TL are distinct and can co-exist in the same thymus. It is paradoxical that despite the structural similarity between mouse and human CD1, the tissue distribution.
The EMBO Journal vol.7 no.10 pp.3081 -3086, 1988
Mouse CD 1 is distinct from and co-exists with TL in the same thymus
Andrew Bradbury, K.Tertia Belt, Tauro M.Neri, Cesar Milstein and Franco Calabi MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Communicated by C.Milstein
Human CD1 antigens have a similar tissue distribution and overall structure to (mouse) TL. However recent data from human CD1 suggest that the mouse homologue is not TL. Since no human TL has been conclusively demonstrated, we have analysed the murine CD1 genes. Two closely linked genes are found in a tail to tail orientation and the limited polymorphism found shows that, as in humans, the CD1 genes are not linked to the MHC. Both genes are found to be equally transcribed in the thymus, but differentially in other cell types. The expression in liver, especially, does not parallel CD1 in humans. This demonstrates conclusively that CD1 and TL are distinct and can co-exist in the same thymus. It is paradoxical that despite the structural similarity between mouse and human CD1, the tissue distribution of human CD1 is closer to TL. The possibility of a functional convergence between MHC molecules and CD1 is discussed. Key words: lymphocyte differentiation/antigen/MHC/ T cells/thymus
CD1-like genes in the mouse (Calabi and Milstein, 1986). Figure 1 shows a restriction map of the 18 kb region spanned by two mouse genomic clones. Within it, two separate areas are found which hybridize to a human CD1 a3 exon probe, giving restriction enzyme fragment sizes consistent with those found in genomic DNA (data not shown). An -7 kb Sacl fragment from mCD ILl and a 3.5 kb BglII fragment from mCD1L17 (hatched bars in Figure 1) spanning each of the two hybridization-positive areas were subcloned and sequenced (Figure 2). Exons were located by comparison to human CD1, potential splice sites and, in some cases, by RNase protection experiments and analysis of cDNA clones (F.Calabi and A.Bradbury, unpublished observations). The two mouse CD 1 genes are arranged in a tail to tail orientation 6 kb apart and are organized in the same way as the human genes. Thus, leader a 1, a2 and a3 exons are clearly recognizable. Both mouse a1 exons are two codons longer than in humans. The amino acid sequence encoded by the two mouse genes is presented in Figure 3, aligned with human CD1 and mouse TL sequences. The homology between mCDl. 1 and mCD1.2 (Figure 3) consists of an inverted repeat ( > 95% identity) at least 2.1 kb in length, which spans all mapped exons (leader, a 1, a2 and 03) as well as the intervening introns (Figure 2). Within this repeat, a single major difference is found in the a2/a3 intron, where mCD 1. 1 contains 250 bp extra. Mouse CDl genes are as similar to human CD1a, b and c as the latter are to each other. All of them are equally distinct from mouse Tla (Figure 3). A detailed analysis of the pattern of sequence conservation in the a3 exon suggests a significant selection against amino acid replacements. This is shown by comparing the observed number of non-synonymous changes with the number expected on the assumption of random drift (Table I). This strongly implies that mouse CD Is are expressed as poly-
-
-
Introduction CD 1 are human surface antigens that have aroused interest because of their pattern of expression during T cell development. Thus, within the T lymphoid lineage CDI antigens are expressed only on cortical thymocytes and on some lymphoblastoid neoplasias (Bernard et al., 1984). The human CDI gene family consists of five genes, three of which code for the CDla, b and c antigens (Martin et al., 1986, 1987). On the basis of the pattern of tissue expression (cortical thymocytes and Langerhans cells) and of biochemical analysis, the CDI antigens have long been regarded as the human counterpart of mouse TL (Flaherty, 1981; Rowden et al., 1983). However, the recent cloning of the CD 1 genes has enabled us to show that they do not map to the MHC and are but distantly related in sequence to either TL or to any other MHC genes (Calabi and Milstein, 1986; Martin et al., 1986). Since no other human TL-like system has yet been conclusively demonstrated, we decided that it was important to find out whether CDl and TL could coexist in the same thymus and that the best system for this purpose was the mouse.
Results Two
mouse
CD 1 genes
Southern blotting analysis has suggested the existence of two ©IRL Press Limited, Oxford, England
peptides. Mouse CD 1 polymorphism and MHC linkage In order to determine the extent of mCDI polymorphism, Southern blotting analysis was carried out with an a3 probe on eleven strains of laboratory inbred mice (data not shown). Out of 85 strain/enzyme combinations tested, only one polymorphic pattern was found. Thus, both the frequency of mCDl polymorphic variants as well as the extent of polymorphism must be limited. No polymorphism has so far been found in humans (F.Calabi, unpublished observations). That the mouse CDl polymorphism does not segregate with the MHC is shown by comparing the patterns given by C3H, BALB/K and BALB/c (Figure 4). The first two strains have MHC haplotypes of common origin, yet only C3H is positive for the polymorphic variant. This clearly shows that in mouse, like in humans, CD1 does not
3081
A.Bradbury et al. mCDlL1 mCDlL17
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Fig. 1. Map of the 18 kb region containing the mCD1 genes. The positions of the a1, (x2 and (3 exons are shown. Restriction sites are indicated (R: EcoRI; H: HindIII; B: BamHI; X: XbaI; S: Sacl; pl: polylinker) and the hatched bars show the region sequenced. Repetitive elements found within this region are indicated by the open boxes. map to the mouse CD1
MHC and unequivocally demonstrates that and TL co-exist and are genetically unrelated.
Pattern of expression Transcription of mouse CD1 genes was investigated both in whole organs and in in vivo cultured cell lines (Figure 5). A nuclease protection assay with a sequence-specific probe was used to distinguish transcripts from either gene. For comparison, a mouse f32m probe was also used, which detects the expected quantitative differences amongst the different samples (see legend to Figure 5). Highest levels of CD1 transcription were fou.id in the thymus, liver and spleen as well as in WEHI3B (myelomonocytic) and FDCP1/G (myeloid), 18-81 (pre-B) and BW5147 (thymoma). Much lower levels of dubious significance were found in kidney and brain as well as in a diverse range of cell lines including 745 -586 (erythroleukaemia), NSO and J558 (myeloma), JAX NULL1 (teratocarcinoma), EL4 (T cell lymphoma), P815/1.1 (mast cell) and NIH-3T3 (fibroblast) (Figure 5 and data not shown). mCDI.2 transcripts are approximately 5-fold less abundant than those of mCD1.1 in all tissues and cells with the interesting exception of the thymus where the levels appear to be equal for both genes. Since CD1s are predicted to be membrane proteins, if the CD1 mRNA is translationally functional it would be expected to be found mostly on membrane-bound polysomes. To test this, membrane-bound polysomal RNA as well as free cytosolic RNA were prepared from mouse thymus and analysed by Northern blotting with a CD1 probe. The results (Figure 6) clearly suggest that mouse CDI genes are expressed as polypeptides. Two main CD1 RNA species are found, with sizes of 1.4 and 1.6 kb, respectively, similar to the pattern reported for human CDla (Calabi and Milstein, 1986).
Discussion There are two mouse CD1 genes, which are closely linked and in opposite orientation. Within each gene, a 2.1 kb segment shows 107 years ago. The cell type pattern of mouse CD1 gene transcription does not conform to the known tissue expression of human CDI antigens. Whilst CD1 transcription in a myelomonocytic, a myeloid and a pre-B cell line may be of questionable 3082
significance, the presence of mCDl transcripts in liver is surprising. There are three possible explanations: first, as transcription of human CD1a, b and c has so far been assayed mainly in the lymphoid lineage, it remains possible that these genes are also transcribed, but not translated in human liver. In addition there are two human CD 1 genes for which no serological data are available [R3 and R2 (Martin et al., 1986)]. In this context, it is interesting to mention that R3 is the human gene which is most closely related to mouse CD1 (Martin et al., manuscript in preparation). A second explanation is that, at least at some developmental stage, mouse liver may differ from human in the content of specific CD1+ cell types (e.g. thymic precursors and/or dendritic cells). Finally, CD1 may indeed be expressed in different tissues in different species, possibly fulfilling different functions. Precedents for such a situation are known (Crocker et al., 1987; Williams and Gagnon, 1982). On the other hand, it may be very significant that mCD1.2 is essentially thymus-specific on the basis of the ratio of transcripts from the two genes. It is intriguing that, whilst the two mouse CD1 genes are almost identical over all the protein coding and leader exons, as well as the intervening introns, they diverge significantly in the 5' flanking regions, which presumably contain transcriptional control signals. A similar situation has been found in the case of the mitochondrial ATP synthase proteolipid genes (Gay and Walker, 1985) as well as of other nucleus-encoded mitochondrial genes (J.E.Walker, personal communication). Whilst a few mouse antigens have been described which could fit the CD1 transcription pattern, no polypeptide product of the mouse CDl genes is as yet known. The immunoprecipitation of mouse CD8 (Lyt2,3) shows a consistent association with a minor component of 50-55 kd (Jay et al., 1982). This parallels the described CDla/CD8 association in humans (Ledbetter et al., 1985; Snow et al., 1985) and prompts the provocative speculation that the 50-55 kd component is the mouse CDI polypeptide. Sequence inspection does not reveal any feature incompatible with polypeptide expression. The only possible exception is found in mCD1.2, where a conserved cysteine is replaced by a tryptophan. A similar replacement (cysteine to phenylalanine) has been reported in an HLA class I gene (Malissen et al., 1982), the status of expression of which is not known. Whilst the structural requirement for a cysteine in the homologous position of MHC class I molecules is not clear, an immunoglobulin has been found in which one of the invariant cysteines, which are components of the immunoglobulin fold core, is replaced by a tyrosine (Rudikoff and Pumphrey, 1986).
Mouse CD 1 and TL in the
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I D S A T I S F T K P W S Q G K L S N Q Q W E K L Q H M F Q V Y R V S F T R D I Q AC TCAGC CAC CAT CAGC TT CAC GAAGCCATGGTC CCAGGGCAAGTT GAGTAACCAGCAGTGGGAGAAGT TGCAGCATAT GT TTCAAGTCTATC GAGT CAGCT TTACCAGGGACATACAGG 60 0 T
E L V K M M S P K E D AATTAGT CAAAATGATGTCACCTAAAGAAGACTGTGAGTGGAGGGGTTGGAACCCTGAA CCCAGTGGGCAGGAGTG.ATCCAGzACCCCAGCTGGATAACTGTGGGCATCGGGCA
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TGGCTA TTTTCACCCTGGCTCTCCACTTGGA TCCA TCTAGGTTTTTA CA TCACTGTCAGCTGTAGAGGAAGGGCAAGGACTAGGMGAGA.TGAGTAAGGCCTGTGGGGTTTA TTTTCTCA 1320 ------------T C CTGA CAGCCA CTCTTGGGTTCTA TAAATTGAGTTAAA TAAAACTGGGC.ATGAAAACTGAGCCATGCCATA TTTC.AGGCAGGCTGTACCAGCTGAAA TTCAGA TTGTTCCAGAGTA CAGAG 144 0 GA GGGCTA CAGC.AGACTCAGA TCAGGC.AGGGGGGTAGAAGTATCAGGGA TGGTCTTCCTAGCCAGAGGGAC.ACAAA TGGATTAAACTAATAGGCAACTTCATCTCCAGAGGTGGACCTGT 15 60 -------------------------------------------------------------__--------------__-----------------------------------------
ax 3 E K P V A W L S S V GGG,ZA GCA TGAACTTGA TTGC.AGACCTGGAGCCCCTAATATAAAATGTCTTGCTTTAAATTTTT- -TTTCTTA TTCTCTTGATGAGTACA(GAGMGCCAGTGGCCTGGTTGTCCAGTGTC 1 68 0 --~~~ATT L P S S A H G H R Q L V C H V S G F Y P K P V W V M W M R G D Q E Q Q G T H R G D C CCAG CT CTGCACATGGCCATAGGCAGCTGGTGTGTCATGT CT CTGGCTTCTACCCAAAACCTGT GTGGGTGATGTGGATGCG.GGGTGACCAGGAGCAACAGGGTACTCACAGAGGTGAT 180 0 CT
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Fig. 2. Nucleic and predicted amino acid sequence of the mCDl genes. The main sequence is that of mCD 1.1 with the amino acid sequence written above the respective exons. Nucleic and amino acid differences between mCDl1.2 and mCD1. 1 are indicated below and above the line respectively. Dashes indicate gaps introduced to maintain the alignment.
Two strong pieces of evidence suggest that the mouse CD1 genes are not only expressed as polypeptides, but also serve an important function: first, CD1 mRNA is found on membrane-bound polysomes, implying that it is actively translated and that the translation product is targeted to the cell
membrane; second, comparison of replacement versus silent substitution rates between mouse and man reveals the existence of selective pressures to retain the amino acid sequence, particularly notable in the (3 exons and presumably related to the structural constraint to bind , 2m.
3083
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Fig. 3. Comparison of mCD1 sequences to human CD1 and TL Asterisks are used when gaps are required to maintain the alignment and double dashes when residues are identical to mCDl1.1. The position of the amino terminal residue of the mature protein is based on a comparison with rabbit CD1 (Wang et at., 1988). Residues in black are cysteines. In mCD1l.2, the tryptophan at position 76 in the cr2 domain replaces a cysteine, which is conserved in all other CD1s as well as in the corresponding position of MHC class Tax chains, where it is involved in an intradomain disulphide bond (Bjorkman et at., 1987). In the same domain, neither of the mouse CD1s contains a third cysteine, which is conserved in all human CD1s and is postulated to mediate the CD1a/CD8 disuiphide bond. However, an unpaired cysteine is present in both mouse sequences at the beginning of the al domain. Boxed and stippled residues are asparagines which can, potentially, be glycosylated. Five potential N-linked glycosylation sites are found in both mouse CD1s, none of which are in cr3, a feature shared by most CD1 and MHC class I molecules. Only one site is shared with CD1a, b and c, whilst a second one is close to a site present in CDla only and the remaining two are mouse-specific. The consensus, given at the bottom of each domain, corresponds to the amino acid present in at least four of the five genes illustrated. The TL sequence corresponds to Tiab (Klein 1986), which gives the best ALIGN score to mCD1 (Dayhoff et al., 1983). Only the cr3 domain is shown for TL as no significant alignment is possible over the cr1 and cr2 domains.
Table I. Evidence for negative selection in mouse CD1 cr3 domains.
mCDl.1 mCDl.2
CD1b
0
0
E
10 10
17
11 10
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Experiments are currently in progress in our laboratory, using peptides and a fusion protein, in an attempt to raise antisera against the predicted amino acid sequence of mCD1. The most important conclusion of this paper is that CD1
is unequivocally distinct from TL. This conclusion, originally based on sequence comparison and on chromosomal mapping data (Calabi et al., 1986), is now clearly established
3084
Fig. 4. Restriction fragment length polymorphism of mCD1. DNA from the strains indicated was blotted on to Hybond N and probed with an mCD1.2 cr3 probe after digestion with Sacl. BALB/K is an H-2-congenic strain which has the C3H MHC haplotype (H-2k) on a BALB/c background.
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by the finding that mouse CDls are also not MHC encoded and coexist with TL. The mouse CDl family, however, is clearly less complex than that in humans, both in gene number and in the extent of sequence diversity between genes. Now that we have established that there is a CDl homologue in the mouse, the apparent absence of a TL homologue in humans acquires a new significance. It is
mnCD 1.2
paradoxical that in spite of the sequence similarities, human CDl is closer to TL than to mouse CD1 in tissue distribution.
mC D1 .1
If there is no genetic homologue of TL in humans or indeed in other species, it may be concluded that TL is of no functional significance (Rogers, 1985). A more attractive
B
m
NL Fig. 5. RNase protection assays on cytoplasmic RNA from different organs and cell lines. (A) (mCDl) and (B) (132m) represent the sizes of the full length unprotected probes. The mCDl probe was derived from the a3 domain of mCD 1.2 and gives two protected bands corresponding to the two genes, labelled mCDl l and mCDl .2 in the figure. The ,B2m protected fragments are labelled accordingly. The panels showing the mCDl and (82m bands have been exposed for different times in order that the intensities of the bands are similar. Counting the radioactivity in the bands after excising them from the dried-down gels shows that in the organs and cell lines that express mCD1, there is 40-60 times more radioactivity in the ,B2m bands than in the mCDl.I bands. The lines used are derived from the following cell types: BW 5147 and EL4, T cell leukaemia; NSO and J558, myeloma; 18-81, pre-B cell; J774A.1, macrophage; PU5/1.8, monocyte/macrophage; WEHI3B, myelomonocyte (Ralph et al., 1976); FDCP1/G, myeloid (Lang et al., 1985); 745;586, erythroleukaemia (Scher and Friend, 1978); JAX NULLI, teratocarcinoma (Stern et al., 1975); NIH-3T3, fibroblast. The organs were obtained from young BALB/c mice, known to express TLC (Michaelson et al., 1986). It was difficult to obtain RNA from spleen without a small degree of degradation and for this reason we believe that the faintness of the protected bands seen in the spleen lane represent an underestimation.
alternative is that in the mouse the complexity of human CDl functions is subserved by a combination of mouse CD and TL. This may not be as improbable as it sounds if we assume that CD functions have structural requirements close to those of MHC class I and class II antigens. Should this be the case, class I homologues (TL) could in the mouse have convergently evolved to fulfil CDl functions. In other words, although TL is not the genetic homologue of CD1, it may still be a functional homologue. Clearly, it will be interesting to investigate further the reciprocal interactions between the two systems.
Materials and methods Bacterial strains and vectors Escherichia coli strain TG1 was used for routine transformation and subcloning. Ecoli Q358 and Q359 were used for the genomic libraries. Plasmids Bluescript (Stratagene), M13 derivative mp8 and pUC18 and 19 were used for subcloning and sequencing (Bankier et al., 1987). Enzymes and chemicals Restriction enzymes and T4 ligase were purchased from New England Biolabs: Klenow from Boehringer and T3 RNA polymerase from Stratagene. Hybond-N membranes and 32P- and 35S-labelled compounds were obtained from Amersham. Pall Biodyne membranes were obtained from Pall Ultrafine Corp. Standard laboratory reagents were obtained from Sigma and BDH.
Genomic libraries Two libraries were made using standard methods (Maniatis et al., 1982). The first from DNA derived from the thymic cell line BW5147, partially digested with Sau3AI and cloned into the BamHI site of X2001 (Karn et al., 1984). This was screened with the insert from clone FCB6 (Calabi and
3085
A.Bradbury et al. Milstein, 1986) as a probe to yield the clone mCD1L1. The second library was made with DNA from BALB/c liver digested with MboI and cloned into the BamHI site of EMBL3 (Frischauf et al., 1983). This was screened with a subclone from mCDlL1 which spans the c3 domain of MCDl1.1 to yield mCDlL17. Blot hybridization and nucleotide sequence analysis Southern and Northern blots were performed as described by Maniatis et al. (1982). Membrane and free cytosolic cellular RNA were prepared essentially as described in Wall et al. (1977). Sequencing was done using the dideoxy-method after 'shotgun-cloning' (Bankier et al., 1987); at least 2-fold redundancy was achieved on both strands throughout. The DBUTIL, Analyseq, Analysep and Diagon programs (Staden, 1986) as well as the ALIGN program (Dayhoff et al., 1983) were used for sequence analysis.
Probes Nick translated probes were made according to Maniatis et al. (1982). High specific activity hexamer-primed probes were prepared after gel purification of inserts using the Amersham hexamer priming kit. High specific activity RNA probes were made using the Bluescript system, after gel purification of linearized plasmids. RNase protection assays These were performed as described by Zinn et al. (1983) with minor modifications. The probe was prepared as described above and 0.25-0.5 x 106 c.p.m. were hybridized to 50 tg cytoplasmic RNA. Total cytoplasmic RNA was prepared by lysis of cells in NP40 lysis buffer (150 mM NaCl, 10 mM Tris 7.4, 0.5% NP40, 1 mM MgCl2, 10 mM Vanadyl Ribonuclease Complex, Anglia) and one extraction in phenol containing 2% SDS and 0.1% 8-hydroxyquinoline followed by repeated extractions in phenol containing 0.1 % 8-hydroxyquinoline.
Acknowledgements We are deeply grateful to R.Staden for help with computer programs, to J.H.Rogers for a kind gift of mouse DNAs and to P.Kourilsky for providing a mouse ,B2m clone. A.B. was supported by an MRC Research Fellowship, T.B. by a Beit Memorial Research Fellowship, T.M.N. by an EMBO short term fellowship and F.C. by a special fellowship from the Leukemia Society of America.
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Note added in proof Antisera have recently been derived which appear to recognize an mCD1 protein.
