III Region Found in a 30-Kb Segment of the MHC Class Dimethylaminohydrolase Homologue

Genes Encoding Three New Members of the Leukocyte Antigen 6 Superfamily and a Novel Member of Ig Superfamily, Together with Genes Encoding the Regulatory Nuclear This information is current as Chloride Ion Channel Protein (hRNCC) and of October 2, 2021. a Nω-Nω-Dimethylarginine Dimethylaminohydrolase Homologue, Are Found in a 30-kb Segment of the MHC Class III Region Downloaded from Gloria Ribas, Matt Neville, Joanne L. Wixon, Jianhua Cheng and R. Duncan Campbell J Immunol 1999; 163:278-287; ; http://www.jimmunol.org/content/163/1/278 http://www.jimmunol.org/

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Genes Encoding Three New Members of the Leukocyte Antigen 6 Superfamily and a Novel Member of Ig Superfamily, Together with Genes Encoding the Regulatory Nuclear Chloride Ion Channel Protein (hRNCC) and a N␻-N␻-Dimethylarginine Dimethylaminohydrolase Homologue, Are Found in a 30-kb Segment of the MHC Class III Region1,2

Gloria Ribas,3* Matt Neville,* Joanne L. Wixon,4* Jianhua Cheng,* and R. Duncan Campbell5*† Downloaded from Many of the genes in the class III region of the human MHC encode proteins involved in the immune and inflammatory responses. We have sequenced a 30-kb segment of the MHC class III region lying between the heat shock protein 70 and TNF genes as part of a program aimed at identifying genes that could be involved in autoimmune disease susceptibility. The sequence analysis has revealed the localization of seven genes, whose precise position and order is cen-G7-G6-G6A-G6B-G6C-G6D-G6E-tel, five of which are fully encoded in the sequence, allowing their genomic structures to be defined. Three of them (G6C, G6D, and G6E) encode

putative proteins that belong to the Ly-6 superfamily, known to be GPI-anchored proteins attached to the cell surface. Members http://www.jimmunol.org/ of the family are speciﬁcally expressed and are important in leukocyte maturation. A fourth gene, G6B, encodes a novel member of the Ig superfamily containing a single Ig V-like domain and a cytoplasmic tail with several signal transduction features. The G6 gene encodes a regulatory nuclear chloride ion channel protein, while the G6A gene encodes a putative homologue of the enzyme N␻,N␻-dimethylarginine dimethylaminohydrolase, which is thought to be involved in regulating nitric oxide synthesis. In addition, three microsatellite markers, 9N-1, 82-2, and D6S273 are contained within the sequence, the last two of which have been reported to be strongly associated with the autoimmune disease ankylosing spondylitis. The Journal of Immunology, 1999, 163: 278–287.

eukocyte Ag 6 (Ly-6)6 Ags are a group of leukocyte Ags anchor. The site of attachment is an Asn residue, based on known by guest on October 2, 2021 first identified in the mouse that consist of 70–80 aa con- attachment sites of similar GPI-anchored proteins (1, 3). The GPI L taining 10 Cys residues (1, 2). One distinctive feature of anchor is directly involved in signal transduction (4, 5). Members these Ags is that they are attached to the cell surface by a GPI of the Ly-6 family are differentially expressed in several hemo- poietic lineages, especially in T lymphocytes, and appear to function in signal transduction and cell activation (1, 3, 6–8). Chromosome mapping studies using somatic cell hybrids, re- *Medical Research Council Immunochemistry Unit, Department of Biochemistry, Oxford University, Oxford, United Kingdom; and †HGMP Resource Centre, Hinxton, combinant inbred mouse lines, and in situ hybridization have re- Cambridge, United Kingdom vealed that all members of the mouse Ly-6 multigene family oc- Received for publication February 2, 1999. Accepted for publication April 14, 1999. cupy a single genetic locus on chromosome 15 that is closely The costs of publication of this article were defrayed in part by the payment of page linked to the sis and myc proto-oncogenes and to loci that mediate charges. This article must therefore be hereby marked advertisement in accordance susceptibility to radiation-induced lymphoid malignancy (9–11). with 18 U.S.C. Section 1734 solely to indicate this fact. The physical map spans 630 kb and contains at least 18 distinct 1 This work was supported by a Formacion Personal Investigator postdoctoral fel- lowship from the Spanish Government (to G.R.) and a Medical Research Council Ly-6-related sequences (12). Six expressed Ly-6 mouse genes are studentship (to J.L.W.). known (Ly-6A/E, -B, -C, and –G; TSA-1/Sca-2; and ThB) and a 2 The nucleotide sequence reported in this paper has been submitted to the EMBL seventh, Ly-6F, has been identified at the molecular level (3, 7, databank with accession number HSA012008. 13–18). The sequences of the leader peptide and membrane-an- 3 Current address: HGMP Resource Centre, Hinxton, Cambridge, U.K. CB10 1SB. choring portions show high similarities, but the sequences of the 4 Current address: Department of Biomolecular Sciences, The Mill Building, Univer- sity of Manchester Institute of Science and Technology, P.O. Box 88, Manchester, middle segments apart from the invariant cysteine residues are U.K. M60 1QD. much more variable. The middle segments probably play an im- 5 Address correspondence and reprint requests to Dr. R. Duncan Campbell, HGMP portant role in the functional diversity that may distinguish the Resourse Centre, Hinxton, Cambridge, CB10 1SB, U.K. E-mail address: Ly-6 gene products. To date, three human members of this family, [email protected] E48 (homologue of mouse ThB), TSA-1/Sca-2 (homologue of 6 Abbreviations used in this paper: Ly-6, leukocyte Ag 6; Tsa-1/Sca-2, thymic shared Ag-1/stem cell Ag-2; ThB, thymocyte B Ag; E48, monoclonal antibody E48; GML, mouse TSA-1), and GML, have been mapped in chromosome 8 GPI-anchored molecule-like protein; RA, rheumatoid arthritis; AS, ankylosing spon- (8q24-qter region) (19–21), which is syntenic to mouse chromo- dylitis; DDAH, enzyme N␻,N␻-dimethylarginine dimethylaminohydrolase; uPAR, urokinase plasminogen activator receptor; SH2–3, Src homology 2–3; ADMA, asym- some 15. There are other Ly-6-related molecules with diverse metric dimethyl-L-arginine; EST, expressed sequence tag. functions, such as the three domain urokinase plasminogen

Table I. Transcript-speciﬁc RT-PCR primers

Genomic cDNA Genes Primers TM GϩC (%) Positiona Length (kb) Lengthb (bp)

G6A F 5Ј-TGCTGGGAGGTAAACTGAGG 60.2 55 12397 0.75 356 R5Ј-GACTGGCACAGTGGAGACG 60.5 63 13153 G6B F 5Ј-TCCTTTACTGGCCTCTGGG 60.2 58 16615 1.10 575 R5Ј-GAGGAGTCTCTCTCATCCCATCC 60.0 57 17735 G6C F 5Ј-GCTGTCACTCCTGCTACAAGG 60.1 57 21221 1.01 200 R5Ј-GTTGCAGTTGTCCTTGTTGC 58.0 60 22235 G6D F 5Ј-TGGCAAGAGACAGGTCAGG 60.0 58 23629 2.2 300 R5Ј-AGCAGTTCTTGCAAAGAGGC 58.0 60 25873 G6E F 5Ј-TCATCGTCCCGACACTCG 62.4 60 27608 1.1 198 R5Ј-CCTCCAGCATCTTCCTCTGC 61.9 61 28720 Actin F 5Ј-CTTCGCGGGCGACGATGG 60.0 72 500 R5Ј-TGGTGGTGAAGCTGTAGCC 60.0 58

a Positions of primers (5Ј) within the genomic sequence generated in this study. b PCR conditions: 1 mM MgCI2, 60°C annealing time, 30 cycles. Downloaded from activator receptor (uPAR) or CD87, and CD59 (complement pro- markers, which achieved the highest LOD scores of all markers tein-blocking assembly of the membrane attack complex) that ex- tested, were strongly associated with susceptibility to the disease. hibit a low degree of sequence identity and are encoded by genes that are not in close linkage with any of the Ly-6 family. No human Materials and Methods homologue of the Ly-6 gene cluster has been identiﬁed to date. Sequencing strategy

Attempts to do so have been hampered by an apparently rapid http://www.jimmunol.org/ The EL3 cosmid clones were sequenced using an M13 shotgun strategy divergence of the genes between species that has prevented de- (46) with fluorescent dye primer and dye terminator sequencing chemis- tectable cross-hybridization of Ly-6-like genes much beyond a tries (Amersham, Arlington Heights, IL). Cosmid DNA was sonicated, and subset of the rodentia. fragments from 1–1.5 kb were selected for cloning into M13 mp18. Re- We report here sequence analysis of a cosmid (cosEL3) from the combinant M13 mp18 phage DNA was purified from culture supernatants using a Vistra DNA Labstation and was cycle sequenced using Thermo- human MHC class III region that has revealed the presence of a Sequenase (Amersham) in a 96-well format on a Hybaid Omnigene ther- cluster of three Ly-6 superfamily members. MHC occupies a seg- mocycler (Middlesex, U.K.; 95°C for 5 min followed by 20 cycles of 95°C ment of approximately 4 Mbp on the short arm of chromosome 6 for 30 s and 60°C for 30 s) in the presence of the fluorescent dye-labeled (6p21.3) and contains the highly polymorphic class I and class II M13 universal primer (5Ј-TGACCGGCAGCAAAATG-3Ј). The sequenc- genes (22). These genes are responsible for coding polymorphic ing reactions were run on an Applied Biosystems 377 automated DNA by guest on October 2, 2021 sequencer (Foster City, CA), and sequence data were analyzed with the cell surface proteins involved in the presentation and recognition Applied Biosystems 377-dedicated software. Individual sequence traces of foreign Ags during immune responses (23–25). In man, these were processed and reassembled using the programs PREGAP and GAP two gene clusters are separated by about 1100 kb of DNA termed v4.0-␤4 from the Staden suite of software (Medical Research Council Lab- the class III region (26, 27), which contains a number of unrelated oratory of Molecular Biology, Cambridge, UK). Ambiguities within the sequence were resolved, and the sequences genes, including those encoding the complement proteins C2, fac- across areas of single orientation read were confirmed with dye terminator tor B, and C4 (28); the related cytokines TNF and lymphotoxin- sequencing chemistries, while gaps between contigs were closed by either ␣/␤ (26, 27, 29); and three genes encoding members of the major sequencing the reverse strand of long clones (Ͼ800 nt) that extended into heat shock protein (hsp70) family (30, 31). the gap or by sequencing of PCR products covering the gaps. Susceptibilities to a wide range of diseases have been linked to Transcript profiling the MHC. These include Behçet disease, systemic lupus erythem- atosus, orchitis, celiac disease, insulin-dependent diabetes melli- The expression of transcripts was investigated in the cell lines Jurkat 6 and Molt 4 (T cell), Raji (B cell), HL60 (monocyte), U937 (monocyte), HepG2 tus, rheumatoid arthritis (RA) and ankylosing spondylitis (AS) (hepatocyte), HeLa (epithelial), HT1080 (epithelial), and SW620 (adeno- (32–41). Although many disease susceptibilities appear to be due carcinoma) with RT-PCR using total RNA and the Promega RT system, to allelic differences in the class I and class II Ags, some additional according to the manufacturer’s protocol (gene-specific primers are shown loci within the central class III region may contribute to disease in Table I). PCR primers were designed to give products containing more than one exon so that amplification products arising from genomic DNA susceptibility as well. contamination were easily discernible. The first-round cDNA synthesis was In addition to the three new members of the Ly-6 superfamily performed in a final volume of 20 ␮l with 1 ␮g of total RNA; 5 ␮l of this the cosmid was found to contain a gene (G6B) encoding a putative reaction mix was used per 25 ␮l of PCR reaction using the transcript- new member of the Ig superfamily and two other genes. The G6 specific primers and amplification conditions listed in Table I. Each transcript-specific RT-PCR reaction was performed in at least triplicate to al- gene encodes a regulatory molecule of a nuclear channel protein low for any variation between reactions. Control amplification reactions described by Valenzuela et al. (42), while the G6a gene encodes a with primers derived from ␤-actin were conducted for each first-round putative homologue of the human enzyme N␻,N␻-dimethylargin- cDNA synthesis reaction. The identities of PCR products were confirmed ine dimethylaminohydrolase (DDAH) first described in rat kidney by direct dye terminator sequencing. by Kimoto et al. (43). This important enzyme may regulate the Sequence analysis L-arginine-NO pathway by governing the degradation of endoge- nous inhibitors of NO synthases (43, 44). In addition, the cosmid The Wisconsin Package version 9-UNIX (GCG), maintained at the Uni- was found to contain the microsatellite markers D6S273 and 82-2. versity of Oxford Molecular Biology Data Center, was used for the majority of the sequence analysis and database interrogation. The DNA se- In a genome-wide screen for susceptibility loci in AS, the data quence generated was screened against the EMBL, SwissProt, PDB, obtained by Brown et al. (45) clearly indicated that these two EMBL-EST, and TIGR-EST ([email protected]) databases to position 280 GENES ENCODING THE Ly-6 SUPERFAMILY IN HUMAN MHC

FIGURE 1. Location of cosEL3 in the MHC class III region, ϳ150 kb centromeric of the TNF locus. Open boxes indicate the boundaries of the genes. Differently filled boxes indicate the exons of the genes found in cosEL3. Black boxes represent repeat elements. Hori- zontal arrows show the orientation of the genes, and vertical double arrows show the locations of the three microsatellites detected in cosEL3. Downloaded from http://www.jimmunol.org/ known genes and identify possible new coding regions. Repetitive ele- Neck, NY). Ten-microliter beads were washed in 10 ␮l of binding buffer ments were identified with the aid of the Repeat Masker server (A. F. A. (supplied by the manufacturer) before mixing with the hybridization mix Smit and P. Green, RepeatMasker at http://ftp.genome.washington.edu/ for 15 min. The beads were then washed twice with 1ϫ SSC/0.1% (w/v) RH/RepeatMasker.html), and potential coding regions were defined using SDS and three times for 15 min in 0.1ϫ SSC/0.1% (w/v) SDS at 65°C to the NIX exon prediction program (http://www.hgmp.mrc.ac.uk/registered/ remove nonspecifically bound cDNAs. The selected cDNAs were eluted Webapp/nix/) at the HGMP Resource Center, Hinxton (Cambridge, U.K.). with 1 M Tris-HCl (pH 7.5). Predictions of protein secondary structure, solvent accessibility, and trans- The secondary selected cDNA products were amplified with linker membrane regions were conducted using the Jpred-consensus secondary primers with adaptors containing dump bases to allow UdG cloning using structure prediction server (http://circinus.ebi.ac.uk:8081/) or PredictPro- the Cloneamp kit (Life Technologies, Grand Island, NY). PCR products tein program ([email protected]). The GCG program SIG- (50–100 ng) were mixed with 25 ng of prepared pAMP 18 vector in the by guest on October 2, 2021 CLEAVE and the SMART (Simple Modular Architecture Research Tool) presence of1Uofuracil DNA glycosylase in UdG annealing buffer. The server (http://coot.embl-heildelberg.de/SMART) were used to identify ligation products were transformed into competent TG1 cells plated out leader peptides. Sequence motifs and protein domains were identified using onto L plates (with ampicillin) at low density, and the cDNA inserts were a combination of the GCG program MOTIF, the Prosite Profilescan server characterized by either dye primer or dye terminator sequencing using the (http://␮lrec3.unil.ch/software/PFSCAN-form.html), and the SMART M13 –21 forward primer. server. Multiple alignments of amino acid sequences were performed using Clustalx software, making use of protein structure information from sequences within the PDB database to aid in the alignment wherever possi- Results ble. Alignments were hand-edited using the GCG9 SeqLab multiple align- The complete genomic sequence of the cosmid cosEL3 was ob- ment editor. tained from a total of 700 templates. This sequence can be found cDNA selection in the EMBL database under accession number AJ012008/ HSA012008. Analysis of the sequence was performed by database Five nanograms of cosmid F9N DNA (overlapping the centromeric part of EL3), obtained by the alkaline lysis method, was biotinylated with 16- searching using BLAST and NIX (@hgmp.mrc.ac.uk). A sche- dUTP (20 ␮M) and radioactively labeled with [␣-32P]dCTP (0.06 ␮M) matic representation of the exons identified, the repeat elements, using the nick translation kit from Promega (Madison, WI) (47). A fetal and the microsatellites present in the cosmid is shown in Fig. 1. brain cDNA library (gift from M. Lovett, Department of Biochemistry, The location of exon coordinates and other sequences, such as University of Texas Southwestern Medical Center, TX) was supplied li- polyadenylation signals, can be found in the EMBL database, gated to a linker, to facilitate amplification using primer 3Ј-CTCGAGAAT TCTGGATCCTC. The reaction was heated to 94°C for 10 min and then submission HSA012008. The derived amino acid sequences of subjected to 30 cycles of 45 s at 94°C, 45 s at 58°C, and then 2 min at 72°C the five proteins encoded by genes located in the cosmid are before a final stage at 72°C for 10 min. The PCR products were ethanol shown in Fig. 2. precipitated and resuspended in 7.5 ␮l of distilled water. Two micrograms of the selected cDNA was mixed with 2 ␮g of COT-1 DNA and 10 ng of BamHI-digested Lorist 4 vector in a total volume of 10 ␮l. This mixture The Ly-6-like genes was boiled for 5 min, 10 ␮l of warmed 2ϫ cDNA hybridization solution The G6C gene. Six different human and three mouse ESTs from (50% deionized formamide, 1 M NaCl, 50 mM Tris-HCl (pH 7.4), 0.2% the EST database aligned over the G6C genomic region. Only one BSA, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, 0.1% sodium pyro- phosphate, 10% dextran sulfate, and 0.5% SDS) was added, and the cDNA of six human EST entries (H03135) matched exactly the G6C sample was preannealed at 65°C for 4 h. The preannealed starting cDNAs gene, while the others are only partial transcripts containing the were mixed with denatured template and 2ϫ cDNA hybridization solution last exon (accession nos. H03945, W56634, R27318, W56597, and in a final volume of 20 ␮l. This mixture was then incubated at 65°C for R25237). One of three mouse EST entries (W12301) is almost a 50 h. After recovery of the template and elution of the selected cDNAs, the selected cDNAs were preannealed and hybridized to the remaining tem- complete transcript, while the other two contain only exons 1 and plate in the same way as the starting cDNA. cDNAs were recovered from 2 (accession nos. AA93044 and AA500454). The G6C gene spans the hybridization mixes using streptavidin M-280 beads (Dynal, Great about 3 kb and contains three exons. RT-PCR analysis using The Journal of Immunology 281 Downloaded from

FIGURE 3. RT-PCR analysis of total RNA from M (Molt 4), J (Jurkat 6), R (Raji), U (U937), H (HeLa), E (HL60), G (HepG2), and the following IFN-␥-activated cells: U937 (U), HeLa (H), HT1080 (T), and Sw620 (S) or PMA-activated cells (U9370). The ﬁrst lane (L) in each panel is a size marker (1-kb ladder). To the left of each panel is the gene analyzed: G6A, ␤ G6B, G6C, G6D, G6E, and the positive control -actin. For the analysis of http://www.jimmunol.org/ G6B, G6C, G6D, and G6E, 8 ␮l of the PCR was loaded onto the gel, while for G6A and ␤-actin, 4 ␮l was loaded.

plete, the first two exons encode the putative signal peptide, and the first 24 aa of the Ly-6 domain containing the first four cysteines. A stop codon was found at the end of the second predicted exon, which might indicate that G6E is a pseudogene. In support of this, G6E was found not to be expressed in any of the cell lines by guest on October 2, 2021 FIGURE 2. Deduced amino acid sequence of the polypeptides encoded tested, whether activated or not by IFN-␥ and PMA (Fig. 3), even by different genes found in cosEL3. The different putative posttranslation though two different primer pairs were used. modifications predicted in the polypeptides are: shadowed, glycosylation; dotted line, PEST sequences; underlined, phosphorylation sites; bold, The G6C/D/E proteins myristylation sites. The predicted sequences of the mature cell surface The G6C and G6D genes encode polypeptides of 125 and 133 aa, proteins encoded by the three Ly-6-like genes are indicated. The N-terminal amino acids Ileu ϩ1 in G6C, Met ϩ1 in G6D, and Leu ϩ1 in G6E were respectively. G6E is an incomplete gene and encodes a partial assigned on the basis of homology to the other Ly-6 sequences, and the polypeptide sequence of only 51 aa. The G6C and G6D polypep- C-terminal amino acid was assigned according to the GPI attachment con- tides each contain a leader peptide, a Ly-6-like domain, a trans- sensus sequences. membrane region (type 1a), and sequences that indicate that they could be anchored to the cell membrane via GPI anchors. Cleavage of the putative signal peptide and the hydrophobic transmembrane primer sequences located in exons 2 and 3 has revealed expression region after the Asn residue next to the last Cys residue would of G6C only in the T cell lines Molt 4 and Jurkat 6 (Fig. 3). yield mature proteins of about 79–85 aa in length. Alignment with Sequencing of the PCR product has allowed the exon boundaries all protein members of the Ly-6 superfamily is shown in Fig. 4 to be confirmed. together with the predicted secondary structural elements (indicat- The G6D gene. Only one mouse cDNA (accession no. ed by arrows (␤-sheets), cylinder (␣-helix), and lines (loops)). The AA794551) was found in the EST databases matching the internal percent similarity and identity between the different members of exons of G6D. The gene spans about 3.5 kb and contains only the Ly-6 family are shown in Table II. G6D lacks two main cys- three exons in reverse orientation to those of G6C. The microsat- teines obstructing two different disulfide bonds. This is rare in the ellite marker D6S273 lies in between the second and third exons family because the 10 cysteines are vital to maintain the structure. very close to a highly repetitive region of about 1.5 kb in length However, one other member of this family is found to have two (see Fig. 1). This microsatellite has recently been found to be cysteines missing (uPAR1, see alignment in Fig. 4). Phylogenetic strongly associated with susceptibility to AS (45). RT-PCR anal- analysis of the Ly-6 alignment is shown in Fig. 5, which reveals ysis using primer sequences located in exons 2 and 3 has revealed that both G6C and G6D belong to the Ly-6 superfamily, although that the G6D transcript is expressed in all the cell lines analyzed they are differently related to mouse Ly-6 proteins. (Fig. 3). Sequencing of the PCR product has allowed confirmation of the exon boundaries. The G6B gene The G6E gene. No EST sequence matching G6E was found in The G6B gene, which spans about 2 kb, has been predicted by EST database searches. Although the gene appears to be incom- several exon prediction programs in reverse orientation to the G6A 282 GENES ENCODING THE Ly-6 SUPERFAMILY IN HUMAN MHC

Table II. Similarity (S) and identity (I) between members of the Ly-6 superfamily and G6C/D, and G6B and Ig V domains

G6C G6D G6B

S (%) I (%) S (%) I (%) S (%) I (%)

G6D 24.36 20.51 Mouse Ly-6-a 29.73 27.03 25.97 24.68 Mouse Ly-6-c 30.56 25.00 26.67 22.67 Mouse Ly-6-g 24.32 21.62 28.95 25.00 Mouse Ly-6-f 28.38 22.97 25.97 23.38 Human GML 34.62 24.36 34.65 20.51 Human Tsa-1/Sca-2 24.66 21.92 23.68 19.73 Human E48 28.57 25.71 25.00 22.22 Human CD59 34.33 26.87 30.44 24.64 Human uPAR-1 32.39 26.76 25.00 22.22 Human Ig-V (ph1672) 41.5 29.79 Human Ig-V (U77554) 37.87 25.24 C. plumbeus (U50610) 37.3 27.66 Mouse Ig-V (L21019) 40.5 26.19 Downloaded from and G6C genes and contains six exons tightly packed between two sets of repetitive elements (Fig. 1). A cDNA selection experiment using fetal brain cDNA as template identiﬁed one clone (3/1B7), which contained the end of exon 2, exons 3 and 4, and the begin- ning of exon 5 (48). One EST entry (accession no. AA699838) was also found containing only the last exon with the predicted poly- http://www.jimmunol.org/ adenylation signal. We were unable to determine any mRNA expression when performing RT-PCR analysis on a number of cell lines, both unactivated and activated. The predicted G6B protein has 241 aa and contains several in- teresting features corresponding to a putative signal transduction receptor. The translated sequence immediately downstream from the initiator methionine codon shows a high proportion of hydrophobic amino acid residues, characteristic of a posttranslationally

cleaved leader sequence from position 1–18. Exon 2 encodes an by guest on October 2, 2021 external domain with high similarity to Ig variable domains and contains four cysteines, two of them in good consensus sequences to interact in a disulﬁde bond, while the other two might interact with another molecule and induce dimerization, a feature common in Ig receptors (49). G6B also contains two key residues that form a salt bridge, Arg85 in strand D with Asp102 in strand F. Alignment with some Ig V domains is shown in Fig. 6A (50). The percent similarity and identity between protein sequences shown in the alignment are given in Table II. G6B also appears to have two putative Asn glycosylation sites at amino acid positions 77 (NQTN) and 88 (NTTC). Another hydrophobic region predicted as a transmembrane region (PSORT II, Nakai, PHD) is positioned in the translated sequence from aa 143 to 165. Residues 168–175 correspond to a putative signal transduction domain, containing a proline-rich sequence motif with a consensus sequence X-X-Pro-Pro-X-Pro-X-X for a SH3 binding domain (reviewed by Cohen et al. (51) and Yu et al. (52)) (Fig. 6B), and two putative phosphorylated tyrosine motifs (53). One of them seems to have the consensus sequence for an SH2 binding domain (Y-hydrophobic-X-hydrophobic: YADL

FIGURE 4. Alignment of the extracellular region of G6C (1–79) and G6D (1–82), generated by the Clustalx program, with the corresponding sequences of the following proteins are from SWISSPROT (mouse Ly-6 region of the related sequences from the Ly-6 superfamily members are genes (P05533, P09568, P35460, P35461), human CD59 (P13987), rat shown. Residues that are common or similar between the different se- CD59 (P27274), GML (Q99445), human uPAR (Q03405), mouse uPAR indicates that all residues are identical; Pret- (P35456), rat uPAR (P49616), Cobratoxin, Naja siamaensis (P01391)) and ء) quences are shown boxed typlot program). The three Ly-6 modules in uPAR are labeled 1–3 starting EMBL (E48 (X82693), ThB (X63782), human Tsa-1/Sca-2 (U56145), from the outermost, N-terminal domain. Positions of ␤-strands (A–E) are mouse Tsa-1/Sca-2 (U04268), chicken Tsa-1/Sca-2 (L34554), rat Ly-6 indicated by arrows, and the single ␣ helix is indicated by a cylinder above (M30692, M30689, M30690), SP-10 from Palfree et al. (68), and Squid the sequence. Disulﬁde bonds between cysteines are shown by lines. The Sgp2 from Williams et al. (79)). The Journal of Immunology 283

The results obtained in the RT-PCR experiments showed that G6A was expressed widely (Fig. 3). However, Spanjaard et al. (54) only detected expression of the mouse mRNA in large intestine, with low levels in lung and brain. In rat, expression was detected in kidney, pancreas, liver, brain, lung, and heart (56), and the protein was clearly shown to exist in peritoneal neutrophils and mac- rophages (56). Recently, the DDAH has been characterized in bovine brain tissue, and the protein is detected in total brain and kidney, at low levels in heart, liver, and lung, but not in blood, muscle, spleen, or intestine extracts (57). The PCR product obtained from the RT-PCR experiment has been sequenced, and the exon boundaries checked.

The G6 gene The gene G6 was originally deﬁned using a 2.4-kb HindIII genomic fragment lying adjacent to a CpG island, which hybridized to mRNA species of 1.5 and 1.4 kb in the cell lines U937, U937 stimulated by PMA, HepG2, and Molt4, and mRNA species

of 1.5 and 1.45 kb in the cell line Raji (31). A fragment containing Downloaded from the 5Ј untranslated sequence in exon 2 was only detected in one of the mRNA species (58). Two mRNA species have also been described by Valenzuela et al. (42) (1.0 and 1.2 kb) that are differently regulated by PMA, retinoic acid, IFN-␥, and IL-2. They also detected a difference in the 5Ј untranslated region between the two species. The gene spans 6 kb and contains seven exons (Fig. 1). http://www.jimmunol.org/ The microsatellite marker 82-2 which has been found to be strongly associated with susceptibility to AS (45), is located be- FIGURE 5. Phylogenetic tree of the Ly-6 superfamily members. The program Clustalx was used to build up the alignment, and the phylogenetic tween the second and third exons. The longest open reading frame tree is based on the neighbor-joining (NJ tree) option rather than maximum of G6 starts in exon 2 and codes for a polypeptide of 241 aa with parsimony. Numbers at the nodes indicate bootstrap conﬁdence levels in a predicted Mr of 26.9 kDa. The amino acid sequence is identical percentages, obtained from 1000 bootstrap replications. The scale and with the polypeptide described by Valenzuela et al. (42), which number above it indicate the number of amino acid substitutions per they claimed could be a putative nuclear chloride ion channel position. protein.

Two PEST regions have been found in G6 (wwwserver at JMB by guest on October 2, 2021 Jena). These regions are rich in proline (P), glutamic acid (E), at position 211), and the second motif Thr-X-Tyr at position 235 serine (S), and threonine (T) and begin and end with relatively is common in mitogen-activated protein kinases. A schematic rep- charged residues, but most commonly lysine, arginine, and histi- resentation of the different motifs in G6B is shown in Fig. 6C.It dine. Those regions are characteristic of proteins that are rapidly is likely that G6B might be involved in some kind of signal trans- degraded within eukaryotic cells (59). PEST sequences have been duction events reported in a number of proteins that are located in the nucleus, The G6A gene including c-Fos, c-Myc, p53, Bmil, and Upl (59–61). Interestingly there are two basic sequences (KRR and KKYR) at The G6A gene contains eight exons that span about 3 kb of DNA positions 49 and 192 in the protein that satisfy the criteria for Ј (Fig. 1). The 5 untranslated region of the mRNA is encoded in the nuclear localization signals (62). In support of this, there are four first two exons and part of the third (from positions 12,052 to putative casein kinase II sites near these nuclear localization sig- 12,257). When either the first or second exon is used, the splicing nals at positions 44 (TTUD), 174 (TLAD), 198 (TIPE), and 222 of the third exon changes, and it starts at position 12194. The most (TCPD) (63). Casein kinase II sites have been located close to ϳ common ESTs ( 30 entries in total) correspond to an mRNA many confirmed nuclear localization signal sequences found in starting at exon 1, although other splice variants are also apparent other proteins, and phosphorylation at these sites has been impli- (accession no. of ESTs starting at exon 2: H87910, AA133714; cated in the rate of nuclear transport. Other post-translation mod- accession no. of ESTs starting at exon 3: AA298342, AA134375, ifications that could occur in G6 are shown in Fig. 2. and R11949). The longest open reading frame from all splice vari- No N-terminal signal peptide is found in the G6 protein se- ants starts at position 12,258 in exon 3 and codes for a 285-aa quence (PsortII and Nakai server). None of the programs used (Tm polypeptide of 31.4 kDa. prediction, PHD) predicted a good transmembrane region in the Database searches revealed that the putative human polypeptide protein, suggesting a globular three-dimensional structure for G6. shared 96.7% identity with the derived amino acid sequence encoded by the mouse cDNA clone (7u) isolated from a malignant melanoma cDNA library (54), and 48.75% identity (70.46% sim- Discussion ilarity) with the rat kidney enzyme DDAH (43) (EC 3.5.3.18; Fig. Ly-6-like genes 7). The active center of the enzyme is probably between residues The genes G6C and G6D, and possibly also G6E, encode small 119 and 166, which are highly conserved (see Fig. 7). Three small Cys-rich proteins of 79–82 aa that appear to be novel members of motifs common to the prokaryote enzyme family that catabolize the Ly-6 family. They are probably anchored in the cell membrane L-arginine to L-citruline (55) are found in the DDAH family as well by a C-terminal GPI moiety, a post-translational modification in (highlighted in Fig. 7). common with each member of the Ly-6 family described to date. 284 GENES ENCODING THE Ly-6 SUPERFAMILY IN HUMAN MHC Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. A, Alignment of the putative Ig-like domain of the predicted G6B polypeptide with some other related Ig variable domain sequences: Carcharhinus plumbeus IgW-V region (U50610), Mus musculus Ig-V chain (l21019), human Ig-V domain (U77554), and human Ig-V domain (ph1672). ␤-strands are represented as lines above the sequence. Eighteen of the 20 key residues of the I set described by Harpaz and Chothia (50), characteristic of the cell adhesion molecules and the surface receptors that belong to the Ig superfamily, are highlighted with a triangle. B, Alignment of the proline-rich sequence (X-X-Pro-Pro-X-Pro-X-X) of G6B with some polypeptides able to bind SH3 domains. Filled boxes represent additional consensus amino acids of class I SH3 binding domains. C, Schematic representation of a putative G6B dimer.

The finding of Ly-6-like proteins in humans is important because located in chromosome 11q23-q24 (68). However, it contains of the independent regulation of each member of this family, gen- some unique features that distinguish it from other members of the erating distinct patterns of expression during hemopoiesis and im- superfamily, including G6C and G6D. For example, more than half mune responses. This information suggests that they provide dis- of the original protein sequence is unrelated to Ly-6, being en- tinct functions affecting various stages of leukocyte development. coded by a completely different exon not present in any other Ly-6 Only a few human genes with this homology have been described member, and it lacks the GPI anchor, which is a distinctive feature to date, the E48 homologue (19) to the mouse ThB gene (64), the of the Ly-6 Ags. Tsa-1/Sca-2 homologue (20) (or 98604 (65) or RIG-E (66)) to the The G6C, -D, and -E genes are not direct homologues of any of mouse Tsa-1/Sca-2 gene (6), and the GML (21). The E48 (in- the mouse Ly-6 genes given that the level of sequence identity is volved in cell-cell adhesion), Tsa-1/Sca-2 (recently, physically and only 21.62–27.03%, but being members of the Ly-6 superfamily, functionally associated with the TCR via CD3 (67)), and GML (the they might have similar functions. Taking into account that both expression of which is induced by p53) genes have been localized genes, G6C and G6D, have different expression patterns in the cell in human chromosome 8 (19–21), which is the syntenic region of lines tested, an in-depth characterization of these genes will help to mouse chromosome 15, where the mouse Ly-6 gene cluster has define a more accurate functionality. been localized (12). No Ly-6 domain has been found in combination with domains of any other superfamily; this may be because The G6B gene the exon structures known for this superfamily are not suited to G6B is an Ig-like receptor with different signal transduction motifs exon shuffling. The SP-10 (sperm acrosomal protein) is another found in the putative cytoplasmic domain of the molecule. The Ig human member of the Ly-6 superfamily that is encoded by a gene superfamily is a large group of related proteins that function, The Journal of Immunology 285 Downloaded from

FIGURE 6. Alignment of the G6A amino acid sequence with mouse AA833494 and rat D86041, which corresponds to the enzyme DDAH. Three small motifs in common with the prokaryotic deiminase enzyme family are highlighted with triangles. http://www.jimmunol.org/ mainly in the immune system, in cell-cell recognition or in the which could indicate the possible existence of G6B as a dimer, structural organization and regulation of muscle (49). The various either disulfide linked to itself or to another polypeptide. members of the superfamily are built of homologous domains of Interestingly, the BG genes within the chicken MHC complex approximately 100 residues and with a structure formed by two have been found to encode molecules composed of a single extra- ␤-sheets packed face to face. A disulfide bond is almost always cellular domain that resembles an Ig V-type domain, a transmem- formed by cysteines localized in strands B and F, which are one of brane region, and a 217-aa cytoplasmic domain (69). We have the most conserved in the Ig domain. Some of the members have compared G6B with the BG molecules and have found that they more than two cysteines, and it is believed that this contributes to share 20% identity (26% similarity). Moreover, the intracellular by guest on October 2, 2021 dimerization. Individual members of the superfamily can differ in segment of sequence is 3 times larger in the BG molecules com- the number and size of strands in the two ␤-sheets and in the size pared with G6B, and they share no similarity whatsoever. Thus, and conformation of the links between the strands. Similarities in although G6B and the BG molecules are members of the Ig su- sequences and structures were found, however, and these allow the perfamily, the lack of sequence similarity indicates that they are different members to be grouped into what Williams and Barclay unrelated to each other. (49) call sets. V set and V-related domains have about 65–75 aa One of the internal motifs corresponds to a short proline-rich residues between the conserved disulfide bond, and there are four sequence of ϳ10 aa that could bind to a SH3 (Src homology 3) ␤-strands in each ␤-sheet plus a short ␤-strand segment across the domain (70) in a left-handed PPII helix conformation (a 3-fold top of the domain. G6B is more related to the V set, as it contains symmetry helix, with proline residues usually in one face). PPII 71 aa in between the two cysteines most likely to form the helices in globular proteins are also ideal mediators of protein- disulfide bond. protein interactions since most of these motifs are located on the Harpaz and Chothia (50) defined a new set called I defined by 20 surface of their proteins (71). key amino acid positions that form the characteristic folds of the G6B also contains two putative phosphorylated tyrosine resi- set. Ig molecules that follow set I seem to be involved in the cell dues with one of them at least in a consensus sequence for a SH2- adhesion and cell surface recognition processes. Some of the pre- binding motif. Thus, G6B contains all the possible features to be a vious V and C2 sets fall into this new set I. Alignment of G6B with signal transduction receptor. some other variable domains in a manner similar to that used by Harpaz and Chothia highlighted the conservation of 18 of the 20 key sites in G6B. This alignment is based on a secondary structure The G6A gene prediction that defined the approximate position of all the The G6A protein is a homologue of the enzyme DDAH that is ␤-strands. Other features that indicate an Ig domain are that the thought to regulate the production of NO by metabolizing N␻- majority are encoded within one single exon, which is always mono-methyl-L-arginine and asymmetric dimethyl-L-arginine to L- phase 1. Both features are found in the predicted variable domain citruline, both of them analogues of L-arginine and direct inhibitors of G6B, providing more evidence for its inclusion in the super- of constitutive and inducible NO synthases (44, 72). Although family. Two residues important for the formation of a salt bridge G6A is probably not the human DDAH enzyme itself, it is likely are also conserved, the arginine residue in strand D with aspartic that it could still function in a similar way as DDAH, but have ␻ acid in strand F. Of the four cysteines found in the predicted se- different specificity. Taking into account that N -mono-methyl-L- quence of G6B, the two in strands B and F most likely form a arginine has a therapeutic effect in different inflammatory pro- disulfide bond. One of the other cysteines is localized after the cesses (73–75) and NO production has been found to contribute to Ig-like domain and before the helical transmembrane region, the development of insulin-dependent diabetes mellitus (76, 77), 286 GENES ENCODING THE Ly-6 SUPERFAMILY IN HUMAN MHC examination of the exact function of G6A might add some valu- 4. Robinson, P. J., M. Millrain, J. Antoniou, E. Simpson, and L. Mellor. 1989. A able information to these processes. glycophospholipid anchor is required for Qa-2-mediated T cell activation. Nature 342:85. Among all arginine-using or -producing enzymes only the di- 5. Su, B., G. L. Waneck, R. A. Flavell, and A. L. M. Bothwell. 1991. The glycosyl methylargininase and arginine deiminase enzyme families contain phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation. three motifs in common, which are thought to be involved in func- J. Cell Biol. 12:377. 6. Classon, B. J., and L. Coverdale. 1994. Mouse stem cell antigen SCA-2 is a tionality or in maintaining their structure (55) (Fig. 7). Both fam- member of the Ly-6 family of cell surface proteins. Proc. Natl. Acad. Sci. USA ilies catalyze the hydrolysis of L-arginine into L-citruline, but the 91:5296. 7. McNeil, I., J. Kennedy, D. I. Godfrey, N. A. Jenkins, M. Masciantonio, C. Mineo, arginine deiminase enzyme is found exclusively in prokaryotes. D. J. Gilbert, N. G. Copeland, R. L. Boyd, and A. Zlotnik. 1993. Isolation of a Interestingly, arginine deiminase is known to be involved in cDNA encoding thymic shared antigen-1: a new member of the Ly-6 family with strongly inhibiting cell growth, suppressing IL-2 production and a possible role in T cell development. J. Immunol. 151:6913. 8. Rock, K. L., H. Reiser, J. Bamezai, J. McGrew, and B. Benacerraf. 1989. The IL-2R expression, and inducing apoptotic cell death in T Ly-6 locus: a multigene family encoding phosphatidylinositol anchored mem- lymphoblasts (78). brane proteins concerned with T-cell activation. Immunol. Rev. 111:195. 9. Huppi, K., R. Duncan, and M. Potter. 1988. Myc-1 is centromeric to the linkage The G6 gene group Ly-6-Sis-Gdc-1 on mouse chromosome 15. Immunogenetics 27:215. 10. LeClair, K. P., M. Rabin, M. N. Nesbitt, D. Pravtcheva, F. H. Ruddle, The genomic localization of NCC27 is in the central part of the R. G. E. Palfree, P. M. Flood, U. Hammerling, and A. Bothwell. 1987. Murine MHC class III region and corresponds exactly with the gene de- Ly-6 multigene family is located on chromosome 15. Proc. Natl. Acad. Sci USA scribed in this paper as G6. NCC27 has been demonstrated to be 84:1638. 11. Meruelo, D., A. Rossamando, S. Scandalis, P. D’Eustachio, R. E. D. Fournier, in the nuclear membrane (42). Although the authors describe the D. R. Roop, D. Saxe, C. Blatt, and M. N. Nesbitt. 1987. Assignment of the protein as a chloride ion channel itself, they do not reject the pos- Ly-6-Ril-1-Sis-H-30-Pol-5/Xmmv-72-Ins-3-Krt-1-Int-1-Gdc-1 region to mouse

chromosome 15. Immunogenetics 25:361. Downloaded from sibility of NCC27 acting as a regulatory subunit of a multiprotein 12. Kamiura, S., C. M. Nolan, and D. Meruelo. 1992. Long-range physical map of the chloride ion complex. We think that this second hypothesis might Ly-6 complex: mapping the ly-6 multigene family by field-inversion and two- be more appropriate due to the presence of several PEST se- dimensional gel electrophoresis. Genomics 12:89. 13. Eckhardt, L. A., and L. A. Herzenberg. 1980. Monoclonal antibodies to ThB quences (seven in bovine p64 and two in G6 or NCC27) that could detect close linkage of Ly-6 and a gene regulating ThB expression. Immunoge- act as signals for the rapid degradation of the protein, and the clear netics 20:47. lack of a transmembrane region. Under physiological conditions, 14. Fleming, T. J., M. L. Fleming, and T. R. Malek. 1993. Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow: RB6–8C5 mAb to gran- nuclear localization must be subject to complicated regulatory ulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J. Im- http://www.jimmunol.org/ mechanisms, since the presence of certain proteins in the nucleus munol. 151:2399. is required only at very specific moments in the cell cycle or only 15. Godfrey, D. I., M. Masciantonio, M. A. Tucek, R. Malin, R. L. Boyd, and P. Hugo. 1992. Thymic shared antigen-1: a novel thymocyte marker discrimi- in response to short-lived stimuli. nating immature from mature thymocyte subsets. J. Immunol. 148:2006. It is apparent that many of the genes located in the MHC class 16. Hogarth, P. M., B. A. Houlden, S. E. Latham, V. R. Sutton, and I. F. McKenzie. III region are good candidate genes for susceptibility to diseases 1984. Definition of new alloantigens encoded by genes in the Ly-6 complex. Immunogenetics 20:57. such as insulin-dependent diabetes mellitus or AS due to the pos- 17. Kimura, S., N. Tada, Y. Liu-Lam, and U. Hammerling. 1984. Studies of the sibility of their involvement in the immune and/or inflammatory mouse Ly-6 alloantigen system. II. Complexities of the Ly-6 region. Immunoge- responses. In a recent genome-wide screening for susceptibility netics 20:47.

18. Palfree, R. G., F. J. Dumont, and U. Hammerling. 1986. Ly-6A.2 and Ly-6E.1 by guest on October 2, 2021 loci in AS, markers D6S273 and 82.2, which lie between the sec- molecules are antithetical and identical to MALA-1. Immunogenetics 23:127. ond and third exons of G6D and G6, respectively, achieved the 19. Brakenhoff, R. H., M. Gerretsen, M. C. Knippels, M. van Dijk, H. van Essen, highest LOD scores of all markers tested (45). In the case of D. O. Weghuis, R. J. Sinke, G. B. Snow, and G. A. M. S. van Dongen. 1995. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a marker D6S273, the LOD score obtained was 3.8 ( p ϭ 1.4 ϫ GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion. 10Ϫ5), while the strongest linkage was observed with marker 82.2, J. Cell Biol. 129:1677. ϭ ϫ Ϫ9 20. Capone, M. C., D. M. Gorman, E. P. Ching, and A. Zlontnik. 1996. Identification which achieved a LOD score of 8.1 ( p 1 10 ). Given that through bionformatics of cDNAs encoding human thymic shared ag-1/stem cell some of the genes in the immediate vicinity of these markers could ag-2: a new member of the human Ly-6 family. J. Immunol. 157:969. have putative immune-related functions, it is tantalizing to specu- 21. Kimura, Y., T. Furuhata, T. Urano, K. Hirata, Y. Nakamura, and T. Tokino. 1997. Genomic structure and chromosomal localization of GML (GPI-anchored mole- late that one of them could be a major susceptibility factor in AS. cule-like protein), a gene induced by p53. Genomics 41:477. The evidence generated from the EST databases and RT-PCR has 22. Campbell, R. D., and J. Trowsdale. 1993. Map of human MHC. Immunol. Today also highlighted the complexity of this region, with many of the 14:349. 23. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell genes being differently and specifically expressed. Also, the MHC recognition. Nature 334:395. class III region remains among the most gene-dense regions of the 24. Strachan, T. 1987. Molecular genetics and polymorphism of class I HLA anti- human genome with, on the average, one gene per 10 kb of DNA. gens. Br. Med. Bull. 43:1. 25. Trowsdale, J. 1987. Genetics and polymorphism: class II antigens. Br. Med. Bull. Indeed, the gene density of the 28.1-kb region discussed here is 43:15. now one gene per 5.6 kb of DNA. 26. Carroll, M. C., P. Katzman, E. M. Alicot, B. H. Koller, D. E. Geraghty, H. T. Orr, J. L. Strominger, and T. Spies. 1987. Linkage map of the human major histo- Acknowledgments compatibility complex including the tumor necrosis factor genes. Proc. Natl. Acad. Sci. USA 84:8535. We are grateful to the DNA Sequencing Facility at the Department of 27. Dunham, I., C. A. Sargent, J. Trowsdale, and R. D. Campbell. 1987. Molecular Biochemistry (Oxford, U.K.), to Dr. M. Lovett for the cDNA selection mapping of the histocompatibility complex by pulsed-field gel electrophoresis. Proc. Natl. Acad. Sci. USA 84:7237. reagents, to Dr. Begonã Aguado for providing total RNA samples and 28. Carroll, M. C., R. D. Campbell, D. R. Bentley, and R. R. Porter. 1984. A mo- helpful advice and assistance, and to Dr. R. E. March, Dr. J. Broxholme, lecular map of the human major histocompatibility complex class III region link- Dr. S. Jenkins, Dr. M. Albertella, and K. Browne for helpful discussions. ing complement genes C4, C2 and factor B. Nature 307:237. 29. Spies, T., C. C. Morton, S. A. Nedospasov, W. Fiers, D. Pious, and References J. L. Strominger. 1986. Genes for TNF ␣ and ␤ are linked to the major histocompatibility complex. Proc. Natl. Acad. Sci. USA 83:8699. 1. Shevach, E. M., and P. E. Korty. 1989. Ly-6: a multigene family in search of a 30. Milner, C. M., and R. D. Campbell. 1990. Structure and expression of the three function. Immunol. Today 10:195. MHC-linked HSP70 genes. Immunogenetics 32:242. 2. Williams, A. F. 1991. Emergence of the Ly-6: a multigene family of GPI-an- 31. Sargent, C. A., I. Dunham, and R. D. Campbell. 1989. Identification of multiple chored molecules. Cell Biol. Int. Rep. 15:769. HTF-island associated genes in the human major histocompatibility complex 3. Fleming, T. J., C. O’Huigin, and T. R. Malek. 1993. Characterization of two class III region. EMBO J. 8:2305. novel Ly-6 genes: protein sequence and potential structural similarity to ␣-bun- 32. Benjamin, R., and P. Parham. 1990. Guilt by association: HLA-B27 and anky- garotoxin and other neurotoxins. J. Immunol. 150:5379. losing spondylitis. Immunol. Today 11:137. The Journal of Immunology 287

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