers discovered S. hispidus M3 not found with the original primer set. rine M3 and I-a genes. The phylogeny of , the largest extant family, has Cloning PCR products been studied intensively. Four subgenera of Mus (, shrew PCR products of the expected size were gel-purified using the Qiagen mice; Mus; Nannomys, African pygmy mice; and , spiny (Valencia, CA) Qiaquick Gel Extraction kit and eluted products cloned mice) diverged ϳ9 MYA (21). The Old World subfamily with the Zero Blunt TOPO PCR Cloning kit (Invitrogen). Plasmids were includes Mus and Rattus, which diverged 14–40 MYA (22, 23). sequenced by Lone Star Labs (Houston, TX) or the DNA sequencing core Two genera of the New World subfamily Sigmodontinae, Sigmo- facility (Baylor College of Medicine) using an Applied Biosystems (Foster ϳ City, CA) ABI PRISM 377 DNA Sequencer. Most sequences were con- don (cotton rats) and Peromyscus (deer mice), diverged 12 MYA firmed from independent clones and/or sequencing in the reverse direction. (24). Subfamilies Murinae and Sigmodontinae diverged 50–65 M3 and cytb sequences have been deposited at GenBank with Accession MYA (22). We isolated 43 unique M3 sequences from 22 species nos. AY263509-AY263623. of these two subfamilies. We tested these for evidence of selective Phylogenetic trees pressures, and the most disparate members for N-formyl specific- ity. Finally, we asked whether the origins of M3 from class I-a A well-aligned database of exons 2 and 3 of 160 class I genes, including genes could be discerned by phylogenetic analysis. the new M3 sequences, sampled four murid subfamilies, diverse mamma- lian orders, and five vertebrate classes. The transitional/transversional bias was 0.7 in the exon 2 and 3 data set. It was aligned using the Clustal Materials and Methods method (33) by MegAlign (DNAstar, Madison, WI) and adjusted manually Downloaded from Genomic DNA templates for PCR with BioEdit (34) to maintain codon alignments. Distant class I sequences such as CD1 and FcRN were excluded as they were difficult to align and Genomic DNA was isolated from tissue samples or cell lines. Cell lines often generated “long branch” effects. None of these exclusions affected the from Mus dunni, Mus macedonicus and Mus spicilegus, Mus Coelomys conclusions described. The databases included the new M3 genes and five pahari, M. Nannomys minutoides and M. Nannomys setulosus, M. Pyromys M3 sequences from the National Center for Biotechnology Information platythrix and M. Pyromys shortridgei were from Dr. S. Chattopadhyay database: M3a (C57BL/6, U18797); M3b (Mus musculus castaneous, (National Institutes of Health, Bethesda, MD). Cell lines of Mus praetextus f sp

b M62844); M3 (B10.SHH, L36074); M3 (Mus spretus, L36072), and http://www.jimmunol.org/ (mice from R. Sage; Ref. 25) and B10.CAS2 (bearing H2-M3 ) were made RT1-M3 (Wistar, AJ249342). Trees were built in Clustal X (35) and as described (26, 27). Rattus norvegicus DNA was from outbred Holtzman MEGA2 version 2.1 (http://watson.hgen.pitt.edu) (36) using neighbor-join- Sprague-Dawley (Harlan Breeders, Indianapolis, IN) and outbred Wistar ing (NJ), minimum evolution, and unweighted pair-group method with rats (Harlan Breeders) and from the Fischer strain CREF cell line (28). arithmetic means (UPGMA) algorithms, with significance estimated from DNA from African murine species, Hybomys uvivittatus (Eastern Black- 1000 bootstrap trials. Maximum parsimony methods were run with 100 striped field mouse), Mastomys (Praomys) natalensis (African multimam- bootstraps. Using the Tamura-Nei substitution model, the ␥ parameter was mate rat) and Nannomys gratus were provided by P. d’Eustachio (New varied from 0.2 to 3.5 (37) without affecting our conclusions. The inclusion York University School of Medicine, New York, NY) (29). Murine species of long branch murid class I-b genes, such as M10, Qa-1, and TL, had no Apodemus agrarius (Asian Black-striped field mouse), Diplothrix legata effect on the relevant conclusions. Divergence times were estimated by (Ryukyu long-tailed giant rat), Rattus tanezumi (Sladen’s rat), and Toku- linear regression of genetic distance vs multiple reference divergence daia osimensis (Ryukyu spiny rat) DNA were from H. Suzuki (Hokkaido times. by guest on September 27, 2021 University, Sapporo, Japan; Ref. 30). Peromyscus attwateri, Peromyscus leucopus, and Peromyscus pectoralis DNA were from R. Pfau (Tarleton Comparisons of genetic distances State University, Stephenville, TX). A Sigmodon hispidus tissue sample was from P. Wyde (Baylor College of Medicine, Houston, TX). We au- Unless otherwise noted, genetic distances used the Tamura-Nei NJ method, thenticated every sample by sequencing cytochrome b (cytb) and phylo- ␥ parameter ϭ 2. We tested a variety of distances estimating algorithms genetic comparison with published sequences. cytb primers were 5Ј-TYT and parameters. To avoid overweighting heavily represented rodent genera YCW TYT TNG GTT TAC AAR AC (forward) (where Y ϭ C ϩ T, W ϭ in calculating genetic distances using monosubstitution models (e.g., A ϩ T, N ϭ A ϩ T ϩ G ϩ C, R ϭ A ϩ G) and 5Ј-TGA AAA AYC ATC Jukes-Cantor), between-group distances were averaged from between-gen- GTT GT (reverse). These modifications of Ref. 31 were suggested by S. J. era distances. For example, Mus M3 vs nonmurid eutherian I-a distances Steppan (Florida State University, Tallahassee, FL). were averaged as a single observation. Assuming constant rates of evolu- tion, we used Tajima’s (38) relative rate test to assess which of three se- High fidelity PCR quences was the outgroup. Thirty-five sets of the sequences were selected ␹2 High fidelity PCR used the Invitrogen (Carlsbad, CA) Platinum Pfx kit and randomly and analyzed in MEGA2. values were summed and signifi- manufacturer’s protocol for 35 cycles with a 30 s melt at 94°C, 30 s of cance was tested with 35 degrees of freedom. annealing with optimized temperature (usually 55°C), and 1 min per kilo- Nonsynonymous and synonymous substitution rates base for extension time at 68°C and MgSO4 usually at 1 mM. Most primers were within introns. Primers for exons 2 and 3 introduced SalI and HindIII Pair-wise nonsynonymous (dN) and synonymous (dS) substitution rates for 5Ј and 3Ј ends, respectively (exon 2) and HindIII and XhoI (exon 3). (39) in the ARS and NARS were calculated using SNAP (http://hiv-we- b Mus-specific intron primers were based on H2-M3 (32). Exon 2 primers b.lanl.govl) (40). The ARS, based on HLA-A2 (41), was residues 5, 7, 9, were 5Ј-GTC GAC CAA TGC TTG TTC ACT GGC CC (forward) and 22, 24, 26, 57–59, 61–77, 80–82, 84, 95, 97, 99, 114, 116, 143, 145–147, 5Ј-AAG CTT TGG ACC TAA ACT GAA AGT GA (reverse); exon 3 149–152, 154–159, 161–163, 165–167, 169, 171 where residue 1 is the primers were 5Ј-AAG CTT TCA CTT TCA GTT TAG GTC CA (forward) first Gly of ␣1. Class I-a controls were from pairwise comparison of Db, and 5Ј-CTC GAG TGG TTC CTA GTT GTT CCT CA (reverse); rat- Dd,Df,Dk,Dp,Ds,Dx,Kd,Kdv,Kf,Kj,Kk,Ks,Ku, RT1.A1o, RT1.A1b, specific primers were based on putative rat M3 intron sequences (National RT1.A1c, RT1.A1f, RT1.A1h, RT1.A1k, RT1.A1n, RT1.A1q, RT1.A2o, Center for Biotechnology Information database, Accession no. RT1.A2b, RT1.A2c, RT1.A2n, and RT1.A2q (all available from the Na- AC112568). Rat-specific primers for exon 2 were 5Ј-GTC GAC GGT TAT tional Center for Biotechnology Information). CAG TGA AGG GTT (forward) and 5Ј-AAG CTT GGC TAA TCT AGC When multiple taxa are studied, variation in time of divergence has a TTA GCA GTA (reverse); exon 3 primers were 5Ј-AAG CTT TGG TTT heavy impact on variation in substitution rates (42). Therefore, we re- CAC TTT CAG TTT (forward) and 5Ј-CTC GAG CCC AGA CAA CAA gressed (one parameter) the rate of nonsynonymous substitution (dN)on GCC TCA CTT (reverse); M3 sequences from non-Mus species were ob- the rate of synonymous substitution (dS) and compared the slopes (43) to tained initially using a degenerate forward primer based on an alignment of the value of 1 expected under neutral selection using exon 1 in Mus and Rattus M3:5Ј-GGT CKC TYT GGC TGT TA. The reverse primer was based on a similar region at the beginning of exon 4: m Ϫ ␮ d ϭ ͩ ͪͱn 5Ј-CAC ATG TGC CTT TGG GGG AT. The S. hispidus sequence allowed s us to design intronic primers to clone S. hispidus M3 into our expression vector. The intronic primers to amplify exon 2 were 5Ј-GTC GAC GCC where m is the slope, ␮ ϭ 1, s is the SD of the one-parameter slope and n CAG GTT CTT GGA GGA A (forward) and 5Ј-AAG CTT GGA CAT is the number of sequences (not the number of comparisons). 838 EVOLUTION OF THE MHC CLASS I-b MOLECULE M3

To test for hyperconservation of the 10 residues coordinating N-formyl

binding, the dN/dS ratio of those residues was compared to the non-ARS (NARS) ratio using 2 ␴2 ␴2 R1 Ϫ R2 ER N S d ϭ where ϭ ϩ E E R2 d2 d2 ͱ R1 ϩ R2 N S n1 n2 ␴ ␴ and R is the ratio, ER its relative error and N and S are the SD of dN and dS, respectively. Construction of M3-Ld chimeric expression vectors Correct ligation of exons 2 and 3 of M3 in an H2-Ld␣3 expression vector (44), which carries the epitope for mAb 28-14-8S (HB-27; American Type Culture Collection, Manassas, VA), was confirmed by sequencing. Be- cause the null CTL activity of M3b mapped to a Leu95Gln substitution (12), only exon 3 of M3b was inserted into pM3aLd to test for M3b N-formyl specificity. Functional assay of N-formyl peptide specificity

d

DAP-3 cells (45), cotransfected with pSVneo and pM3-L in Lipo- Downloaded from fectamine Plus (Invitrogen), were selected in 750 ␮g/ml geneticin (Invitro- gen) and sorted with a Beckman-Coulter (Fullerton, CA) Altra flow cy- tometer after overnight culture at 37°C with N-formyl-MLF-benzylamide (Sigma-Aldrich, St. Louis, MO) and staining with mAb 28-14-8S. pM3Ld transfectants were cultured overnight with 20 ␮M peptide or DMSO ve- hicle before staining with 28-14-8S and FITC-goat anti-mouse Ig (Baxter, Mundelein, IL) (46, 47). Cells fixed in 1% paraformaldehyde were ana- lyzed on an EPICS XL-MCL flow cytometer (Beckman-Coulter, Fullerton, http://www.jimmunol.org/ CA) with Beckman-Coulter System II version 3.0 software. Peptides (Bay- lor College of Medicine Protein Core Facility) from L. monocytogenes (fMIVIL), or influenza hemagglutinin (fHA; fMLIIW) (48) and their non- formyl forms were dissolved in DMSO.

Results Confirmation of sample identity using cytb To confirm sample identity, we constructed phylogenetic trees

from sample cytb sequences (Fig. 1) and published sequences. cytb by guest on September 27, 2021 sequences for M. Nannomys gratus and R. tanezumi were not pub- ءء lished. As expected, M. N. gratus and R. tanezumi cytb clustered FIGURE 1. Phylogeny of cytb. , Six cytb sequences previously re- significantly (with high bootstrap values) with other Mus Nanno- ported by this laboratory. Underlined sequences were determined for this study. The accession number indicates GenBank sequences. Percent (%) mys and Rattus sequences, respectively. M. P. shortridgei clus- values represent the percentage of bootstrap trials supporting the branch; tered with M. C. pahari (98% of bootstraps) rather than with other only values Ͼ50% are shown. Pyromys (49), consistent with our results for cytb and TL (19), suggesting Pyromys might be polyphyletic. Rattus and Diplothrix sequences formed a single cluster with 100% bootstrap values. All samples of subfamilies Murinae and Sigmodontinae clustered orders. Third, in trees using an algorithm (UGPMA) that assumes appropriately. constant rates of substitution, M3 appeared to have evolved before the radiation of placental mammals (Fig. 2C). Thus, all tree-build- Sequence analysis of M3 in murid ing methods suggested that M3 evolved before the eutherian radi- From 22 murid species we sequenced 43 distinct genes with high ation, or evolved unusually rapidly. homology to H2-M3 in exons 2 and 3. These genes were not well- Thirty-nine of 43 new M3 sequences were easily aligned with differentiated from class I-a-like sequences in exon 4 (data not class I-a genes in exons 2 and 3. Four sequences (marked insertion shown). To confirm orthology with M3, we constructed phyloge- and/or deletion (indel) in Fig. 2A) from M. C. pahari and M. P. netic trees of exons 2 and 3 with candidate sequences, five known shortridgei are altered near the 5Ј end of exon 2 (Fig. 3). In each M3, and over 100 other class I genes. All M3 candidates were of these, a 9-bp tandem duplication encoding the N-formyl inter- isolated together (Ͼ98% of bootstraps) in all tree-forming methods action residue His9 is closely followed by an “indel”, predicting a used (Fig. 2, A and B), justifying their classification as “M3”. The polypeptide with a net gain of one amino acid without a frameshift. M3 species tree (Fig. 2C) resembled that of cytb, except for T. The indelϩ M. C. pahari sequences have premature stop codons osimensis, which has one copy of M3 very similar to that of A. and exhibit a nonfunctional Asn Pro Ser glycosylation motif (50) agrarius, as expected (30), and one highly divergent copy. at positions 86–88, a site critical for peptide binding by H2-Ld Large-scale trees (Fig. 2, B and C) revealed three notable fea- (51). In contrast, indelϩ M3 from M. P. shortridgei are otherwise tures regardless of the algorithm used. First, the M3 branch never intact and might encode functional proteins. clustered significantly with other murid class I genes even when Multiple copies of M3 have not been reported in laboratory (as in most NJ algorithms) it did cluster with eutherian class I-a mice. However, R. norvegicus has at least three copies (52). We genes in general (Fig. 2B). Second, M3 genes were on a “long isolated four sequences from M. C. pahari, three from M. P. shor- branch,” indicating a larger genetic distance between M3 and class tridgei, and five from M. N. gratus. Assuming monomorphism, I-a genes, than between class I-a genes of different mammalian these sequences represent distinct loci. Two distinct M3 sequences The Journal of Immunology 839 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Phylogeny of M3. Sequences for exons 2 and 3 were aligned as described in Materials and Methods. A, Representative “close” phylogeny M3 sequences previously published. B, Large-scale tree representative of non-UPGMA methods. Most branches are collapsed for ,ء .of M3 sequences clarity. M3 clusters with eutherian I-a genes, but not with murid I-a, suggesting an origin at or prior to the eutherian radiation. C, UPGMA methods place M3 outside the eutherian cluster (86%), suggesting an origin prior to the eutherian radiation.

were found in T. osimensis, each with multiple defects. We cannot residues among 42 sequences (99% identity), suggesting hyper- rule out the possibility that this species carries another copy of M3 conservation (Table I). All three variants, at residues 7 and 159, in that is functional. two genes from S. hispidus, are at sites highly conserved among most MHC molecules; two were identical conservative Tyr159Phe Conservation of the N-formyl coordination residues substitutions. There was complete codon conservation for His9, Ten residues of M3a (Y7, H9, Y22, S24, L63, K66, V67, I70, V99, Leu63, and Val99, and only synonymous substitutions at Lys66, Y159) coordinate N-formyl binding (53) of which four (Y7, Y22, Val67, and Ile70 sites (Table I). Thus, the N-formyl-coordinating S24, Y159) are frequent in other class I molecules. Excepting the residues are extremely well-conserved and the N-formyl coordi- six indelϩ sequences, there were only three variants in these 10 nating residues should be under negative selection. 840 EVOLUTION OF THE MHC CLASS I-b MOLECULE M3 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Amino acid alignment of M3 genes. Nucleotide sequences predicting identical protein sequences are listed together to the left of the ,& ;stop codon; large arrows: N-formyl coordinating residues; #, deletion; @, insertion of HYFH ء ;sequence. A, ␣1, B, ␣2. Key:—identity with M3a insertion of HFFH; $, insertion of RAPWMETR (a near tandem duplication).

Synonymous and nonsynonymous rates of substitution in M3 Conservation of N-formyl peptide binding specificity To test whether the ARS of M3 has been under positive (diversi- Hyperconservation of N-formyl coordination residues suggested fying) or negative (purifying) selection, we regressed nonsynony- conservation of N-formyl binding specificity. To test this, the mous substitution rates against synonymous rates (Fig. 4). As ex- ␣1␣2 domains of “null” H2-M3b and both rat and S. hispidus M3 pected, mouse and rat I-a ARS were positively selected (1.48 Ϯ were expressed in an Ld␣3 expression vector and tested for pep- 0.03), but the M3 ARS was under strong negative selection tide-induced surface expression in transfected cell lines (46, 47). (slope ϭ 0.236 Ϯ 0.002, p Ͻ 0.001 versus a slope of 1). As ex- fMIVIL and fHA but not nonformylated peptides induced surface pected, the slope of the M3 NARS, 0.162 Ϯ 0.002, was also neg- expression of M3a,M3b, RT1-M3, and Sihi-M303 (Fig. 5A), dem- atively selected ( p Ͻ 0.001; Fig. 4B); this slope is similar to that onstrating conservation of N-formyl specificity. of many murine non-MHC genes (54). Negative selection of mu- The indelϩ M. C. pahari sequences had multiple debilitating rine class I-a NARS sequences (0.59 Ϯ 0.01) was consistent with mutations in exon 2 but the indelϩ Mush-M302 was intact except other reports for class I-a genes (15). The 10 N-formyl-coordinat- for two nonconservative substitutions in the N-formyl coordinating ϭ Ϯ Ͻ ing residues were hyperconserved (dN/dS 0.025 0.003; p site (Fig. 3A). This suggests the indel was acquired in the ancestral 0.001) compared to the NARS (19). species before other mutations accumulated in M. C. pahari. Null The Journal of Immunology 841

Table I. Conservation of the 10 N-formyl coordinating residuesa

Residues in Other Amino Acid Codon Used Frequency Class I

Tyr7 TAT 43/44 Tyr Cys7 TGT 1/44 His9 CAC 44/44 Variable His rare (HLA-E) Tyr22 TAT 32/44 Tyr or Phe TAC 12/44 Ser24 TCT 39/44 Ser or Ala TCC 5/44 Leu63 CTG 44/44 Variable Lys66 AAG 26/44 Variable AAA 18/44 Lys rare (H2-D) Val67 GTC 25/44 Variable GTA 8/44 Val rare (HLA-A) GTG 11/44 Ile70 ATT 39/44 Variable ATC 5/44 Val99 GTT 44/44 Variable Tyr159 TAT 40/44 Tyr Downloaded from Phe159 TTT 4/44

Coordinating residues identified in the crystal structure of M3 (53) compared with respect to codon usage (frequency among 44 sequences). The most commonly used residues for class I-a molecules are shown in the far right column. When usage is variable, class I molecules sharing a residue with the M3 residue are indicated in parentheses. http://www.jimmunol.org/ duplicates can be fixed in a population but are also rapidly lost (42); the presence of indelϩ M3 in two species suggests it may retain some function, and allowed us to test experimentally and statistically whether the indel event neutralized M3. To test whether indelϩ M3 retains N-formyl specificity, we ex- pressed the ␣1 and ␣2 domains of Mush-M302 with in the Ld vector. In contrast to our results using M3b, previously considered ϩ a “null” allele, the indel M3 was not induced by any tested pep- by guest on September 27, 2021 tide (not shown). N-formyl peptides did induce a chimeric mole- cule with M3a exon 2 and exon 3 from the indelϩ gene (data not shown). This negative experimental result suggests the indel of exon 2 destroyed N-formyl specificity but cannot rule out the pos- sibility that the indel simply altered peptide specificity. Compared Ϯ with all other M3, the dN/dS slopes of both the ARS (0.27 0.01) and NARS (0.40 Ϯ 0.02) of the indelϩ genes were below 1, but FIGURE 4. ARS and NARS residues of M3 are under negative selec- tion. Pairwise rates of nonsynonymous (d ) substitution are plotted against significantly higher than the ARS (0.21 Ϯ 0.01) and NARS N synonymous rates (d ). F, M3; ᮀ, mouse H2-K and H2-D and rat RT1-A. (0.20 Ϯ 0.01) of indel-negative genes of the same two species, S A, ARS. The slope of M3 was 0.236 Ϯ 0.002; the slope of I-a was 1.48 Ϯ consistent with a relaxation of negative selection after indel ac- 0.027. B, NARS. The slope of M3 was 0.162 Ϯ 0.002; of I-a, 0.589 Ϯ ϭ quisition. Moreover, in direct comparisons (n 2) between exons 0.006. All four slopes differed from neutrality (dashed line, slope ϭ 1, p Ͻ 2and3ofM3 of M. C. pahari and M. P. shortridgei, the dN/dS 0.01). ratio of indelϩ M3 was 1.4 Ϯ 0.22 compared to 0.27 Ϯ 0.01 for indel-negative M3. Clearly, only the indel-negative genes have been under negative selection. The experimental and statistical re- Because ARS and NARS residues can evolve at different rates sults are consistent with the interpretation that the indel in exon 2 in class I genes, we analyzed NARS residues, which are under inactivated M3. negative selection and should exhibit more uniform evolutionary rates. As expected, genetic distances between M3 and murine I-a Three models of M3 evolution before the sigmodontine/murine genes were less using the NARS alone, compared with ARS ϩ divergence NARS. However, this shortening also affected distances between Despite the long branch length of M3 genes (Fig. 2, A and B), the I-a genes as well, such that NARS genetic distances between M3 dN/dS ratios and functional data indicated M3 evolved conserva- and murid I-a-like genes were significantly greater than between tively under negative selection since the murine/sigmodontine split murid and eutherian I-a-like genes (data not shown). Moreover, as ϳ65 MYA. Phylogenetic trees failed to join M3 to a murid branch, in Fig. 2, the NARS of M3 genes remained on a long branch (not and UPGMA trees put M3 completely outside eutherian I-a genes. shown) and did not cluster with other murid I-a genes. However, UPGMA models assume constant evolutionary rates, To test whether M3 and/or I-a evolution rates were constant over and all models assume a uniform or monophasic ␥ distribution of time, we plotted NARS genetic distance against estimated taxonomic rates at different sites along the gene. Therefore, the origins of M3 divergence dates (Fig. 6B). Using a variety of distance-estimating al- within murid I-a genes (Fig. 6A, model I) might be obscured by gorithms, and the range of published rat/mouse, murine/sigmodon- nonuniformity or inconstancy of substitution rates. tine, eutherian/marsupial, and /bird divergence times, both 842 EVOLUTION OF THE MHC CLASS I-b MOLECULE M3

FIGURE 5. Rat, cotton rat, and the null H2-M3b proteins are N-formyl specific. Cells transfected with indicated M3Ld vectors were incubated with 20 ␮M of the indicated peptide derived from L. monocytogenes (MIVIL) or influenza hemagglutinin (MLIIW) and stained the next day for expres- sion of M3Ld. Cells transfected with a neoplasmid served as a control. Values are the average and SD of three independent experiments. Downloaded from

M3 and I-a genetic distances were linear since the mammal/bird di- vergence. Moreover, M3 and I-a rates were very similar, though rates estimated for M3 ranged from 0 to 30% faster than those estimated for I-a genes.

Three models might explain why M3 looks old (Fig. 6A). In FIGURE 6. Evolution of NARS residues in M3 and I-a genes. A, Three http://www.jimmunol.org/ model I, exons 2 and 3 of M3 arose from murid I-a genes, through models of M3 evolution. I. M3 was a recent offshoot of murid I-a genes. II. a “leap” (saltation) involving rapid divergence after gene duplica- M3 arose before the eutherian radiation. III. Murid rodents were an early tion, during which M3 evolved N-formyl specificity and, after offshoot of eutherian mammals. B, M3 and I-a genes have evolved linearly which, M3 evolution became conservative. The saltation generated since the mammal/bird divergence. The average between-group protein a large genetic distance, giving M3 the appearance of old age. This p-distances were plotted against estimated divergence time for different taxa. “Young” estimates (Ͻϳ65 MYA) were obtained from between-taxa model predicts M3 will be found only in murids, that transitional comparisons of M3 or of murid I-a genes; older estimates used marsupial forms should occur in other murids, and the genetic distance be- and birds as outgroups. For example, the average pairwise distance be- tween M3 genes and such transitional forms should follow the gray tween Peromyscus and Mus M3 was plotted against the divergence time line indicated in Fig. 6B. In model II, M3 looks older because it is (50–65 MYA) of Murinae and Sigmodontinae. “Old” estimates were com- by guest on September 27, 2021 older, arising before the eutherian radiation. This model predicts puted from genetic distances between M3 (or I-a) and marsupial or bird M3 orthologs should be found in other rodent or mammalian class I genes, the latter treated as outgroups. F, M3 distance ϭ 0.043 ϩ groups. Model III combines the first two: M3 arose in murids, but 0.0024/MY, r2 ϭ 0.98. ᮀ, I-a, distance ϭ 0.068 ϩ 0.0019/MY, r2 ϭ 0.96. murids are older than nonmurid mammals (55, 56). This plot is representative of plots produced using protein and nucleotide Model I predicts M3 and murid I-a genes are most closely re- data, and using Poisson or Tamura-Nei corrections for multiple substitu- lated, while model II predicts that murid I-a and eutherian I-a tions. Substitution rates for I-a related genes ranged from 0.0013 to 0.0019/MY (r2 from 0.80 to 0.98). Substitution rates for M3 were 10–30% sequences are more closely related. However, the distance between 2 ⌬ Ϯ ϭ higher, and ranged from 0.0016 to 0.0022/MY (r from 0.97 to 0.99). , M3 and murid I-a genes (0.23 0.02, SEM; n groups 36) was Distances between M3 and murid and eutherian I-a genes (0.335 Ϯ 0.004) not different from that between M3 and nonmurid eutherian I-a are plotted at the time ϳ75 MYA estimated for the murid radiation under genes (0.21 Ϯ 0.01; n ϭ 18), and longer than the distance between model I or at 131 MYA predicted as the M3/I-a divergence (assuming murid and nonmurid eutherian I-a genes (0.14 Ϯ 0.004; n ϭ 8). linear evolution under model II). Values using other distance measures and Assuming equal substitution rates (Fig. 6B), we tested whether divergence times ranged from 100 to 200 MYA, but were linear in all the eutherian I-a genes are a likely outgroup for M3 and murid I-a models. A saltation is indicated by the gray line leaving the I-a line after genes (model I) or if M3 is the outgroup (model II). We used the eutherian radiation, and returning to the M3 line by the time of the Tajima’s (38) relative rate test to assess these two models using murine/sigmodontine divergence. Transitional forms would be predicted to randomly selected sequence sets from our database. The results fall along this line. C, No evidence for positive selection in the NARS ϭ ϫ Ϫ15 driving rapid evolution of M3 from murid I-a genes. Nonsynonymous sub- were significantly different from model I ( p 2 10 ) but not F ϭ ϭ stitution rates are plotted against synonymous rates. , M3 pairs, slope with model II ( p 0.58). Thus, NARS genetic distances and 0.17; ᮀ, I-a pairs; slope ϭ 0.6; ⌬, M3-I-a pairs, slope ϭ 0.20. Dashed line Tajima’s test both favor model II. It might be argued that the indicates a slope of 1 (neutral selection). NARS of M3 expresses an altered function, and that positive se- lection for new functionality (17) drove rapid evolution of M3

ARS. Thus, both models I and III predict an elevated dN/dS slope identified M3 orthologs in diverse murine and a smaller subset of in comparisons of M3 NARS with murid I-a-like genes. However, sigmodontine murid rodents. We did not find the expected transi- this slope was very low (Fig. 6C), consistent with model II. tional forms, but genes representing the missing link between M3 and I-a might have been missed using PCR probes biased toward Discussion detecting M3-like genes. Detecting such transitional forms, if they M3 is a highly conserved MHC class I-b molecule with unusual exist, will require a different strategy. There is a second caveat: we specificity for N-formyl peptides—a PAMP. Using the criteria of have assumed throughout that M3 is a “I-b” gene in all the species close phylogenetic relationship, presence of residues characteristic studied. From these data, we see that M3 appears essentially mono- of the N-formyl coordination site and N-formyl specificity, we morphic in R. norvegicus,asitisinM. musculus. This leaves open The Journal of Immunology 843 the possibility that M3 is polymorphic—much more I-a-like—in not at all closely related by sequence and differ in many other murids, consistent with model I. other ways. M3 is specialized in other ways: it lacks conventional side chain F. M. Burnet (66) asserted that it was their polymorphism that specificities, and binds peptides both shorter and longer than the made MHC genes biologically significant. Certainly this is true for canonical range of 8–10 amino acids. In most other respects, M3 I-a function, but modern PRR-like I-b molecules suggest an alter- behaves like a class I-a molecule: widespread tissue distribution, nate model for MHC origins. The duplication model of MHC or- TAP dependency, presentation to diverse ␣␤ T cell receptors. Like igins (67) hypothesizes that a primitive locus expanded to become many I-b genes, M3 has not been known outside a narrow taxo- the modern polymorphic MHC, leaving unspecified whether the nomic range. This is consistent with a model of frequent “birth and original MHC gene was itself polymorphic. Because most genes death” in which class I-b genes are derived by duplication from I-a are monomorphic or minimally oligomorphic, and most class I-like genes but rarely survive long enough to transcend the boundaries genes not linked to the MHC are monomorphic (68), parsimony of taxonomic families or even genera (15). Therefore, we expected suggests the ancestral MHC locus was also monomorphic. This isolation of M3 from murid species distantly related to mice and rat primitive MHC molecule, functioning as a PRR, would have been species to offer molecular clues as to how M3 evolved from I-a preadapted for the evolution of polymorphic class I-a molecules in genes. However, the ␣1␣2 domain of M3 from sigmodontine ro- the evolving adaptive immune system. dents maintains N-formyl specificity and scarcely differs from that of murine rodents. In particular, the N-formyl coordination resi- Acknowledgements dues, ARS and NARS of M3 have all evolved under intense pu- We thank J. Levitt, P. d’Eustachio, H. Suzuki, and S. Steppan for advice, rifying selection and no part of M3 exons 2 and 3 appears closely the laboratories of J. Richards, J. Rosen, and S. Marriott, P. d’Eustachio, Downloaded from related to other murid I-a-like genes. and P. Wyde for murid DNA, H. Suzuki for DNA samples of the endan- Assuming standard models of gene evolution, in which muta- gered Japanese species, T. osimensis and D. legata, and two anonymous tions occur singly and at random, M3 appears older than the radi- reviewers who challenged us to mount a deeper analysis of the three ation of eutherian (placental) I-a genes and therefore appears to models. have evolved well before the eutherian radiation (57). This model

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