with Spondyloarthropathies Express an MHC Class I Molecule with Only Limited Sequence Similarity to HLA-B27 that Binds Peptides with Arginine at P2 This information is current as of September 27, 2021. Julie A. Urvater, Heather Hickman, John L. Dzuris, Kiley Prilliman, Todd M. Allen, Kevin J. Schwartz, David Lorentzen, Clare Shufflebotham, Edward J. Collins, Donald L. Neiffer, Raphael, William Hildebrand, Alessandro Sette and David I. Watkins Downloaded from J Immunol 2001; 166:3334-3344; ; doi: 10.4049/jimmunol.166.5.3334 http://www.jimmunol.org/content/166/5/3334 http://www.jimmunol.org/

References This article cites 59 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/166/5/3334.full#ref-list-1

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Gorillas with Spondyloarthropathies Express an MHC Class I Molecule with Only Limited Sequence Similarity to HLA-B27 that Binds Peptides with Arginine at P21

Julie A. Urvater,2* Heather Hickman,† John L. Dzuris,‡ Kiley Prilliman,† Todd M. Allen,* Kevin J. Schwartz,* David Lorentzen,§ Clare Shufflebotham,3* Edward J. Collins,¶ Donald L. Neiffer,4ʈ Bonnie Raphael,§§ William Hildebrand,† Alessandro Sette,‡ and David I. Watkins5*§

The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloar- thropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several Downloaded from nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the ␣1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 http://www.jimmunol.org/ demonstrate that, like HLA-B27, this MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27. The Journal of Immunology, 2001, 166: 3334–3344.

he strongest known association between the MHC and these theories, the arthritogenic peptide hypothesis, postulates that disease susceptibility is that of HLA-B27 and inflamma- B27 plays a direct role in pathogenesis by binding an arthritogenic tory spondyloarthropathies (SpAs)6 in humans. Several peptide (or peptides) and presenting it to autoreactive CTLs. Re-

T by guest on September 27, 2021 theories have been proposed to explain the role of B27 in the cent transgenic mouse and in vitro studies suggest that the role of development of SpAs and have been reviewed elsewhere (1–3). B27 in the mechanism of disease may be distinct from its primary The primary function of MHC class I molecules is to present en- function as an Ag presentation and CD8ϩ T cell restriction mol- dogenously produced peptides to CTLs. Thus the most intuitive of ecule (4–7). However, the transgenic rat model provides evidence that the specificity of peptides bound to B27 significantly influ- ences the prevalence of arthritis in these animals (8). Thus, *Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, WI 53715; †Department of Microbiology and Immunology, University of Oklahoma whether the disease mechanism involves B27 functioning in its Health Sciences Center, Oklahoma City, OK 73190; ‡Eppimune, San Diego, CA conventional role of peptide binding molecule remains in question. 92121; §University of Wisconsin Histocompatibility Laboratory, Division of Labo- There are several features of B27 that make it unique among ratory Medicine, Department of Pathology and Laboratory Medicine, Madison, WI 53792; ¶Department of Microbiology and Immunology, University of North Carolina, MHC class I molecules. Crystal structures and molecular models Chapel Hill, NC 27599; ʈPittsburgh Zoo, Pittsburgh, PA 15206; and §§Wildlife Health have demonstrated that B27 is unique in its possession of an un- Sciences, Bronx, NY 10460 usually deep B pocket when compared with other MHC class I Received for publication July 24, 2000. Accepted for publication December 11, 2000. molecules (9). Indeed, many groups have now eluted and se- The costs of publication of this article were defrayed in part by the payment of page quenced peptides bound to B27 and found that the peptides con- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tained the bulky and positively charged amino acid, arginine, at the 1 This research was supported by a Biomedical Science Award from the Arthritis second position (10–12). Additionally, the combination of amino Foundation and by National Institutes of Health Grant RR00167 (to the Wisconsin acids that make up the B27 B pocket is unique to and conserved in Regional Primate Research Center). all B27 subtypes (13), all of which bind peptides with arginine at 2 Current address: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue the second position. Of these residues, glutamic acid at position 45 North, Seattle, WA 98109. (E45), and cysteine at position 67 (C67) have been shown to be 3 Current address: Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, critical for peptide binding, cell surface expression, and CTL rec- Cheshire, SK10 4TG, U.K. 4 ognition (13, 14). C67 has also been implicated in triggering au- Current address: Disney’s Animal Kingdom, P.O. Box 10000, Lake Buena Vista, FL ␤ 32830. toimmunity (15) and in the formation of unique 2-microglobulin ␤ 5 Address correspondence and reprint requests to Dr. David I. Watkins, Wisconsin ( 2m)-free B27 heavy chain homodimers (7). Regional Primate Research Center, University of Wisconsin, 1220 Capitol Court, Although B27 is the primary genetic factor determining suscep- Madison, WI 53715. E-mail address: [email protected] tibility to SpAs, not all B27-positive individuals develop disease; 6 Abbreviations used in this paper: SpAs, spondyloarthropathies; AS, ankylosing this remains one of the mysteries of these disorders. In general, spondylitis; ReA, reactive arthritis; P2, second amino acid of peptides bound by MHC ϳ class I molecule; PBR, peptide binding region; nano-ES-MS/MS, nanoelectrospray 0.2% of the general population will develop ankylosing spon- tandem mass spectrometry. dylitis (AS), whereas 2% of B27-positive individuals will develop

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3335

the disease (16). Although there is no direct data showing what from primers in Lawlor et al. (23). We designed additional locus-specific percentage of B27-positive individuals will develop reactive ar- primer sets that were also used for these animals. These were: 5ЈGGAXHO Ј Ј thritis (ReA), extrapolation from data summarized by Keat indicate (5 -CGGCCTCGAGATGGCGCCCCGAACCCTCSTCCTGCTA-3 ), 3ЈGGAH3 (5Ј-CGGCAAGCTTCACACAAGGCAGCTGTCTCAC that 20–30% of B27-positive individuals that contract Shigella ACTTTA-3Ј), GGCF (5Ј-CKCCCCGAACCCTCA-3Ј), and GGCR (5Ј- should develop ReA (17). Sixty to 80% of ReA patients are B27- AGGCTTTACAAGYGATGAGAGACT-3Ј). Each primer was used at a ␮ positive, a lower association than is seen in AS where as many as final concentration of 0.25 M. The PCR mixture contained 2 mM MgCl2, 96% of patients are B27-positive. However, this allele is only 50 mM Tris, pH 8.3, and 2.5 U Amplitaq DNA polymerase (Perkin-Elmer ϳ Cetus) in a final volume of 100 ␮l. The reactions were denatured initially present in 7% of Caucasian populations. for 2 min at 94°C followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, Although SpAs are a common phenomenon in nonhuman primates, 72°C for 1.5 min, and a single final extension at 72°C for 10 min. RNA was very little is known about the relationship between disease occurrence not available for Harry, so MHC typing was performed on genomic DNA and expression of MHC class I molecules in these species. Previously, extracted from 200 ␮l of blood using the QIAmp Blood kit (Qiagen, Santa ␮ other groups described AS in the gorilla, Beta (18), and post shigel- Clarita, CA). DNA (175 ng) was amplified in a 50- l reaction using the Invitrogen PCR optimizer kit, Buffer F (Invitrogen, Carlsbad, CA) with the losis ReA in Holli and Husani (19). Another gorilla, Harry, also de- MHC class I generic primer NA1STAR1 (5Ј-GCGAATTCGCTCY veloped ReA (D. Neiffer, manuscript in preparation). By examining CACTCCWTGARGTATTTC-3Ј) and the B friendly primer 3ЈGGBA2 skeletal remains, Rothschild and Woods have also observed SpAs in (5Ј-GTCCGCCGCGGTCCAGGAGCT-3Ј) at a final concentration of 20% of gorillas (20). Gorillas are one of humans closest relatives, last 0.5 ␮M. sharing a common ancestor ϳ10 million years ago (21, 22) and they Subcloning and sequencing express homologs of the HLA-A, B, and C loci (23, 24). Given the

association between B27 and SpAs and the remarkable similarity be- Amplified products were gel purified using QIAEX II suspension (Qiagen) Downloaded from tween SpAs in gorillas and humans, we asked whether gorillas with and subcloned into the pCR2.1 vector using the TA cloning kit from In- vitrogen. Plasmid DNA (200 ng) was sequenced using the ABI PRISM SpAs expressed a MHC class I molecule with sequence and func- Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq tional similarity to B27. DNA Polymerase, FS, according to the manufacturer’s instructions (Per- kin-Elmer-Applied Biosystems, Foster City, CA). The sequencing primer Materials and Methods used for screening, 62P: 5Ј-GTGGGCTACGTGGACG-3Ј, anneals to nu- cleotides 73–88 in exon 2 of MHC class I cDNAs. The sequence generated Animals by the 62P primer (near nucleotide 109 of exon 2 to nucleotide 216 of exon http://www.jimmunol.org/ Whole blood was obtained opportunistically by venipuncture from gorillas 3) was aligned to a database of published gorilla MHC class I cDNAs using anesthetized for routine exams (Gorilla gorilla) at the Yerkes Regional MacVector (Scientific Imaging Systems, New Haven, CT). Sequences that Primate Research Center (Atlanta, GA), the (Wildlife Conser- did not match to the database were assigned a new allele name according vation Society, Bronx, NY), the Brookfield Zoo (Chicago Zoological So- to established convention (27). Briefly, the MHC of the lowland gorilla is ciety, Brookfield, IL), the Toledo Zoo (Toledo Zoological Society, Toledo, designated by a four-letter abbreviation of the scientific names of the spe- OH), and the Pittsburgh Zoo (Zoological Society of Pittsburgh, Pittsburgh, cies, Gorilla gorilla, or Gogo. This is followed by the locus designation PA). Description of AS in Beta and ReA in Holli and Husani was reported based on homology to human class I loci. Allele numbers were assigned in previously (18, 19). The clinical report of ReA in Harry is in preparation the order they were isolated. Three copies of each new MHC class I cDNA (D. Neiffer et al., manuscript in preparation). were sequenced full length to minimize reporting of PCR-generated nu-

cleotide substitutions and to confirm locus identity. Primers used for se- by guest on September 27, 2021 Cell culture quencing were reported previously (28). Sequences of new cDNAs shown in this paper have been deposited with GenBank and have accession num- PBL were separated from whole blood using Ficoll/diatrizoate gradient bers AF157406–AF157411. centrifugation. These cells were cultured with 5 ␮g/ml Con A (Sigma, St. , MO) and 20 U/ml rIL-2 (a gift from Roche, Nutley, NJ). PBLs from Antibodies Beta and Holli were also transformed with EBV by culturing PBL with supernatant of the B95-8 cell line (25) in the presence of cyclosporin A at The W6/32 hybridoma was obtained from the American Type Culture Col- 0.2 ␮g/ml using a protocol modified from Lawlor et al. (26). Transformed lection (ATCC) (Manassas, VA). This mouse mAb is directed against hu- and activated lymphocytes were cultured at 1 ϫ 106 cells/ml in RPMI 1640 man MHC class I proteins. W6/32 from hybridoma supernatant was puri- medium (Life Technologies, Grand Island, NY) supplemented with 15% fied over a Sepharose CL4B column (Sigma) for use in purifying MHC heat-inactivated FBS (Sterile Systems, Logan, UT), 2 mM L-glutamine, class I molecules. The 949 (anti-class II Ab) hybridoma obtained from 5 ϫ 10Ϫ5 M 2-ME, 20 mM HEPES, 50 U/ml penicillin, and 50 ␮g/ml ATCC was used to generate mouse 949 ascites according to established streptomycin. protocols (29).

DNA/RNA extraction, cDNA synthesis, and PCR Molecular modeling of Gogo-B*0101 6 Total cellular RNA was extracted from 2–7 ϫ 10 lymphocytes using A molecular model of Gogo-B01 was constructed using the crystallographic RNAzol (Tel-Test, Friendswood, TX). cDNA was synthesized from 0.1–1 coordinates of HLA-B27 (PDB1HSA) (30). Substitutions in the extracellular ␮g of RNA in a 20-␮l reaction containing 50 mM Tris, pH 8.3, 5 mM portion of B27 to make it into Gogo-B01 were made using the graphical MgCl2, 1 mM each of dATP, dGTP, dCTP, and dTTP (Gene AMP-Perkin- program O (31). The substituted side chains were placed in the rotamer loca- Elmer, Foster City, CA), 0.5 ␮g random primers (Promega, Madison, WI), tion (32) that minimized steric clashes with neighboring residues. Local min- 50 U of SuperScript II reverse transcriptase (Life Technologies, Gaithers- imization was performed to remove bad contacts, and the model was examined burg, MD), and 20 U of RNase inhibitor (Gene AMP-Perkin-Elmer). visually for poor interactions. The resulting model was subjected to 40 cycles cDNA was synthesized at room temperature for 10 min, 42°C for 15 min, of energy minimization refinement using X-PLOR (33) with a small harmonic 99° for 5 min, and 5° for 5 min in a Perkin-Elmer Cetus 9600 thermocycler constraint placed on the ␣ carbon positions. (Norwalk, CT). PCR was then conducted in a Perkin-Elmer Cetus 9600 using several sets of locus-specific primers. All of the B locus typing in Stable transfection of Gogo-B*0101 into the 721.221 cell line Table III was conducted with the B locus-specific primer set GG5ЈBXHO: 5Ј-CGGCCTCGAGATGCCTCCTCCTGCTGCTCTCGGC-3Ј; GG3ЈBH3: A clone containing the consensus cDNA for Gogo-B*0101 was subcloned 5Ј-CGAAGCTTCCCTCACAACACAGCTGTCTCAGGCTTTT-3Ј. For A, into the pKG5 expression vector (a gift from Andrew McMichael, Oxford B, and C locus typing of Beta, Holli, and Husani, additional locus-specific University, Oxford, U.K.). This vector was then electroporated into the primer sets were used. They were as follows: The 5Ј generic primer, 721.221 cell line, a cloned EBV-transformed B lymphoblastoid cell line BETA2H3ϫHO (5Ј-CGCTCGAGGACTCAGAATCTCCCCAGACGCCG (BLCL) with homozygous deletions of the MHC class I loci (34). Cell line AG-3Ј) paired with either 3PALOCH3 (5Ј-CCGCAAGCTTTTGGGG 721.221 cells (7.5 ϫ 106) were transfected in a 0.4-cm electroporation AGGGAGCACAGGTCAGCGTGGGAAG-3Ј), the B locus-specific primer cuvette with 25 ␮g of plasmid DNA. Electroporation was conducted with BETA2H3, (5Ј-CGAAGCTTGGAGGAAACACAGGTCAGCATGGG a Bio-Rad Gene Pulser II Electroporation System (Bio-Rad, Hercules, CA) AAC-3Ј), or 3PCLOCH3 (5Ј-CCGCAAGCTTTCGGGGAGGGAACA at 200 V and a capacitance of 950 ␮F. The cells were then put into 50 ml CAGGTCAGTGTGGGGAC-3Ј). The latter four primers were derived of RPMI 1640 culture medium supplemented with 50 U/ml penicillin, 50 3336 GORILLA MHC MOLECULE BINDS PEPTIDES WITH ARGININE AT P2

␮g/ml streptomycin, 2 mM L-glutamine, 5% defined FBS (HyClone, Lo- technology Information (National Institutes of Health, Bethesda, MD) web gan, UT), and 10% defined/supplemented bovine calf serum (HyClone) server to identify homology with currently catalogued sequences. and plated at 1 ml/well in 24-well plates. The cells were incubated for 2 125 days at 37°C. On day 3, the cells were placed under selection by adding 1 Peptides and iodine labeling ml of culture medium containing G418 (Life Technologies, Gaithersburg, MD) for a final concentration of 650 ␮g/ml. About 4 wk later, viable Peptides were obtained as lyophilized products from Chiron Mimotopes transfectants were tested for MHC class I surface expression by flow cy- (San Diego, CA) or synthesized at Epimmune using standard t-Boc or tometry with the W6/32 mAb directly conjugated to FITC (Sigma). The F-moc solid phase synthesis methods (43). Peptides were stored in stock transfectant with the highest level of MHC class I expression was selected solutions at either 10 or 20 mg/ml in 100% DMSO and then diluted in RPMI 1640 for use in the cellular assays. HPLC-purified peptides were to be grown up for peptide elution studies. 125 We also produced soluble Gogo-B*0101 transfectants to produce higher radiolabeled with I according to the chloramine-T method (44). amounts of bound peptides for sequencing. This method was described Live cell binding assays previously (35). Peptide binding to gorilla MHC class I molecules was measured by the Affinity purification of Gogo-B*0101 ability of test, unlabeled peptides to inhibit binding of a radiolabeled peptide probe in live cell binding assays The live cell binding assay was MHC class I molecules were purified from 721.221 transfectants according performed as previously described (36, 45). Briefly, gorilla MHC class to a modified protocol as previously described (36, 37). Briefly, 6 ϫ 109 I-transfected 721.221 cells (106 cells/ml) were preincubated overnight in cells were washed in cold HBSS (Life Technologies), harvested, and fro- RPMI 1640 supplemented with 15% FBS, L-glutamine, penicillin (100 zen until needed. Thawed cells were then resuspended in 100 ml of 1% IU/ml), and streptomycin (100 ␮g/ml) at room temperature. Then, cells Nonidet P-40 lysis buffer containing 0.25% sodium deoxycholate, 174 ␮ ␮ ␮ ␮ were washed twice in RPMI 1640 without supplements and resuspended to g/ml PMSF, 5 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin ϫ 7 ␮ ␮ a final concentration of 1.25 10 cells/ml in RPMI 1640 supplemented

A, 20 g/ml iodoacetamide, 0.2% sodium azide, and 0.003 g/ml EDTA. Downloaded from with 3 ␮g/ml ␤ m (Scripps Clinic and Research Foundation, La Jolla, CA). Cell lysates were incubated at 4°C for 1 h, centrifuged at 100,000 ϫ g at 2 Aliquots of 2 ϫ 106 cells/well were incubated in a 96-well U-bottom 4°C to remove cellular debris, and then filtered sequentially through 0.8- microtiter plate with 105 cpm (10 ␮l) of specific radiolabeled peptide and and 0.22-␮m Nalgene filters to remove any remaining lipids. Filtered ly- in the presence of a protease inhibitor cocktail (final concentrations of 250 sates were then passed twice over a 949-coupled protein A-Sepharose col- ␮g/ml PMSF, 1.07 mg/ml EDTA, 62.5 ␮g/ml pepstatin A, 60 ␮g/ml umn to preclear the lysate. The flowthrough was passed twice over two 1-chloro-3-tosylamido-7-amino-2-heptanone-hydrochloride and 325 ␮g/ml consecutive W6/32-coupled columns to specifically bind MHC class I mol- phenanthroline). Following a 4-h incubation at 20°C, unbound peptide was ecules. The protein A beads of the W6/32 columns were then washed removed by washing three times in serum-free medium, followed by pas- separately, twice with lysis buffer (without protease inhibitors), twice with http://www.jimmunol.org/ sage through a FBS gradient. The amount of a bound, labeled peptide was a high salt buffer (1 M NaCl, 20 mM Tris pH 8.0), and twice with a no salt then determined by counting pelleted cells on a gamma scintillation buffer (20 mM Tris, pH 8.0). Purification of soluble Gogo-B*0101 was as counter. For inhibition assays, a dose range of unlabeled peptide inhibitors described previously (35). was coincubated with the radiolabeled probe and the cells, and the con- Purification of Gogo-B*0101-bound peptides centration of peptide yielding 50% inhibition of the binding of the radio- labeled probe peptide was calculated (IC50). Peptides were eluted from Gogo-B*0101 as described previously (36). Briefly, the protein A beads were incubated in 0.2 N acetic acid. The beads Results were then briefly centrifuged and transferred to a new tube and the process Gorillas that develop SpAs express an MHC class I molecule was repeated. Glacial acetic acid (100 ml) was added to each tube to allow

␤ with limited similarity to B27 by guest on September 27, 2021 for dissociation of the MHC heavy chain/ 2m/peptide complexes. MHC ␤ class I heavy chains, 2m, and W6/32 Abs were then separated from the Gorillas develop signs of SpAs indistinguishable from clinical signs peptides by centrifugation through an Ultrafree-CL filter (500 NMWL; Millipore, Bedford, MA). Peptide yields were determined by quantitation seen in humans with SpAs. The case description of Beta’s AS was the of Gogo-B*0101 heavy chain using SDS-PAGE. Purification of peptides first in a nonhuman primate (18) (Table I). She was initially diagnosed from soluble Gogo-B*0101 was as described (35). with arthritis in 1979 when radiographs were taken due to a stiffness HPLC fractionation and automated Edman degradation in her gait. Later her condition worsened further; she moved with sequencing of peptides great difficulty and used an awkward, rigid gait. Under anesthesia, board-like rigidity of the dorsal spine and extreme bilateral contrac- ϫ The filtered peptide eluate was purified by reverse phase HPLC on a 1.0 tures of the hips were noted. There was also evidence of chronic 150 mm C18 column (Michrom Bioresources, Auburn, CA) using the fol- lowing gradient conditions at a flow rate of 40 ␮l/min: 2–10% acetonitrile synovitis of the larger peripheral joints, and swelling and/or contrac- in 0.02 min, 10–60% acetonitrile in 2 min. The entire region correspond- tures involving the shoulders, elbows, wrist, knees, and ankles in a ing to UV absorbance at 215 nm was collected during the gradient and symmetric fashion. Radiographs showed hallmark and progressive subjected to pooled Edman degradation on a model 492A pulsed liquid changes typical of AS, including advanced sacroiliitis and lumbar phase protein sequencer (Perkin-Elmer Applied Biosystems Division, Nor- spondylitis. Beta’s son, Kwashi developed inflammatory synovitis of walk, CT) with underivatized cysteine. Nonpeptide material, which copu- rified with the peptides in the first experiment (from the cell-bound mol- the right wrist (18) (Table I). Holli and Husani developed signs very ecules), eluted as two peaks on the HPLC analysis. One of these peaks typical of ReA (19). Both developed Shigella flexneri enteritis and obscured threonine and aspartate for the majority of the cycles and glycine subsequently developed inflammatory joint disease. Harry also devel- for the first three cycles. Only data from the second run (peptides eluted oped signs of ReA following shigellosis (Table I). from soluble molecules) is used for these amino acids. Raw data analysis was performed according to established protocols (38–40). The average Given the remarkable similarity between SpAs in gorillas and relative frequency table was generated according to Kubo et al. (38). humans and the close association of B27 with these diseases in humans, we cloned and sequenced all of the MHC class I mole- Nanoelectrospray tandem mass spectrometry (nanoES-MS/MS) cules expressed in the four affected gorillas (Table II). Of all the Sequencing of individual peptide ligands was as previously described (41). MHC class I molecules isolated from these animals, Gogo-B01 Typical nanoES-MS/MS runs involved gating for an ion with the first was the most similar to B27 (Figs. 1 and 2). However, at the amino quadrupole and scanning a range with the third quadrupole of 30–1200 m/z using a step size of 0.2 atomic mass units and a dwell time of 1.5 ms with acid level, Gogo-B*0101 is most similar to HLA-B*1513, differ- underivatized lysine; the collision gas (Ar) was adjusted in each case to ing by 21 residues. HLA-B*2702 is the next most similar human optimize fragmentation for the ion examined. Nano-ES-MS/MS data were class I molecule, differing from Gogo-B*0101 by 22 aa (Fig. 1 and evaluated and interpreted using the Predict Sequence algorithm (BioMul- data not shown). Fifteen of those differences occur in the poly- tiView software; PE Sciex, Boston, MA) as well as PeptideSearch 3.0.2 ␣ ␣ (42) in instances of low ligand ionization/concentration or poor fragmen- morphic 1 and 2 domains that comprise the peptide binding tation. Advanced Basic Local Alignment Search Tool searches were per- region (PBR). Interestingly, Gogo-B01 is identical with HLA- formed against databases available through the National Center for Bio- B*2702 from residues 71 to 90 at the end of the ␣1 domain (Figs. The Journal of Immunology 3337

Table I. Clinical signs in gorillas that developed SpAs

Name Sex Diagnosis Reference Description/Comment

Beta F AS 18 Stiffness, awkward, rigid gait; board-like rigidity of the dorsal spine and extreme bilateral contactures of the hips; chronic synovitis of the larger peripheral joints; swelling and/or contractures involving the shoulders, elbows, wrist, knees, and ankles in a symmetric fashion; advanced sacroiliitis and lumber spondylitis Kwashi M Inflammatory 18 Swollen, painful right wrist; local inflammatory synovitis (Beta’s male synovitis offspring) Husani M ReA 19a Five months following Shigella flexneri enteritis at 6 mo of age, developed weight-bearing lameness of right leg; swelling of right stifle and tarsus; arthrocentesis revealed acute to subacute inflammatory joint disease; joint fluid cultures negative; symptoms resolved after 4 wk of treatment; swelling in stifle and lameness recurred within 3 days after medication was discontinued; right stifle was then enlarged and firm; radiography showed increased soft tissue swelling; medication was changed, symptoms resolved and no further occurrences were observed. Holli F ReA 19a Six weeks following appearance of profuse watery diarrhea (S. flexneri type 2 isolated from feces), animal developed lameness of the right Downloaded from leg and left arm; increasing severity of depression, lethargy, and lameness leading eventually to non-weight bearing on the left arm and right leg; swelling of the left carpus and right tarsus with increased heat and painful response to palpation of joints; blood count showed presence of leukocytosis with regenerative left shift; cytologic examination of the joint fluid showed neutrophilia but

bacterial culture was negative; lameness resolved but intermittent http://www.jimmunol.org/ joint tenderness was observed for 3 mo. Nine to 10 mo later, mild lameness of left forelimb noted. No recurrences of lameness noted for the next 2.5 years. Harry M ReA D. Neiffer et al., Febrile with loose watery stool at 6 and 11 mo of age; lameness with manuscript in malodorus, grayish stool at 15 mo of age (Shigella sp. type B—most preparation likely flexneri isolated from feces). Placed on course of ciprofloxacin. One month later developed left leg lameness (foot held up with toes clenched); placed on anti-inflammatory; three further episodes between ages 21 and 39 mo characterized by lameness of legs and/or arms; on one occasion animal unwilling to move; pain elicited by extension of left elbow; episodes resolved with or without anti- by guest on September 27, 2021 inflammatory therapy; examination at 41 mo of age revealed effussion around metacarpal-phalangeal joint of 2nd digit; radiographs revealed roughened femoral heads and acetabular margins, and bilateral metaphyseal thickening of distal radii and ulnas with medial deviation of radial diaphysis; at 48 mo of age severe left arm and right leg lameness with palpable hot swelling present over right stifle; arthrocentesis resulted in 12 ml viscous yellow fluid with 100% polymorphonuclear cells and no bacteria; blood work showed a leukocytosis with absolute neutrophilia. Radiographs revealed progressive joint bone/cartilage changes and soft tissue swelling around right stifle. Placed on long-term sulfasalazine therapy with no clinical signs of over 3 years.

a In Ref. 19, case 1 refers to Holli, case 2 refers to Husani.

1 and 2). The amino acid sequence in this region is not unique to ond amino acid of peptides bound by the MHC class I molecule B27 and also is not conserved between all the subtypes of B27 (see (P2) (13, 14). However, examination of the amino acid residues Fig. 1, HLA-B*2705) and so may not be important in disease that make up the P2 environment (47) in other HLA molecules that pathogenesis. However, this region has been shown to influence have been shown to accommodate arginine at the second position peptide binding specificity (46). Importantly, there are several dif- suggested that Gogo-B*0101 should bind peptides with R2 at least ferences between B27 and Gogo-B01 in the B pocket of the PBR as well as HLA-Cw*0602 (Table III). Gogo-B01 is expressed in (Figs. 1 and 2). In particular, the E45 and C67 residues of B27 are most animals that develop SpAs. Therefore, we wished to deter- replaced with M45 and S67 (Figs. 1 and 2). mine what peptides are bound by Gogo-B*0101. When we defined the peptide binding motif of Gogo-B*0101 by Gorillas that develop SpAs express an MHC class I molecule pool sequencing of bound peptides, we found that arginine was the that binds peptides with arginine at position 2 dominant anchor residue at position 2 (Tables IV and V). Sequenc- We found that Gogo-B01 has only limited similarity to B27, pri- ing of individual ligands confirmed the presence of R at P2 in marily at the end of the ␣1 domain. Additionally, there are key peptides bound by Gogo-B*0101 (Table VI). Individual ligand differences between the B pocket residues of this molecule and sequences also revealed a strong preference for tryptophan (W) at B27. Moreover, previous studies have shown that substitution of the C terminus, which was not apparent from the pool peptide data. E45 for M45, the substitution present in Gogo-B01, greatly dimin- This is consistent with previous work showing that HLA-B15 pep- ished the ability of B27 to bind peptides with arginine at the sec- tide ligands are preferentially anchored at their C termini while 3338 GORILLA MHC MOLECULE BINDS PEPTIDES WITH ARGININE AT P2

Table II. Molecular typinga of the MHC class I loci in gorillas that developed AS or ReA

Name Clinical Symptomsb Gogo-A Gogo-B Gogo-C

Beta AS A*0401, A*0501 B*0101, B*0301 C*0101, C*0103 Husani ReA A*0401, A*0501 B*0101, B*0201c C*0101, C*0202 Holli ReA A*0101, A*0501 B*0201, B*0401 C*0202, C*0204 Harry ReA A*0401c,? B*0101, B*0201 ND Kwashi Signs of peripheral ND B*0101, B*0103 ND (Beta’s male offspring) arthritis

a MHC class I cDNAs from affected gorillas were amplified by PCR and cloned as described in Materials and Methods. b See Table I for references. c Only one copy isolated by cloning and sequences. exhibiting relative variation in N-proximal amino acids and overall found in B27, such that the charge of the B pocket remains predom- peptide length (41). Additionally, one of the P9 anchor residues of inantly negative like that of B27 (Fig. 3, A–C). At a more detailed B*2702 is W (12). Thus, Gogo-B*0101 shares both a P2 and a level, the E45/M45 substitution changes the charge of that side of the C-terminal anchor residue with B27. B pocket from negative to neutral. This substitution appears to make We found it curious that substitutions that were shown to abrogate the pocket smaller on that side, pushing the P2 residue away from binding of R2 peptides in B27 (13, 14) did not result in this effect on M45 toward the aspartate (D) at position 9 (Fig. 3, D and E). How- Downloaded from Gogo-B*0101. Therefore, we sought to determine through computer ever, the change in Gogo-B*0101 from H9 to D9 at this position modeling how the B pocket might accommodate R at P2. The mo- results in a smaller and now positively charged residue that can in- lecular model of the Gogo-B*0101 molecule suggests that changes in teract with P2. The substitution of I66 for the smaller and more polar other residues of the B pocket compensate for the lack of E45 such T66, may also allow a positively charged P2 residue to interact more that the B pocket should still preferentially bind peptides with posi- closely with D9 (Fig. 3, D and E). Thus, compensatory changes in and

tively charged residues at P2 (Fig. 3). The substitutions in and around around the B pocket of Gogo-B*0101 allow for binding of peptides http://www.jimmunol.org/ the B pocket of Gogo-B*0101 are similar in charge and size to those with R at P2. by guest on September 27, 2021

FIGURE 1. Predicted amino acid translations of MHC class I A, B, and C locus cDNAs isolated from gorillas have limited sequence similarity to B27. Predicted amino acid sequences of the ␣1 and ␣2 domains of gorilla MHC class I A, B, and C locus cDNAs are aligned to a gorilla consensus sequence. HLA-B*2702 and B*2705 are included for comparison. B pocket residues are denoted by the symbol F. The critical B pocket residues E45 and C67 are boxed. Regions of similarity between human and gorilla sequences are also boxed. The new gorilla cDNA sequences in this figure and Table III (Gogo-B*0301, Gogo-B*0401, Gogo-B*0501, Gogo-B*0502, Gogo-C*0103, Gogo-C*0204) are available from EMBL/GenBank/DDBJ under accession numbers AF157406-AF157411. The GenBank accession numbers for other sequences used in this paper are: HLA-B*2705 L20086, HLA-B*2702 X03664, Gogo-B*0101 X60255, Gogo-B*0102 X60693, Gogo-B*0103 X60254, Gogo-B*0201 X60253, Gogo-C*0101 X60252, Gogo-C*0202 X60249, Gogo- A*0101 X60258, Gogo-A*0401 X54376, and Gogo-A*0501 X60256. The Journal of Immunology 3339 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Similarities and differences between Gogo-B*0101 and HLA-B*2702 mapped onto the ribbon structure of HLA-A*0201, as described by Bjorkman et al. (56). A, Residues that make up the P2 environment (B pocket) of MHC class I molecules are highlighted in yellow (57). B, Differences between the Gogo-B*0101 and HLA-B*2702 P2 environments are colored red. Similarity at the end of the ␣1 domain between the two molecules is shown in purple.

Gogo-B*0101 can bind peptides known to be bound by B27 ing of peptides with substitutions at P2, we found that the arginine Given the similarity in the peptide binding motifs of Gogo-B*0101 at P2 is crucial for binding to Gogo-B*0101 (Table VII). and B27, we asked whether Gogo-B*0101 could bind peptides that are known to bind to B27. Live cell binding assays using a variety Gogo-B01 is present in nearly every gorilla of B27 and B27-like ligands demonstrated that Gogo-B*0101 can Because such a high rate of SpAs are found in gorillas, we rea- bind B27 ligands (Table VII). Furthermore, when we tested bind- soned that a candidate susceptibility allele would be similarly 3340 GORILLA MHC MOLECULE BINDS PEPTIDES WITH ARGININE AT P2

Table III. Amino acid residues of the P2 environment for alleles with motifs that show arginine occupancy at P2a

7 9 24 25 26 34 35 36 45 62 63 66 67 70 99 159 163 167 P2 Amino Acid(s)

HLA-B27 Y H T V G V R F E R E I C K Y Y E W Rb,c HLA-B*1401 – Y S –––––––N––N– – T –RKb,d HLA-B*3501 – Y A –––––T–N–FN– – L –PAVYRDc HLA-B*39011 – Y S –––––––N––N– – T –RHb,c HLA-B*7301 – – ––––––––N–––– – – –Rb,e HLA-Cw0301 ARc HLA-Cw0602 – D S –––––G––KYQ– – T –PRc HLA-Cw0702 – D S ––––––––KYQS – T –YPRDc Gogo-B*0101 – D ––––––M––TSQF – T –RQb

a One letter amino acid code for residues in HLA-B27 are shown in the top row. Identity with HLA-B27 is indicated by a –. P2 environment residues are from Ref. 47. b Molecules for which arginine is the dominant anchor residue. c Ref. 12. d Ref. 58. e Ref. 59. present at a high frequency. Therefore, we determined the MHC can bind B27 ligands. This molecule is expressed in the majority

type of 10 additional unrelated, unaffected gorillas and found that of gorillas. Downloaded from Gogo-B01 is expressed in nearly every animal (Table VIII). These We have not shown that B01 is associated with the development results could account for the high rate of cross-reactivity between of disease in gorillas. Due to the small number of affected gorillas gorilla lymphocytes and human B27 heteroantisera previously ob- and the high frequency of this allele in the gorilla population, this served by other groups (18, 23). We also tested human B27 anti- kind of study was not feasible. However, the high prevalence of sera against PBLs from ␤ and T cell blasts from another gorilla, disease in these animals (20) suggests that an associated MHC , and observed similar cross-reactivity (data not shown). class I molecule would also have to be present at a high frequency. http://www.jimmunol.org/ If 20% of Shigella-infected humans were to develop SpAs, all of Discussion them would have to be B27-positive (17). If Gogo-B01 is associ- In this study, we show that most gorillas that develop SpAs express ated with disease, the fact that a very small percentage of Gogo- an MHC class I molecule, Gogo-B01, that has only limited se- B01-positive animals develop disease suggests that other genes quence similarity to B27. Gogo-B01 and HLA-B27 differ at sev- may be involved in susceptibility, as is also observed in humans. eral residues that make up the B pocket. However, compensatory Studies currently under way in humans to discover the nature of substitutions change the B pocket of Gogo-B*0101 such that it can non MHC genes associated with development of SpAs (55) may accommodate arginine at P2. In fact, Gogo-B*0101 has a remark- provide further insight into the mechanism of disease in humans ably similar peptide binding motif to that of B27; furthermore, it and gorillas. by guest on September 27, 2021

Table IV. Pool sequencing of peptides eluted from Gogo-B*0101 molecules

Cycle Number (amount of amino acid in pmols)b Average Rel. Frequency)d

Amino Acida 1c 234567891012345678910

A 92.10 9.26 43.97 115.79 68.22 42.24 36.44 27.85 17.30 9.59 0.33 1.22 1.99 1.30 1.11 0.99 0.96 0.81 0.73 De 1.64 3.91 3.85 4.33 4.52 4.47 4.71 4.90 5.81 6.94 0.46 0.34 0.37 0.38 0.42 0.47 0.54 0.91 1.61 E 5.65 3.44 6.55 29.62 32.89 27.47 42.79 30.12 17.56 14.81 0.31 0.29 1.17 1.05 0.96 1.57 1.37 1.08 1.16 F 5.96 7.76 61.66 33.69 64.54 40.57 33.36 23.02 67.88 35.99 0.25 1.30 0.57 1.58 0.93 0.83 0.65 1.74 1.54 G 24.66 9.79 31.46 84.24 110.46 66.49 92.07 71.53 34.41 18.54 0.20 0.47 1.22 1.57 1.06 1.54 1.33 0.91 0.73 H 2.70 1.84 3.54 7.37 12.43 11.08 10.04 8.83 6.96 4.21 1.56 1.16 1.10 1.35 1.20 1.11 1.05 1.14 1.00 I 28.78 18.63 49.82 25.22 41.05 40.72 75.51 37.83 19.55 13.20 0.57 1.18 0.54 1.05 1.24 2.61 1.30 1.10 1.16 K 254.13 41.36 65.61 63.71 45.73 78.69 44.98 42.31 21.68 14.05 0.52 1.01 0.96 0.68 1.27 0.74 0.80 0.61 0.54 L 26.34 9.40 27.38 66.88 50.86 52.94 40.81 43.89 26.94 12.64 0.26 0.65 1.36 0.95 1.39 1.10 1.25 1.27 0.99 M 30.01 2.28 11.48 8.99 24.30 13.91 13.87 11.73 7.96 3.91 0.33 1.12 0.89 2.28 1.06 1.22 1.37 1.23 0.90 N 7.75 2.92 38.66 15.32 24.53 21.26 25.47 40.05 22.70 10.22 0.26 1.41 0.58 0.90 0.83 1.04 1.83 1.40 1.02 P 2.51 3.40 137.12 97.54 70.88 60.51 35.17 21.48 11.09 5.96 0.13 2.81 2.03 1.52 1.51 0.92 0.62 0.50 0.43 Q 21.84 5.64 10.49 23.31 32.90 60.42 42.43 32.59 20.45 62.24 0.75 0.25 0.47 0.51 1.10 0.84 1.31 1.07 2.10 R 80.72 354.74 103.26 68.96 61.29 46.25 31.19 29.69 27.72 13.55 5.94 1.05 0.65 0.56 0.47 0.36 0.40 0.56 0.42 S 82.54 15.52 22.69 28.93 28.76 34.57 32.32 28.79 19.14 11.45 0.34 0.78 0.91 0.78 1.12 1.01 1.06 0.95 0.87 T 119.91 20.56 22.42 17.21 24.39 32.26 33.90 88.97 49.32 22.27 0.61 0.50 0.37 0.52 0.77 0.85 2.47 1.95 1.31 V 16.89 5.16 24.21 27.82 40.25 36.40 27.68 24.33 12.59 6.57 0.27 0.97 0.89 1.43 1.33 1.23 1.14 1.35 1.04 W 3.15 7.33 8.27 8.45 8.79 10.01 7.81 8.05 2.13 2.12 0.37 0.39 0.40 0.40 0.48 0.61 0.69 0.72 1.01 Y 15.35 30.72 73.83 34.85 31.00 13.71 29.58 19.88 26.86 13.30 1.22 1.86 0.87 0.81 0.36 0.79 0.65 1.10 1.42

a One letter codes for amino acids. b Raw data of pool sequencing of peptides extracted from Gogo-B*0101 interpreted according to previously outlined methods (39). Values increasing Ͼ50% over the previous (or pre-previous) cycle are underlined. Values increasing Ͼ100% are bolded. c Cycle 1 is not usually considered due to heavy contamination by free amino acids. d See Materials and Methods for formula used to calculate average relative frequency values. Underlined values indicate those which exceed 1.00 by Ͼ2 SDs. Average SD ϭ 0.26 Ϯ 0.26. e Average relative frequencies for aspartate (D), glycine (G), and threonine (T) could not be calculated because signals were obscured in the first experiment by a nonpeptide contaminating peak. Values shown for these amino acids are the relative frequencies from the second experiment. Cysteine residues were not modified prior to pool sequencing and, therefore, were not detected. The Journal of Immunology 3341

Table V. Peptide binding motif of Gogo-B*0101

Residue

C Terminusa

12 3 45678910 7/8/9/10

Gogo-B*0101 Dominantb R PE W N F M A V G Stronga LGM YTFQ IA YL Q H Weaka YH F QI N

EHKE Downloaded from QI KN HLA-B*2702c Anchor residue RF Y I L http://www.jimmunol.org/ W Other preferred residues K F G I I Y K LPKVLV Xd KE YVD DV RTE EMDFR QT H TE SQ

a C terminus preference was evident from sequence of individual ligands. See Table VI. by guest on September 27, 2021 b Determination of peptide binding motif according to previously established methods (39–41). Dominant: Ͼ3.5-fold increase over the previous cycle; anchor residue is shown in bold and is applied if a position reveals a strong signal for only one residue as is the case with R at P2 (see Table IV); Strong: Ͼ2-fold increase over the previous cycle; Weak: 1.5- to 2-fold increase over the previous cycle. c Motif for HLA-B*2702 is shown for comparison (12). d X indicates that several additional residues were detected at lower levels.

The question of whether HLA-B27 acts as an Ag-presenting viewed in Refs. 3 and 48). Although the results presented here do molecule in the mechanism of SpAs remains a central and, as yet, not show that Gogo-B01 is associated with susceptibility to dis- unanswered question. The peptide binding capabilities of the var- ease, they are consistent with recent data from transgenic rats (8) ious B27 subtypes either associated with disease susceptibility or that suggest that the peptides bound by the B27 molecule play a not associated with disease susceptibility have been described (Re- role in the mechanism of disease.

Table VI. Peptides bound by Gogo-B*0101

Position

Source 123456 7 8910

No match SDTCSMDTW Human uridine nucleotide receptor, gi 1117913 P E D S S C R W No match PVGPGSMPTW Human ␦-6 fatty acid desaturase, gi 4406528 I R F L E S H W Human serine/threonine protein kinase pLK-1 ARLPYL R TW 506–514, gi423046 No exact match R P M A F Y S W No exact match S D A Q P A L/I T S W Human tubulin ␣-4 chain, AA722099 SRCHLGA Chromosome 7, HELA cDNA library KRCSPY AA078075.1 Similar but not exact match to serine threonine TRFEGY F kinase No exact match WPGPAVSZW Homo sapiens htgs, working draft AC007683.2 A S L G A K W 3342 GORILLA MHC MOLECULE BINDS PEPTIDES WITH ARGININE AT P2

Table VII. Capacity of Gogo-B*0101-positive cell lines to bind a panel of peptidesa

IC50 (nM)

721.221- Peptide Origin Sequence Beta Gogo-B*0101 Reference

153.07 SIV (B27-like) IRYPKTFGW 34 54 60 155.01 SIV (B27-like) IRFPKTFGW 12 30 60 330-1 Human Fau protein (B27) RRFVNVVPTF 64 196 11 330-3 HBNPB␤-subunit 35–43 (B27) SRDKTIIMW 31 128 11 405.1 Human actin (B27) KRGILTLKY 316 668 11 405.2 Unknown (B27) GRFKLIVLY 1572 3091 11 405.5 HIV env 584–592 (B27) ERYLKDQQL 7972 14579 61 405.6 Flu Histone H3.3 (B27) RRYQKSTEL 26 53 10 5423.3 HFPS 191–199 (B27) KRYKSIVKY 36 100 11 5423.4 Human cytochrome C oxidase (B27) KRKKAYADF 3267 6115 11 155.01 SIV (B27-like) IRFPKTFGW 12 30 60 Chiron 50 SIV with substitution IKFPKTFGW 1836b 3274 Chiron 51 SIV with substitution ILFPKTFGW 423 998 Chiron 52 SIV with substitution IFFPKTFGW 2108 3174 Chiron 53 SIV with substitution INFPKTFGW 17064 23443

Chiron 54 SIV with substitution IDFPKTFGW 65757 107383 Downloaded from

a Experiments performed with radiolabelled 153.07 IRYPKTFGW. b Numbers in bold indicate three times or greater decrease in binding capacity.

Interestingly, a strong C-terminal W residue was revealed in the peptides or T cell epitopes identified to date contain this C-terminal

sequence of individual ligands eluted from Gogo-B*0101. This residue (12, 52). Further studies are necessary to elucidate a possible http://www.jimmunol.org/ observation is consistent with results from peptides sequenced role in disease pathogenesis for peptides containing a C-terminal W. from HLA-B15 allotypes that showed that a S116 residue correlated The role of the free C67 in the B27 B pocket in disease mechanism with a strong C-terminal anchor residue (41, 49). Although the motif has been under investigation. Studies suggest that the free C67 can be of HLA-B*2702 demonstrates a dominant W at P9 (11) and this is modified due to its chemical reactivity (53), and homocysteine-mod- also a preferred residue in the motif of HLA-B*2703 (50, 51), few ified B27 can be recognized by homocysteine-specific CTL in vivo by guest on September 27, 2021

FIGURE 3. Molecular model of Gogo-B*0101 compared with that of HLA-B27. In A–C, the top view of the PBR is shown with negative charge in red, pos- itive charge in blue. In C, substitutions that result in a difference in charge or size are shown in pink, changes that remain similar in charge and size are shown in light blue. In D and E, the B pocket environment is shown with the peptide and it’s P2 side chain in pink. The Journal of Immunology 3343

Table VIII. Eleven of 13 unrelated gorillas express Gogo-B01a,b 5. Khare, S. D., J. Hansen, H. S. Luthra, and C. S. David. 1996. HLA-B27 heavy ␤ chains contribute to spontaneous inflammatory disease in B27/human 2-micro- ␤ ␤ glogulin ( 2m) double transgenic mice with disrupted mouse 2m. J. Clin. Invest. Name (ancestry) Gogo-B 98:2746. 6. Khare, S. D., M. J. Bull, J. Hanson, H. S. Luthra, and C. S. David. 1998. Spon- Beta (wild born) B*0101, B*0301 taneous inflammatory disease in HLA-B27 transgenic mice is independent of Husani (Pattycake ϫ Barney) B*0101, B*0201c MHC class II molecules: a direct role for B27 heavy chains and not B27-derived Hollid (Huerfanita ϫ Kongo) B*0201, B*0401 peptides. J. Immunol. 160:101. Harry (Kweli ϫ ) B*0101, B*0201 7. Allen, R. L., C. A. O’Callaghan, A. J. McMichael, and P. Bowness. 1999. Cutting ␤ Kwashi (Beta ϫ Carlos) B*0101, B*0103 edge: HLA-B27 can form a novel 2-microglobulin-free heavy chain homodimer Choomba (wild born) B*0103, B*0501 structure. J. Immunol. 162:5045. Katoomba (wild born) B*0101, B*0401 8. Zhou, M., A. Sayad, W. A. Simmons, R. C. Jones, S. D. Maika, N. Satumtira, M. L. Dorris, S. J. Gaskell, R. S. Bordoli, R. B. Sartor, et al. 1998. The specificity Rann (wild born) B*0103, B*0401 of peptides bound to human histocompatibility leukocyte antigen (HLA)-B27 Shamba (wild born) B*0301, B*0401 influences the prevalence of arthritis in HLA-B27 transgenic rats. J. Exp. Med. e Banga (wild born) B*0101, B*0103 188:877. Calabare B*0102, B*0103 9. Guo, H. C., D. R. Madden, M. L. Silver, T. S. Jardetzky, J. C. Gorga, Inaki (wild born) B*0101, B*0201 J. L. Strominger, and D. C. Wiley. 1993. Comparison of the P2 specificity pocket Oko (wild born) B*0201f in three human histocompatibility antigens: HLA-A*6801, HLA-A*0201, and Paki (wild born) B*0101, B*0502 HLA-B*2705. Proc. Natl. Acad. Sci. USA 90:8053. Ozoum (wild born) B*0101,orB*0103g 10. Jardetzky, T. S., W. S. Lane, R. A. Robinson, D. R. Madden, and D. C. Wiley. 1991. Identification of self peptides bound to purified HLA-B27. Nature 353:326. a MHC B locus alleles in bold indicates unrelated animal positive for a B01 11. Ro¨tzschke, O., K. Falk, S. Stevanovic, V. Gnau, G. N. Jung, and H.-G. Ram- subtype. mensee. 1994. Dominant aromatic/aliphatic C-terminal anchor in HLA-B*2702 b and B*2705 peptide motifs. Immunogenetics 39:74. Gorillas were typed as in Table I. Downloaded from c Only one copy isolated by cloning and sequencing. 12. Rammensee, H. G., T. Friede, and S. Stevanoviic. 1995. MHC ligands and pep- d Holli is the half aunt of Husani. tide motifs: first listing. Immunogenetics. 41:178. e Banga, Calabar, Inaki, and Oko were typed by Lawlor et al. (23). 13. Buxton, S. E., R. J. Benjamin, C. Clayberger, P. Parham, and A. M. Krensky. f Oko appears to be homozygous at the B locus. 1992. Anchoring pockets in human histocompatibility complex leukocyte antigen g Ozoum was typed indirectly by typing Rok, Banga’s offspring with Ozoum. (HLA) class I molecules: analysis of the conserved B (“45”) pocket of HLA-B27. J. Exp. Med. 175:809. 14. Rojo, S., F. Garcı´a, J. A. Villadangos, and J. A. L. de Castro. 1993. Changes in the repertoire of peptides bound to HLA-B27 subtypes and to site-specific mu- tants inside and outside pocket B. J. Exp. Med. 177:613. http://www.jimmunol.org/ (15). Furthermore, recent studies suggest that B27 forms nonconven- 15. Gao, X. M., P. Wordsworth, A. J. McMichael, M. M. Kyaw, M. Seifert, D. Rees, tional structures dependent on C67 (7). However, C67 does not appear and G. Dougan. 1996. Homocysteine modification of HLA antigens and its im- to be required for SpA-like disease in transgenic rats because rats munological consequences. Eur. J. Immunol. 26:1443. 16. Khan, M. A. 1992. An overview of clinical spectrum and heterogeneity of spon- made transgenic with a S67 mutant of B27 developed a disease phe- dyloarthropathies. Rheum. Dis. Clin. North Am. 18:1. notype similar to that of the homozygous wild-type B27 rats, although 17. Keat, A. 1983. Reiter’s syndrome and reactive arthritis in perspective. N. Engl. with a lower prevalence of arthritis (54). Gogo-B01, expressed in J. Med. 309:1606. 18. Adams, R. F., G. S. J. Flinn, and M. Douglas. 1987. Ankylosing spondylitis in a most gorillas that develop SpAs, also has a S67 in place of the C67. nonhuman primate: a monkey tale. Arthritis Rheum. 30:956. Thus, like transgenic rats, gorillas develop arthritis in the absence of 19. Raphael, B. L., P. P. Calle, N. Haramati, D. I. Watkins, M. D. Stetter, and an MHC class I molecule that has a free cysteine. The recent obser- R. A. Cook. 1995. Reactive arthritis subsequent to Shigella flexneri enteritis in by guest on September 27, 2021 ␤ two juvenile lowland gorillas (gorilla gorilla gorilla). J. Zoo Wildl. Med. 26:132. vation that B27 can form a C67-dependent 2m-free heavy chain 20. Rothschild, B., and R. J. Woods. 1989. 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