Rat MRC OX-47 activation antigen sequence

Eur. J. Immunol. 1991. 21: 671-679

Sigbjern Fossum.., Susan Mallett. and A. Neil Barclay MRC Cellular Immunology Unit, S i r William Dunn School of Pathology, University of Oxford, Oxford

The MRC OX-47 antigen is a member of the immunoglobulin superfamily with an unusual transmembrane sequence The MRC OX-47 monoclonal antibody recognizes a membrane antigen present at low levels on many lymphocytes but whose expression is markedly increased on activation with mitogens. cDNA clones for the OX-47 antigen were isolated from an expression library and the protein sequence deduced. It contains a leader sequence giving a mature protein of 251 amino acids with a single putative transmembrane region, a cytoplasmic domain of 40 amino acids and an extracellular domain of 187 amino acids that contained two immunoglobulin-like domains.The putative transmembrane sequence includes a glutamic acid residue within the hydrophobic sequence. The presence of acidic residues within the hydrophobic sequence of transmembranesequences usually indicates association with other polypeptides and this is predicted for the OX-47 antigen. A sequence of 37 amino acids that included all the transmembrane region was identical to that of the chicken HT7 antigen present on endothelium in brain and erythroblasts. The level of protein sequence identity in the Ig-like domains was lower but HT7 is almost certainly the chicken homologue of the rat OX-47 antigen.The ligand and function of the molecule are unknown. In addition to lymphoblasts the OX-47 antigen was localized on a variety of other cell types including various immature cells, endothelia and cells with excitable membranes.

1 Introduction The triggering of both T and B lymphocytes by specific antigens or mitogens leads to their transformation from small, metabolically quiescent cells to rapidly dividing metabolically active lymphoblasts. This is accompanied by many changes in the expression of molecules present at the cell surface. These must account for the new properties of the lymphoblast which include increased metabolic rate, altered responses to cytokines, and new homing properties [11. Many cell surface molecules, whose levels of expression increase upon activation have been described [l, 21. An example is the antigen recognized by the MRC OX-47 mAb. This antibody was produced following immunization of mice with rat activated Tcells ([2]; for brevity OX-47 is used to denote the antibody, the antigen recognized and the cDNA clone).The OX-47 antigen qualifies as a lymphocyte activation marker as it was found at high levels on both activated B and Tcells but at low levels on most BM cells and thymocytes and some mature lymphocytes; it was also present on some endothelia and kidney tubules [2]. The OX-47 mAb had no effect upon in vitro Tcell proliferation and failed to give immunoprecipitates from radiolabeled

[I 88921

A

671

Supported by the British Council and the Anders Jahre Foundation. Present address; Institute of Anatomy, University of Oslo, Karl Johans Gate 47, 0162 Oslo 1, Norway. Funded by an MRC research studentship.

Correspondence: A. Neil Barclay, MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3FU3,GB

Abbreviations: IgsF Ig superfamily 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

activated Tcells [2]. In this report we describe the cloning, sequencing and detailed tissue distribution of the OX-47 antigen.

2 Materials and methods 2.1 Isolation of cDNA clones for the OX-47antigen The methods of cDNA library construction were as described for the cloning of the MRC OX-40 antigen and are given in detail in [3]. In brief, RNA was extracted from Con A-activated lymphocytes by the guanidinium isothiocyanate method and a cDNA library prepared in the CDM8 vector [4-61. Purified plasmid from the cDNA library was used to transfect COS cells using DEAE-dextran and cells expressing OX-47 antigen were separated with the OX-47 mAb and magnetic Dynal beads coated with sheep antimouse Ig antibody (Dynal Ltd., Oslo, Norway). Plasmid was recovered by the Hirt procedure from these transfected cells for transformationof E. coli and used to transfect fresh COS cells by spheroplast fusion followed by selection with OX-47 mAb and Dynal beads. After three more rounds of spheroplast fusion and selection with OX-47 mAb, 3 out of 15 cDNA clones selected gave OX-47 expression as analyzed by FCM after transfection of plasmid into COS cells by the DEAE-dextran method. 2.2 Nucleotide sequencing and sequence comparisons

Nucleotide sequences were obtained by subcloning restriction fragments into M13 phage or plasmid vectors and sequenced by the dideoxy chain termination method using a Sequenase sequencing kit (US Biochemical Corporation, Cleveland, OH).The complete sequence was determined in both strands with an average of 3.3 determinations per base. Nucleic acid sequences were analyzed with the 0014-2980/91/0303-0671$3.50+ .25/O

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computer programs of Staden [7]. The NBFG (Protein Identification Resource, National Biomedical Research Foundation, Georgetown, USA) and SWISS-PROT (EMBL Data Library, Heidelberg, FRG) databases were searched using the FASTA program [8] on DEC VAX computers at Oxford University and the Science Research Council, Daresbury, GB. Sequence alignments were with the ALIGN program using the mutation matrix, a bias of 6, a gap penalty of 6 and 100 random alignments for the calculation of standard deviations [9]. The alignments shown were based on the output from the ALIGN program and the MULTALIGN program of Barton and Sternberg [lo].

Eur. J. Immunol. 1991. 21: 671-679

1 2 3 4 5 6

-28s

- 18s

2.3 Immunocytochemistry Unfixed or glutaraldehyde-fixed tissues (0.5% glutaraldehyde in PBS for 10 min at room temperature) were frozen in isopentane cooled in liquid nitrogen. The peroxidase monoclonal anti-peroxidase method was applied on 5-pm thick cryostat sections, which had been fixed in acetone, washed three times in PBS and preincubated for 10 min with normal serum from the species of the linker antibody.The sections were incubated with tissue culture SN containing OX-47 mAb overnight at 4 "C.Thesecondary antibody was a rabbit anti-mouse IgG (raised in the laboratory in Oslo) to which was added 2% rat serum (1 : 10 dilution, 2 h, 4°C). The tertiary layer was a peroxidase mouse monoclonal antiperoxidase complex (Sigma, St. Louis, MO; 1 : 100 dilution, 2 h, 4°C). Peroxidase was demonstrated with 33-diaminobenzidine tetrahydrochloride (Sigma) as substrate.

3 Results and discussion 3.1 Isolation of a cDNA clone for the OX47 antigen

cDNA clones for the OX-47 antigen were isolated from a cDNA library prepared from activated rat T cells in the CDM8 vector by the expression cloning system developed by Seed et al. [4-61. In the final round of cloning 3 plasmids out of 15 tested gave expression of OX-47 antigen on transfection of COS cells. All three positive plasmids contained cDNA inserts of 1.4 kb and sequence analysis showed that they all had identical 5' nucleotide sequences. Northern blot analysis showed that the OX-47 cDNA clone reacted with a mRNA of about 1.7 kb present in liver and activated T lymphocytes with much lower levels on spleen cells and thymocytes (Fig. 1) in accordance with the tissue distribution of OX-47 [2]. The cDNA sequence contained an ATG codon followed by an open reading-frame encoding 308 amino acids (Fig. 2). However, it is probable that a second in-frame ATG is actually utilised as it conforms more closely to the consensus nucleotide sequence found around initiator ATG [ l l ] and also would give a typical leader sequence of 21 amino acids (see also discussion below).This would result in a mature protein of 251 amino acids with a single putative transmembranesequence giving an extracellular domain of 187 amino acids and a cytoplasmic domain of 40 amino acids. The predicted molecular weight of the polypeptide is 27563 but the extracellular domain contains three potential N-linked glycosylation sites (Fig. 2) so the actual M, is probably nearer to 4oOoo.

Figure 1. Distribution of OX-47 mRNA by Northern blot analysis. Samples were electrophoresed on a 1.2% agarose gel in formaldehyde and transferred to Genescreen plus and hybridized with 32P-labeledOX-47 cDNA probe as in [3].The migration positions of 28 S and 18 S RNA are indicated. The numbers above the tracks represent RNA prepared from the following: (1) liver, (2) spleen, (3) Con A-activated LN cells, (4) Con-A-activated thymocytes, (5) LN, (6) thymocytes. A clear band of about 1.7 kb is present in samples from liver and activated Tcells and a faint band in the other samples.

3.2 The OX-47 antigen is a member of the Ig superfamily (IgSF) The extracellular region of the protein predicted from the cDNA could be divided into two domains with sequence similarities to Ig domains. Analysis by the ALIGN program gave good scores against Ig or other Ig-related domains (Table l).The highest scores for both domains tended to be in the CZset of sequences although some good scores were also obtained in the V-set and C1-set sequences.The C2-set of the IgSF is distinguished by having sequence patterns similar to those invdomains but to be closer in size to the Ig C domains, i.e. with a predicted pattern of seven p strands compared to the V domains that have two additional short strands; these extra strands are made up by residues in the middle of the domain sequence (see [12, 131 for discussion of these predictions). Although some good scores were obtained for both OX-47 domains with the C1-set they lack the key residues that are characteristicfor this set (FYP in p strand B and CXVXH in p strand F; see [ 131).The second domain of OX-47 is longer than the first and one would predict that it contained additional p strands to the basic C2-set and is assigned to the V-set. The first domain tends not to score so highly as the second in this analysis but clearly fits the C2-set best. The sequences of the two domains are shown aligned with a selection of other IgSF domains in Fig. 3 together with the predicted positions of the p strands based on the structure of Ig domains [23].The extra sequence in domain 2 is clearly seen and is assigned to p strands C' and C . The overall similarity is typical of IgSF members [12]. One particularly good score was obtained with OX-47 domain 2 and one of the IgSF domains of the twitchin protein from Caenorhabditis elegans that is

Eur. J. Immunol. 1991.21: 671-679

Rat MRC OX-47 activation antigen sequence

673

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440

50

100 150 200 250 300 30 9

involved in myosin contraction [ 171; however, this protein contains 26 IgSFdomains and the scores of OX-47 domain 2 with these other 25 domains varied from 1.7 to 7.6 SD with 8 scores of > 5 SD. Thus the score of 10.1 SD seen with domain 20 is seen as fortuitous rather than implying a special relationship with this protein. Within the sequences it can be seen that some patches of sequence are well conserved such as the motif CXAXNXXG in p strand F that is characteristic of many of the C2 set domains found in brain IgSF molecules and also in many of the twitchin domains [17]. Particularly good scores were also obtained for domains 1 and 2 with the respective domains of the mouse teratocarcinoma antigen gp70 (Table 1 and Fig. 3). The gp70 protein also showed similarities in the transmembrane region (see Sect. 3.3) but not in the cytoplasmic region (not shown). The organization of the Ig-like domains in the OX-47 antigen and the teratocarcinoma antigen gp70, with an NHz-terminal C2-set followed by a V-set domain is unusual because in most other IgSF members that contain both V-set and C2-set domains, the V-set domain is amino terminal to the CZset, e.g. CD2, LFA3, BCM1, MRC

Figure 2. Nucleotide and deduced protein sequence of the OX-47 antigen. (A) shows the OX-47cDNA nucleotide sequence with the two potential initiator ATG, the termination TGA and polyadenylation signal in bold. (B) Protein sequence deduced from cDNA sequence. The second Met in the sequence (boxed) is thought to be the initiator but both are shown here.The probable cleavage site after the leader is indicated with an arrow. The two Ig-related domains are indicated with the solid lines and the potential N-linked glycosylation sites with asterisks. The putative transmembrane sequence is boxed and the glutamic acid residue within this region shown in bold. The nucleotide sequence has been submitted to the EMBLlGenBanklDDJB nucleotide sequence databases under the accession number

x54640.

0 x 2 , carcinoembryonic antigen family, and for proteins withV-set and C1-set, Ig H and L chains and TcR a, p, y and 6 chains [12, 201. There are exceptions, for example the CD4 antigen has a V-like domain at domain 3 but this is thought to have arisen by duplication from a precursor containing a V-set and C2-set domain [24], and the platelet-derived growth factor receptor and related proteins that have four C2 set domains followed by a single V-set domain [12] and the gp70 mentioned above.

3.3 The putative transmembrane sequence of the OX47 antigen contains a glutamic acid residue Proteins that span the lipid bilayer of eukaryotic cells are characterized by containing a sequence of about 20 hydrophobic or other non-polar amino acids. At the cytoplasmic side of the membrane there are usually a group of basic amino acid residues.The OX-47 sequence is unusual in that a glutamic acid residue is predicted within the hydrophobic sequence of the proposed transmembrane segment. Charged residues are rarely found in transmembrane segments apart from in (a) proteins that have multiple

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Eur. J. Immunol. 1991. 21: 671-679

B

A

C

C'

C

.....................

OX-47 D1 OX-47 D2 GP70 D1 GP70 D2 CD4 D1 Ig K a p p a FASC2 D3 TWIT D20 MAG D3 NCAM D2 H L A I1

D ED---[DQTISNG-TEANS-PLETTG-------------NE---TAQVPIDA---HSNE-NQIKILGNQGSFLTKGPSKLND KPGKAPKLLIYDASKLEAGVPS DTARDLT---------------

..................... EPIEQE--------------QEEKBVV-------------

E

D

G

F

OX-47 D1 OX-47 D2 GP70 D1 GP70 D2 CD4 D1 Ig K a p p a FASC2 D3 TWIT D20 MAG D 3 NCAM D2 H L A I1

Figure 3. Alignment of the two Ig-related segments of the OX-47 antigen with other domains from the IgSE Residues identical between either of the two OX-47 domains and at least two of the other sequences are boxed. Dashes indicate gaps introduced to maximize similarities.The domain number indicated after each sequence name refers to the number of the IgSFdomain in the molecule starting at the amino terminus. Sequences are referenced by the NBRFdatabase code in round brackets or literature reference in square brackets. GP70; mouse teratocarcinoma gp70 [21], CD4; human CD4 antigen (RWHUT4). Ig V,-human (KlHURY), FASC2; Fasciclin 11-grasshopper[ 16],Twit,Twitchincytoplasmic protein-Caenorhubdih eleguns [ 171, NCAM; Neural cell adhesion molecule-chicken[ 141, MAG; myelin-associated glycoprotein-rat (BNRT3), MHC 11; MHC class I1 p chain (HLHU3D).

Table 1. ALIGN scores for OX-47 Ig-like domains with other IgSF domainsa) Cl-SET

IgCj.

IgC,

IgGI(1)

OX-d7(I ) OX-47(11)

3.7 3.7

2.1 3.3

4.1 4.4

C2-SET

NCAM(3) AMAL(1)

RR

8

TcR y

82m

MHCI

MHCIIa

MHCIIP

2.9 2.5

5.2 2.1

3.7 3.2

2.7 3.0

5.1 2.5

4.9 5.1

FAS2(2)

MAG(3)

Wit(2O)

LAR(3)

Ll(6)

OX45(2)

GWO(1)

OX-47(1) OX-47(11)

4.5 9.6

4.5 6.0

4.9 7.2

5.9 7.4

4.0 10.1

5.5 6.8

4.2 6.5

5.3 3.8

6.5 1.3

V-SET

IgVx

IgvA

IgVH

lkR a

CD4(1)

CDS a

PIG(1)

LINK

GW0(2)

OX-47(1) OX-47(11)

1.9 4.2

0.8 3.7

2.3 2.4

2.1 5.0

2.2 4.9

1.6 4.7

3.4 5.7

1.6 4.3

2.1 7.3

a) ALIGN scores are given in SDs for alignmentsbetween the two OX-47 extracellulardomains and IgSFdomains. Assuming a normal distribution and no effect of sequence selection, scores of 3.1,4.3,5.2and 6.0 SD indicate probabilities of occurrences by chance of 1 C 5 , lo-', and respectively [9]. In all cases the sequences compared extend from 20 residues before the Cys residue in p strand B to 20 residues after the Cys residue in p strand F [12,13].The sequences are referred by the IgSFdomain number starting from the amino terminus and the data are either from the NBRFdatabase denoted by NBFR code in round bracketsor the original reference in square brackets. C1-SET; I&; Ig h C-region (L2HU), IgC,; Ig x C-region (K3HU), &GI; Ig y , C-region (GHHU),TcR fl; TcR p chain C domain (RWHUCY),TcR y ; TcR y chain C domain (RWMSCI), P2m; 8 2 microglobulin (MGHUB2), MHC I; MHC class I antigen (HLHU12), MHC I1 a; MHC class I1 a chain (HLHUDA), MHC I1 p; MHC class I1 p chain (HLHU3D). C2-SET; NCAM; Neural cell adhesion molecule [ 141, AMAL; Amalgam [ 151, FAS2; Fasciclin2 [ 161, MAG; myelin-associated glycoprotein (BNRT3), Twit; twitchin [17], LAR; LAR glycoprotein [18], L1; LlCAM adhesion molecule [19], OX-45; OX-45 or BCMl antigen (201 GP70; teratocarcinoma gp 70 [21]. V-SET IgV,; Ig x V region (KlHURY), IgVh; Ig h V region (LlMS4E), IgVH; Ig H chain V region (GlHUNM),TcR a;TcR a chain V region (RWMSAV), CD4; Tcell CD4 antigen (RWHUT4), CD8 a; a chain of lymphocyte CD8 antigen [22], PIG; Poly Ig receptor (QRRBG), LINK; Proteoglycan link protein (LKRT2).

Eur. J. Immunol. 1991. 21: 671-679

Rat MRC OX-47 activation antigen sequence

membrane-spanningsegments such as the neurotransmitter receptors and ion transporters (reviewed in [25]) and (b) proteins with a single membrane-spanning region which are often associated with other polypeptides. Fig. 4 shows those single transmembrane-spanningregions that include acidic amino acids such as the CD3 6 , E, y and chains that are associated with the two polypeptide chains of the antigen receptor onTcells, the MB-1 protein that is thought to be associated with a comparable complex on B cells, and the FcyFUII (CD16) that is associated with the chain also found in the CD3 complex.The FcERIis a complex of one a, one p chain and two y chains in which the y and a chains contain an acidic residue in their transmembrane segments; the FceRI y chain is closely related to the CD3 chain (see Fig. 4 and [28]). The mouse teratocarcinoma antigen gp70, which like the OX-47 antigen has two Ig-related domains, has one acidic residue in the transmembrane and shows the closest similarity to the OX-47 antigen with 13 out 21 identities (Fig. 4); however, there is no evidence as yet that this protein associates with other components at the cell surface [21]. The presence of glutamic acid residue in the transmembrane of the neu gene product has a marked functional effect as a single mutation of the valine residue normally at this position to the glutamic acid residue leads to oncogenesis [32]. The molecular basis for this is not known although there is some evidence that the neu oncogene product is more likely to aggregate at the cell surface [33]. It is possible that the glutamic acid residue in OX-47 (and possibly in the other molecules) undergoes post-translational modification as peptide sequencing of the chicken homologue of OX-47 (see below and [34]) failed to give a residue at this position [34]. Many of the transmembrane segments shown in Fig. 4 also have an acidic residue as the first or second charged amino acid on the cytoplasmic side; while this is not true of all the examples shown above, it is not commonly found except for proteins with multiple transmembrane segments, e.g. the acetylcholine receptor [25]. It seems likely that these acidic residues will be important in interactions with other polypeptides in the transmembrane region. The OX-47 transmembrane sequence also contains a proline residue which is uncommon and could disrupt an a helical structure in the membrane. PROTEIN

Extracellular

OX-47

V R S R

Mouse gp70 CD3 DELTA

V V L S

CD3 EPSILON

V E L D M IM D

CD3GAEMA

I E L N

FcERl ALPHA A W L Q FcyRIII CD16 P G Y Q CD3 ZETA L E P X FcERl GAMMA G L P Q nel T K N R NEU S P V T

There is no evidence as to whether the OX-47 antigen is associated with any other components and indeed it has not proven possible to immunoprecipitate the OX-47 antigen with the OX-47 mAb. In the human CD3 and rat FcERI complexes, which contain several polypeptide chains including some with acidic amino acids in their transmembrane segments, reconstitution experiments have shown that the individual components are not expressed at the surface unless all the components are present [26,28].Thus it seems likely that the OX-47 antigen is associated with other polypeptides and that these are presumably expressed on COS cells which allows the cloning system to work for the OX-47 antigen. 3.4 OX-47is the homologue of the chicken endotheliaVerythroblast antigen HT7 and the mouse

gP42 The OX-47 sequence contains a block of 37 residues, including the membrane-spanning region, that is identical to that of the chicken HT7 antigen present on brain endothelium, kidney epithelium and erythroblasts and almost identical (36 out of 37 residues) to that of mouse sequence gp42 (Fig. 5). The cytoplasmic domains are quite similar (rat sequence has 95% and 71% identity with mouse and chicken, respectively). This argues for considerable evolutionary pressure to maintain this sequence and reinforces the idea that OX-47 (and HT7) interact with one or more components in the transmembrane and probably extending either side. The level of similarity in the extracellular domains is less (81% and 39% identity for rat with mouse and chicken, respectively). Ig-related domains often show considerable divergence between species e.g. rat CD8a chain and CD4 are only 42 and 52% similar in their Ig-related segments compared to their human homologues [22, 351 although, some Ig-related domains are highly conserved e.g. domain 5 of NCAM is 98% conserved between chicken and mouse [14,36]. The IgSF domains of HT7 and OX-47 score very highly with each other with the ALIGN programme (13 SD and 14 SD for comparisons of domains 1 and 2, respectively) and in addition there are characteristic patches of sequence identities between the chicken and rodent sequences that are in regions not

Transmembrane

Int racellul ar

C L V M V L L F A V D T G L Y F S V

I P H K Q H K

Y Y I A

R R R K

L A A F L V P P V A

L L A M A

L I F V S F L C L C I F I I

L I T T

W K T S T

P P V V I

F F A A S

L L G T G

G A I I F

I I I V L

V A E V L V L V T I I F I Y L A E V I L L V A I I L L C V T D V I A T L L L A L G V F C F A G I V D I C I T G G L L L L V Y Y W S F A E I V S I F V L A V G V Y F I A G

P S L A V I L F A V D T G L W F S T L L A V

D D E I

G A G G

I I I V

675

L F I Y G V I I T A L Y L L F L Y G I V L T L L Y C I L L F C A V V P G T L L L F L L F L I L V V V V G I L I

K R R K V Y T H

E T G R N D K T A L K R

R G Q N K K R R

K V F I F I W R

A R L R S Q Q Q

Figure 4. Alignment of transmembrane sequences containing acidic residues.T h e putative transmembrane and flanking sequences are shown for cell surface proteins containing a single transmembrane sequence containing either a glutamic acid residue (E)or aspartic acid residue (D) which are shown in bold. Data are from the following sources; h u m a n CD3 6 , E and y chains [26]; mouse CD3 5 chain [27]. FceRl y is the short chain of the rat IgE receptor that is necessary for expression of FceRl a and fl chains [28]. FceRl a is the rat sequence from [29]. FcyRIII (CD16) is the form with a transmembrane sequence found on h u m a n NK cells [30], MB-1 is the B lymphocyte-restricted glycoprotein with similarities to C D 3 [31]. N e u is the transmembrane sequence of the oncogenic form of neu [321.

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Eur. J. Immunol. 1991. 21: 671-679

7

OX-47 MARAL-----LLALAFTFLSGQGACAAAGTIVTSVQEVDSKTQLTCFLNSSGIDIVGHRWMRGGKVLQED-TLPDLQMKYTMA--DDRSGEYS Mgp42 MARAL-----LLALAFTLLSGQGACAAAGTIQTSVQEVNSKTQLTCSLNSSGVDIVGHRWMRGGKVLQED-TLPDLHTKYIVDA--DDRSGEYS HT7 MAAGADVPCAVLALLVLGSLGGDATGPVITGQYSSSADKWLSCNISAPPTLIKGHKWMLGDKVLKTDESDASSYISYTIEGKVEDHSGVYE

***

***

*

*

*

*

* *

* ** ** * *** *

**

* ** *

OX-47 C I F L P E P V G R G N I N V E G P P R I K V G K K S E ~ S E G E F ~ L I C K S ~ S H P P ~ E ~ F K T S D T G D ~ Q T I S N G T ~ S K W I I S T P E L S E L I I S D L D M N D K Mgp42 CIFLPEPVGRSEINVEGPPRIKVGKKSEHSSEGEW(LVCKSDASYPPITDWFWFKTSDTGEEEAITNSTEANGKYVWSTPEKSQLT1SNLDVN HT7 CIYNTNPVAKGNVSIEVEPQWAYKKSEHGNEGDVGVLTCKS-PSYPPWHWAWYKI------YNISRTGNKTELRILKLNIE

**

**

*

*

*****

**

* * * * * **

* *

*

*

* *

*

I I OX-47 VDPGTYVCNATNSQGSARETISLRVRSRLAALWPFLGIVAEVLVLVTIIFIYEKRRKPDQTLDEDDPGAAPLKGSGSHLNDKDKNVRQRNAT Mgp42 VDPGTWCNATNAQGTTRETISLRVRSRMAALWPFLGIVAEVLVLVTIIFIYEKRRKPDQTLDEDDPGAAPLKGSGTHMNDKDKDKNVRQ~AT HT7 QDMGDYSCNGTNMKGSGSATVNLRVRSRLAALWPFLGIVAEVLVLVTIIFIYEKRRKPDEVLDDDDGGSAPLKSNAT--NHKDKNVRQRNAN

** **

**

* ****** . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

** * * * * * * *

* **********

Figure 5. Alignment of sequences of rat OX-47 antigen and homologues in chicken (HT7) and mouse (Mgp42). Residues identical between the three sequences are indicated beneath the alignment. The arrowhead indicates the probable amino terminus of the OX-47 antigen and the known amino terminusof the HTI antigen.Theputative transmembrane region is overlined and the glutamic acid residues in this region are shown in bold. Data for the chicken and mouse sequences are from [34, 421.

typically conserved between IgSF domains. For example compare the sequence similarities between OX-47, HT7 and mouse gp42 in beta strands A to C in domain 2 with comparable regions in other IgSF domains (Fig. 3). This argues for a close relationship between these three sequences and taken together with the sequence similarities in the rest of the protein, indicates that the HT7 antigen is the chicken homologue of OX-47 and not just a related gene. The teratocarcinoma gp70 also showed similarities to both OX-47 (Table 1 and Sect. 3.3 and 3.4) and HT7 [34] but these were less than between HT7 and OX-47. One notable difference between the chicken and rodent sequences is a deletion in the HT7 sequence (the sequence TEANSK in OX-47, see Fig. 5 ) , that correspondsclosely to the proposed beta strand C in domain 2.The HT7 domain 2 would not have sufficient sequence for assignment to the V-set and thus this domain would be assigned to the C2-set as proposed by Seulberger et al. [34].These assignments are slightly arbitrary as there are considerable differences in the lengths of Ig-related domains in the C2-set and V-set and the interpretations will become clearer when the structures of members of these sets of Ig-related sequences become known. Both OX-47 and HT7 nucleotide sequences are unusual in having two potential, in frame, ATG sites for initiation, of which the second is presumed to be used as it conforms closer to the consensus sequence [ll]. The amino terminus has been established for the HT7 antigen (Fig. 5) but that for the OX-47 is predicted from the analysis of cleavage positions by von Heijne [37].

3.5 Tissue distribution of the OX-47antigen In a previous study OX-47 was shown to be present at low levels on most thymocytes and BM cells with lower levels on thoracic duct lymphocytes, but to increase markedly upon activation of both B and T cells [2]. On tissue sections OX-47 was found on endothelium on brain capillaries, kidney tubules and was also present on muscle and heart [2]. Fig. 6 shows the localization of the OX-47 antigen by indirect immunoperoxidase staining of tissue sections. In accordance with these previous findings, peripheral lym-

phoid tissues were weakly stained with only a few scattered weakly positive cells, usually along the rim of germinal centers (Fig. 6 b). Cortical thymocytes were uniformly stained with intermediate staining intensity but medullary thymocytes were much weaker (Fig. 6a). In contrast, a variety of cell types in non-lymphoid tissues were strongly stained, only some of which will be described here (see also Fig. 6). Various epithelial cells exhibited strong staining for OX-47 antigen. In polarcells, the antigen is usually present only on the basolateral membrane e.g. cells from distal tubules and the descending loop of Henle in the kidney and the salivary glands (Fig. 6d). In the kidney the basolateral membranes of cells from distal tubules were labeled with intermediate intensity. In the proximal parts of collecting tubules only scattered cells were labeled, whereas in the medullary parts, all cells were labeled (Fig. 6 c and g).The epithelium of the small intestine was stained both along the basolateral surface and on the apical part of the cells (Fig. 6e). In the liver, OX-47 mAb labeled the sinusoids (Fig. 6f). The basolateral parts of the hepatocytes were clearly labeled, as the staining was seen to follow the sides where the hepatocytes abutted on each other, away from the sinusoids, but did not reach the central part, forming the bile canaliculi (Fig. 6f).Whether the sinusendothelium wasalso stained was difficult to judge from the frozen sections, but in the central venules endothelial cells were seen to be labeled only weakly or not at all. The basal layers of the epidermis were stained with moderate intensity, whereas hair follicles were strongly stained. Various non-epithelial cells were labeled, in particular muscle and nerve cells.The surface membranes of striated skeletal and heart muscle fibers were relatively strongly stained (Fig. 6 i). Most neural cells in the forebrain and cerebellum were also stained (Fig. 6 h). The endothelial cells of brain capillaries were strongly OX-47+, as were the choroid plexus and the perineural cells surrounding peripheral nerves (Fig. 6 h). Follicular cells in the ovaries and Leydig cells in the testis were weakly to moderately OX-47+. In the testis, the convoluted seminiferous tubules showed a striking staining pattern: in the basal layer of some segments the seminiferous cells were strongly stained. The poor nuclear morphology did not permit accurate identification of the matura-

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Figure 6. Localization of OX-47 antigen on tissue sections by the immunoperoxidase technique. a) Thymus,with capsule (cap) to the left, cortex in the centre and medulla (med) to the right (Bar 50 pm). b) LN, with a follicle (foll) surrounded by a few weakly labeled cells, whereas the paracortex (par) is completely unstained (Bar 100 pm). c) and g) Kidney cortex and medulla. Note unlabeled glomeruli (gl) and weakly stained medullary rays (mr); in (g) the upper part represents the proximal parts of the medulla. d) Submandibular gland-note the strongly stained salivary ducts. e) Lower part of the intestinal mucosa with parts of villa (top), intestinal crypts (middle) and muscularis mucosa (mm) (bottom). Note strong staining of smooth muscles. f) Hepatic lobule with central venule in the centre. It can be seen that the staining of the hepatocytes covers the basolateral membrane, whereas the apical membrane, forming the biliary canaliculi is unstained (arrowheads). h) Cerebellum, ml-molecularlayer, pc-layer of Purkinje cells, gl-granularlayer and wm-white matter. Note distinct staining of the capillary endothelium (arrow). i) Skeletal muscle fibers. Note striped staining of the surface membrane. j) Seminiferous tubules. Note staining of basal cells, probably spermatogonia B, in some tubules and not in others. Interstitial (Leydig) cells are also stained. [Bar in lower left of (c) represents 100 pm in (c), (g) and (j).50 Km in (d), (e), (f) and (h) and 25 pm in (i)].

tional stages of these cells, but their location suggests that they are type B spermatogonia, perhaps also early stages of primary spermatocytes. Later stages of primary spermatocytes were weakly labeled, as were secondary spermatocytes and spermatids, whereas spermatozoa were strongly labeled (Fig. 6j).

The range of cells type examined which were positive for the OX-47 antigen illustrates the wide tissue distribution of this antigen which is broader than that of the chicken homologue, the HT7 antigen. This is present on the endothelium in the brain, where it has been proposed to be involved in cell recognition in the blood brain barrier,

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S. Fossum, S. Mallett and A. N. Barclay

erythroblasts, pinealocytes, epithelium of kidney tubules, Received September 6, 1990; in revised form November 27, choroid plexus, retinal pigment layer and neurons [34].The 1990. HT7 antigen has recently been shown to be identical to neurothelin characterized as an inducible cell surface 5 References glycoprotein of blood brain barrier-specific endothelium and some neurons [34,38].The mouse gp42 distribution has Weiss, A . and Imboden, J. B., Adv. Immunol. 1987. 41: 1. been analyzed at the RNA level and is present on several Paterson, D. J., Jefferies,W. A., Green, J. R., Brandon, M. R., tissues including brain, and lymphoid cell lines but surprisCorthesy, P., Puklavec, M. and Williams, A . E, Mol. Immunol. ingly is absent from liver where rat OX-47 is abundant (Figs. 1987. 24: 1281. 1 and 6). The tissue distribution of OX-47 is also similar to Mallett, S., Fossum, S. and Barclay, A . N., EMBO J. 1990. 9: 1063. that of the MRC OX2 antigen in that it is present at low Aruffo, A . and Seed, B., Proc. Natl. Acad. Sci. USA 1987.84: levels on thymocytes and B cells but increases on activation 8573. [2, 391 and is also present on a variety of other cell types Seed, B. and Aruffo, A . , Proc. Natl. Acad. Sci. USA 1987.84: including endothelia, neurons [39] and cells in the repro3365. ductive organs [40].The MRC OX2 antigen is also similar in Seed, B., Nature 1987. 329: 840. overall molecular features, with an extracellular domain Staden, R., Nucl. Acids Res. 1986. 14: 217. consisting of two IgSF domains and a single transmembrane Pearson,W. R. and Lipman, D. J., Proc. Natl. Acad. Sci. USA sequence that is also well conserved between human and rat 1988. 85: 2444. Dayhoff, M. O., Barker, W. C. and Hunt, L. T., Methods. (96% identical) although it does not contain any acidic Enzvmol. 1983. 91: 524. residues [41]. Like the OX-47 antigen, the function of the 10 Barion, G. J. and Sternberg, M. J. E., J. Mol. Biol. 1987.198: MRC OX2 antigen is unknown.

4 Concluding remarks The presence of IgSF domains in the OX-47 antigen suggests that it could react with other cell surface molecules or soluble factors such as cytokines [12]. As discussed above, the presence of a charged amino acid residue in the transmembrane sequence and its remarkablystrong conservation between chicken and rats argues for an important role for this region and interactions with other components within the membrane. The ligand(s) for this molecule as well as its function are at present unknown and can only be a matter of speculation. Seulberger et al. [34] have suggested that the avian homologue HT7 is a receptor involved in cell surface recognition at the blood brain barrier. The wider tissue distribution of OX-47 points to a more far-reaching role. The cell types expressing the OX-47 antigen can be divided into four main groups: (a) immature, activated and dividing cells (BM cells, cortical thymocytes, lymphoblasts, spermatogonia B; basal layers of epidermis and hair follicles may also be included in this group, (b) epithelial cells involved in transepithelial transport, e.g. nephronic tubular cells, salivary duct cells, hepatocytes, intestinal absorptive cells, brain endothelium, choroid plexus, perineural cells, (c) cells with excitable membranes, e.g. nerve and muscle cells and (d) other cells including endocrine cells such as Leydig cells and ovarian follicular cells. It is likely that the OX-47 antigen is part of a complex of surface proteins and it is possible that the other components vary between the different cell types. Even with this reservation, it is difficult to envisage what these cells have in common to account for the shared marker.The similarities to the CD3 and FcR complexes suggest that the OX-47 antigen might have a comparable signalling role. The identification of the ligands of OX-47 antigen and associated proteins is therefore a prerequisite to an understanding of the function of the OX-47 antigen. We are grateful to H . Seulberger, F: Lottspeich and W Risau for providing the H T 7 data prior to publication and to Alan Williams for valuable discussions.

327. 11 Kozak, M., Nucl. Acids Res. 1987. 15: 8125. 12 Williams, A. F. and Barclay, A . N.,Annu. Rev. Immunol. 1988. 6: 381. 13 Williams, A . F., Immunol. Today 1987. 8: 298. 14 Cunningham, B. A . , Hemperly, J. J., Murray, B. A., Prediger, E. A.. Brackenburv. R. and Edelman. G. M.. Science 1987. 236: 7 b . 15 Seeger, M. A., Haffley, L. and Kaufman,T. C., Cell 1988. 55: 589. 16 Harrelson, A. L. and Goodman, C. S., Science 1988. 242: 700. 17 Benian, G. M., Kiff, J. E., Neckelmann, N., Moerman, D. G. and Waterson, R. H . , Nature 1989. 342: 45. 18 Streuli, M., Krueger, N. X., Hall, L. R., Schlossman, S. F. and Saito, H., J. Exp. Med. 1988. 168: 1523. 19 Moos, M., Tacke, R., Scherer, H . , Teplow, D., Friih, K. and Schachner, M., Nature 1988. 334: 701. 20 Killeen, N., Moessner, R., Arvieux, J. ,Willis, A. and Williams, A. F., EMBO. J. 1988. 7: 3087. 21 Ozawa, M., Huang, R.-P., Furukawa,M. and Muramatsu,T.,J. Biol. Chem. 1988. 263: 3059. 22 Johnson, F!, Gagnon, J., Barclay, A. N. and Williams, A . F., EMBO. J. 1985. 4: 2539. 23 Amzel, L. M. and Poljak, R. J., Annu. Rev. Biochem. 1979.48: 961. 24 Williams, A. F., Davis, S. J., He, Q. and Barclay, A. N., Cold Spring Harbor Symp. Quant. Biol. 1989. 54: 637. 25 Betz, H . , Biochemistry 1990. 29: 3591. 26 Clevers, H., Alarcon, B.,Wileman, T. and Terhorst, C., Annu. Rev. Immunol. 1988. 6: 629. 27 Weissman, A . M., Baniyash, M., Hou, D., Samelson, L. E., Burgess, W. H. and Klausner, R. D., Science 1988. 239: 1018. 28 Blank, U., Ra, C., Miller, L.,White, K., Metzger, H. and Kinet, J.-F!, Nature 1989. 337: 187. 29 Kinet, J. F!, Metzger, H., Hakimi, J. and Kochan, J., Biochemistry 1987. 26: 4605. 30 Ravetch, J.V. and Perussia, B., J. Exp. Med. 1989. 170: 481. 31 Sakaguchi, N., Kashiwamura, S.-I., Kimoto, M.,Thalmann, P. and Melchers, F., EMBO J. 1988. 7: 3457. 32 Bargmann, C. I., Hung, M.-C. and Weinberg, R. A . , Cell 1986. 4s: 649. 33 Weiner, D. B., Liu, J., Cohen, J. A.,Williams,W.V.and Greene, M. I., Nature 1989. 339: 230. 34 Seulberger,H . , Lottspeich, F. and Risau,W., EMBOJ. 1990.9: 2151. 35 Clark, S. J., Jefferies, W. A., Barclay, A. N.,Gagnon, J. and Williams, A. F., Proc. Natl. Acad. Sci. USA 1987. 84: 1649. ,I

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36 Barbas, J. A . , Chaix, J. C., Steinmetz, M. and Goridis, C., EMBO. J. 1988. 7: 625. 37 Von Heijne, G., Nucleic Acids Res. 1986. 14: 4683. 38 Schlosshauer, B. and Herzog, K. H., J. Cell Biol. 1990. 110: 1261. 39 Webb, M. and Barclay, A. N., J. Neurochem. 1984. 43: 1061.

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40 Bukovsky, A . , Presl, J. and Zidovsky, J., Immunology 1984.52: 631. 41 McCaughan, G. W., Clark, M. J. and Barclay, A . N., Immunogenetics 1987. 25: 329. 42 Altruda, F., Cervella, F?, Gaeta, M. L., Daniele, A., Giancotti, E,Tarone, G., Stefanuto, G . and Silengo, L., Gene 1989. 85: 445.

The MRC OX-47 antigen is a member of the immunoglobulin superfamily with an unusual transmembrane sequence.

The MRC OX-47 monoclonal antibody recognizes a membrane antigen present at low levels on many lymphocytes but whose expression is markedly increased o...
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