lllllllUtlO-

lmmunogenetics 33: 113-117, 1991

genetics

© Springer-Verlag 1991

The isolation of cDNA clones for CD48 Hilary A. Vaughan, Margaret M. Henning, Damian F. J. Purcell, Ian F. C. McKenzie, and Mauro S. Sandrin Research Centre for Cancer and Transplantation, Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia Received August 14, 1990; revised version received September 18, 1990

Abstract. HuLy-m3 is an M~ 47 000 pan-leukocyte antigen detected by the monoclonal antibody (mAb) 5-4.8. This report describes the isolation and analysis of a cDNA clone encoding HuLy-m3. Serological analysis demonstrated that antibodies of the CD48 cluster also reacted with transfected cells expressing HuLy-m3. The DNA sequence of the clone suggests linkage to the cell membrane through a glycosyl phosphatidylinositol tail and this was verified experimentally. Sequence similarity with the human B-cell activation antigen Blast-1 was noted.

Introduction We have previously described the HuLy-m3 antigen as a pan-leukocyte antigen detected by the 5-4.8 monoclonal antibody (mAb; Vaughan et al. 1983). This M r 47 000 glycoprotein is present on all peripheral blood T, B, and null cells and also on thymocytes, spleen cells, and - 3 0 % of bone marrow cells. In this report we describe the cloning and sequencing of the corresponding cDNA, which indicates nucleotide sequence similarity with the B-cell activation antigen Blast-1 (Staunton and Thorley-Lawson 1987); the latter was however, demonstrated to be absent from resting lymphocytes. In addition, transfected cells reacting with 5-4.8 (HuLy-m3) also react with two antibodies which cluster with CD48 and thus identify the cDNA isolated as coding for CD48.

Materials and methods MAbs. The mAb 5-4.8 was described elsewhere (Vaughan et al. 1983). Other mAbs produced in our laboratory were: HuLy-m2 (CD7; Thurlow et al. 1984), used as an isotype control for HuLy-m3; HUNK2 (CD16; The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 59904. Offprint requests to: I. F. C. McKenzie.

reported in Knapp 1989); HuLy-m4 (CD45; Sparrow and McKenzie 1983); a monomorphic anti-HLA class I (Sparrow 1983). Two samples of the Blast-1 mAb (Staunton and Thorley-Lawson 1987) from the Fourth International Workshop on Human Leukocyte Differentiation Antigens were obtained from G. Pilkington (Peter MaCallum Cancer Institute, Melbourne, Australia) and H. Zola (Flinders Medical Centre, Adelaide, Australia). The antibodies WM63 and WM68, which served to define the CD48 cluster in the Fourth Leukocyte Workshop (Knapp 1989), and WM66 (Henniker et al. 1990) were kindly provided by K. Bradstock (Westmead Medical School, Sydney, Australia). The WM65 pan-leukocyte antibody (Henniker et al. 1989) was obtained from K. Atkiuson (Westmead Medical School, Sydney, Australia) and two CD45 antibodies, CMRF12 and CMRF26 (Starling et al. 1987), from D. Hart (Christchurch Hospital, Christchurch, New Zealand). The CD32 antibody CIKM5 (reported in Knapp 1989) was obtained from G. Pilkington (Peter MaCallum Cancer Institute, Melbourne, Australia). Cells and cell lines. Peripheral blood lymphocytes (PBLs) and granulocytes were prepared as described (Vaughan et al. 1983). COS cells and the cell lines Daudi, U937, CEM, and K562 were maintained in fully supplemented Dulbecco's modified Eagle's Medium (DMEM). cDNA cloning and characterization, cDNA libraries prepared in the CDM8 vector from the JY and Daudi cell lines were a gift from B. Seed (Massachusetts General Hospital, Boston, Massachusetts). DNA prepared from these libraries was transfected into COS cells and expression cloning performed (Seed and Aruffo 1987; Aruffo and Seed 1987). After four cycles of selection using the 5-4.8 mAb and panning, DNA was prepared from 24 single colonies and used for transfection studies. Transfection of COS cells with cloned DNA was by diethylaminoethanol-dextran (Seed and Aruffo 1987); 48-72 h post-transfection the cells were examined for cell surface expression using mAbs and either indirect immunofluorescence or rosetting using sheep anti-mouse immunoglobulin-coated sheep red blood cells (Parish and McKenzie 1978). The HuLy-m3 cDNA insert was sequenced directly in the CDM8 vector. Both double- and single-stranded DNA, produced by transforming XS127 bacteria and superinfecting the resultant transformed bacteria with the VCSm13 helper phage, were sequenced using the Sequenase protocol (US Biochemicals, Cleveland, Ohio). RNA production and northern blots. Cytoplasmic RNA was prepared by a modification of the NP40 lysis technique (Gilman 1989). Total RNA was electrophoresed in a 1% agarose gel containing formamide, the gel was blotted onto nylon membranes, and the membranes were probed with a random-primed HuLy-m3 cDNA insert. The final wash was in 0.2 x standard sodium citrate/0.1% sodium dodeeyl sulfate (SDS) at 42 °C.

114

H.A. Vaughan et al.: Cloning of CD48

Immunoprecipitations and phospholipase C treatment. Cell surface

labeling using ~25I and lactoperoxidase, immunoprecipitation, and analysis on SDS-polyacrylamidegel electrophoresis (PAGE) were performed (Goding 1980). For phospholipase studies, radiolabeled human PBLs were treated with either 5 units phosphatidylinositol-specific phospholipase C (PI-PLC; kind gift of P. Robinson, Mill Hill, UK) or phosphate-buffered saline for 60 min at 37 °C before lysis. Mitogen activation. Phytohaemagglutinin(PHA) at a final concentration

of 2.5 gg/ml or concanavalin A (Con A) at a final concentration of 50 gg/ml was added to 107 PBLs in DMEM supplemented with 10% antologous human serum. After 72 h at 37 °C in 10% CO2 the cells were harvested and tested for the expressionof HuLy-m3and compared to non-activated PBLs.

Results Twenty-three HuLy-m3 cDNA clones of apparently identical size [1.1 kilobases (kb)] were isolated using the Brian Seed COS cell expression system. Serological analysis of COS cells transfected with one of these clones (pHuLym3.7) demonstrated that the c D N A clone encoded a protein that was recognized by the 5-4.8 mAb but not by several other mAbs (Table 1). The complete nucleotide sequence of the clone is shown in Figure 1. The insert of 1137 base pairs (bp) contains a single open-reading frame of 729 bp encoding a protein of 24 837d. At the 3' end there is an 11 bp poly-A tail and the polyadenylation consensus sequence (AATAAA) is located 13 bp upstream of this tail. A homology search with the DNA sequence in the nucleic acid databases (EMBL/GenBank) detected almost complete identity with the Blast-1 antigen over the entire coding region. In this region there were only two nucleotide differences in the sequence of pHuLy-m3.7

Table 1. Serological analysis of pHuLy-m3.7-transfectedCOS cells. Cluster

Antibody

Results

HuLy-m3

++++

CD48

WM68 WM63

++++ ++++

CD45

CMRF26 CMRF12 HuLy-m4

-

CD7

HuLy-m2

-

CD16

HUNK-2

-

CD32

CIKM5

-

Unclustered

WM65 WM66 Blast-1

-

1620

-

HLA class I

+ + + +, strong cell membrane fluorescence on 40%-50% of cells. - , no fluorescence detected above background.

compared with Blast-l: at position 214, which had a T instead of an A, and position 641, which had a C instead of a G. These differences are discussed below. Northern blot analysis using the HuLy-m3 c D N A insert (Fig. 2) revealed a 1.2 kb band detected on R N A from the cell line U937. Identical bands were observed with RNA prepared from PBLs and on R N A from the cell lines Dandi and CEM but not from K562 (data not shown). These results are consistent with our previously described serological findings (Vanghan et al. 1983), but not with the published tissue distribution of Blast-1 (ThorleyLawson et al. 1982). Southern blot analysis of Bgl IIdigested human genomic D N A gave identical results to those described for Blast-1 (Fisher et al. 1989; data not shown). HuLy-m3 was precipitated from the surface of COS cells transfected with the pHuLy-m3 cDNA, and had an apparent relative mass of 47 000. Similar molecules were also precipitated from the surface of PBLs, but not from nontransfected COS cells (Fig. 3A). The mAb to Blast-1 did not immunoprecipitate any molecules from PBLs, from the HuLy-m3 transfectants, or from the cell lines. The HuLy-m3 + molecules on both transfected COS cells (data not shown) and PBLs (Fig. 3B) were shown to be sensitive to phospholipase C treatment, indicating their linkage to the cell surface by a glycosyl phosphatidylinositol tail, as reported for the Blast-1 antigen (Staunton et al. 1989). COS cells transfected with HuLy-m3 c D N A were tested by both immunofluorescence and rosetting with a number of antibodies, where activity was directed against leukocyte antigens which reacted with molecules of M r 40 000-50 000. WM65 was nonreactive, but antibodies WM63 and WM68 were strongly positive on the HuLym3 + transfectant. These antibodies, which recognise different epitopes, are part of the CD48 cluster (Knapp 1989). Thus HuLy-m3, Blast-l, WM63, and WM68 are part of the same cluster group, CD48, the cDNA clones of which are therefore described herein. As Blast-1 has been described as an activation antigen, we examined the effects of blast cell transformation on the expression of HuLy-m3. Activation with either PHA or Con A resulted in the increased expression of HuLy-m3 (Fig. 4), suggesting that HuLy-m3 is upregulated by activation.

Discussion We describe the isolation of c D N A clones encoding HuLy-m3 by use of a high-efficiency eukaryotic expression system. Upon transfection with one of these clones (pHuLy-m3.7) into COS cells, there was a reaction with the 5-4.8 (HuLy-m3) antibody and with two antibodies defining the CD48 cluster (WM63 and WM68; Table 1).

H.A. Vaughan et ai.: Clomng of CD48 M ATG

CTGTGAAAGAAGGAAGC

W TGG

115 S TCC

R AGA

G GGT

W TGG

D GAT

I ATT

+i IQ CAA

S TCG

C TGT

L CTG

A GCT

L CTG

E GAA

L TTG

L CTA 62

L CTG

LCTG

P CCT

L CTG

S TCA

L CTC

L CTG

V GTG

T ACC

S AGC

G GGT

H CAC

L TTG

V GTA

H CAT

M ATG

T ACC

V GTG 122

V

S

G

S

N

V

T

L

N

I

S

E

S

L

P

E

N

Y

K

Q

GTC

TCC

GGC

AGC

AAC

GTG

ACT

CTG

AAC

ATC

TCT

GAG

AGC

CTG

CCT

GAG

AAC

TAC

AAA

CAA 182

L CTA

T ACC

W TGG

F TTT

Y TAT

T ACT

F TTC

D GAC

Q CAG

K AAG

I ATT

V GTA

E GAA

W TGG

D GAT

S TCC

R AGA

K AAA

S TCT

K AAG 242

Y TAC

F TTT

E GAA

S TCC

K AAA

F TTT

K AAA

G GGC

R AGG

V GTC

R AGA

L CTT

D GAT

P CCT

Q CAG

S AGT

G GGC

A GCA

L CTG

Y TAC 302

I

S

K

V

Q

K

E

D

N

S

T

Y

I

M

R

V

L

K

K

T

ATC

TCT

AAG

GTC

CAG

AAA

GAG

GAC

AAC

AGC

ACC

TAC

ATC

ATG

AGG

GTG

TTG

AAA

AAG

ACT 362

G GGG

N AAT

E GAG

Q CAA

E GAA

W TGG

K AAG

I ATC

K AAG

L CTG

Q CAA

V GTG

L CTT

D GAC

P CCT

V GTA

P CCC

K AAG

P CCT

V GTC 422

I ATC

K AAA

I ATT

E GAG

K AAG

I ATA

E GAA

D GAC

M ATG

D GAT

D GAC

N AAC

C TGT

Y TAT

L CTG

K AAA

L CTG

S TCA

C TGT

V GTG 482

I

P

G

E

S

V

N

Y

T

W

Y

G

D

K

R

P

F

P

K

E

ATA

CCT

GGC

GAG

TCT

GTA

AAC

TAC

ACC

TGG

TAT

GGG

GAC

AAA

AGG

CCC

TTC

CCA

AAG

GAG 542

L

Q

N

S

V

L

E

T

T

L

M

P

H

N

Y

S

R

C

Y

T

CTC

CAG

AAC

AGT

GTG

CTT

GAA

ACC

ACC

CTT

ATG

CCA

CAT

AAT

TAC

TCC

AGG

TGT

TAT

ACT 602

C TGC

Q CAA

V GTC

S AGC

N AAT

S TCT

V GTG

S AGC

S AGC

K AAG

N AAT

G GGC

T ACC

V GTC

C TGC

L CTC

S AGT

P CCA

P CCC

C TGT 662

T ACC

L CTG

A GCC

R CGG

S TCC

F TTT

G GGA

V GTA

E GAA

W TGG

I ATT

A GCA

S AGT

W TGG

L CTA

V GTG

V GTC

T ACG

V GTG

P CCC 722

T ACC

I ATT

L CTT

G GGC

L CTG

L TTA

L CTT

T ACC

*** TGA

GATGAGCTCTTTTAACTCAAGCGAAACTTCAAGGCCAGAAGAT 792

CTTGCCTGTTGGTGATCATGCTCCTCAGCAGGACAGAGACTGTATAGGCTGACCAGAAGCATGCTGCTGAATTATCAAC 871 GAGGATTTTCAAGTTAACTTTTAAATACTGGTTATTATTTAATTTTATATCCCTTTGTTGTTTTGTAGTACACAGAGAT 950 TATAGAGATACACATGCTTTTTTCCCAAAATTGTGACAACATTATGTGGAATCTTTTATTATTTTTAAAATAAAAAGAT 1029 ATAATTATAAAAAAAAAAA 1037

Fig. 1. Nucleotide sequence of

pHuLy-m3.7. Underlined

amino acids w e potential N-linked glycosylation sites; *** is the termination codon.

Thus, the pHuLy-m3.7 clone must encode CD48 + molecules. The cDNA sequence contains 1137 nucleotides, with the largest open-reading frame encoding a 243 amino acid

protein (Fig. 1). Comparison of the nucleotide sequence in several databases indicated it to be identical (but for two nucleotides) with that of Blast-1 (Staunton and Thorley-Lawson 1987). Thus, we consider that H u L y -

116

Fig. 2. Northern analysis of U937 poly-A RNA with pHuLym3.7 cDNA.

m3, WM63, WM68, and Blast-1 are all members of the CD48 cluster. Both HuLy-m3 and Blast-1 mAbs were used in the Fourth Leukocyte Workshop but neither clustered with CD48 or with the other. Blast-1 has been extensively described as an antibody nonreactive with resting PBLs, and indeed is considered to be a B cell-specific activation marker, being found on Epstein-Barr virus-transformed cells and more recently on some activated T cells. In the Fourth Workshop, as the antibody was considered to be B cell-specific, it was compared with other B cell-specific antibodies and did not cluster with any of these. It is clear from evidence from the original laboratory, from the Fourth Workshop, and in our hands (Fig. 3A) that Blast-1 is nonreactive with PBLs. This is a major difference between Blast-1 and the three antibodies defining the CD48 cluster and can be ascribed to two possibilities: that Blast-1 and CD48 are expressed on different molecules, or that Blast-1 and CD48 are expressed on the same molecules, but the difference in tissue distribution is due

H.A. Vaughan et al.: Cloning of CD48 to either antibody affinity or to differential epitope expression. It is highly unlikely that CD48 and Blast-1 are on different molecules. The sequences obtained for HuLy-m3 and Blast-1 cDNA are identical but for three minor discrepancies, two of which are single nucleotide differences within the coding region; a difference was also noted in the 5' untranslated region. The first nucleotide difference is at position 214, which has a T in HuLy-m3 and an A in Blast-1 giving rise to the codon ATT for HuLy-m3 (coding for isoleucine) and A A T for Blast-1 (coding for asparagine). It should be noted that in the original Blast-1 study (Staunton and Thorley-Lawson 1987) isoleucine was assigned to this codon, and it is not clear whether there has been a typographical error in the nucleotide sequence or the amino acid sequence. We believe isoleucine to be the correct amino acid. The second nucleotide difference is at position 641, which is a C in HuLy-m3 and a G in Blast-1 giving rise to codons ACC and ACG, respectively; this change in nucleotides does not alter the amino acid threonine at this position. It should be noted that this amino acid was designated alanine in the Blast-1 study (Staunton and Thorley-Lawson 1987). On this point, we should point out that the rat antigen OX45, the homologue of Blast-1 and therefore CD48, also has a threonine at this position (Killeen et al. 1988), making it likely that threonine is the correct amino acid rather than alanine. Incidentally it should be noted that, like HuLy-m3 and CD48 but unlike Blast-l, OX45 is present on resting PBLs. The third difference is in the 5' untranslated regions of HuLy-m3 and Blast-l: the reason for this discrepancy is not clear, but as this is not in the coding sequence it would not affect antibody binding. We conclude that the absence of Blast-1 antibody reactivity on the CD48 + transfectants is probably not due to

Fig. 3A. SDS-PAGEanalysis of immunoprecipitations of cells surface-labeled with 125I:HuLym3+ COS cells (lanes a-c), COS cells (lanes d-f), and PBLs (lanes g-e); imunoprecipitated with 5-4.8 (HuLym3; lanes a, d, and g), CD7 (HuLym2; lanes b, e, and h), and Blast-1 (lanes c, f, and i). B SDS-PAGEanalysis of PI-PLC experiments. Immunoprecipitationfrom untreated PBLs (lanes A, B, and C) and PI-PLC-treatedPBLs (lanes D, E, and F); cell lysates and supernatants from untreated PBLs (lanes G, H, and I) and PI-PLC-treatedPBLs (lanes J, K, and L); using a CD46 mAb (lanes A, D0 G, and J), an Ly-2.1 control mAb (lanes B, E, H, and K) and an HuLym3-specificmAb (lanes C, F, I, and L).

117

H.A. Vaughan et al. : Cloning of CD48 64

support was obtained from the National Health and Medical Research Council of Australia.

PBL

References

10 0

101

10 2

10 3

64

10 4

ConA

10 0

101

10 2

10 3

64

10 4

PHA

10 0

101

10 2

10 3

10 4

Fig. 4. Fluorescence-activated cell sorter analysis of the presence of HuLy-m3 on PBLs (top); PBLs after activation with Con A (center); PBLs after activation with PHA (bottom). Cells were treated with 5-4.8 (HuLy-m3; solid lines), or an isotype control antibody (dotted lines).

alterations in the amino acid sequence. It is likely that the Blast-1 antibody is of low affinity, which could account for a low reactivity on some cells (Staunton et al. 1989); a higher-affinity antibody (like 5-4.8) may react with more cells and give a broader tissue distribution. Finally, the Blast-1 mAb could react with an "activation" epitope which is not exposed on resting PBLs [similar to the T11.3 epitope of CD2, which is only detected when the CD2 molecule undergoes conformational changes when its ligand (LFA-3) is bound (McMichael 1987)]. We are currently investigating whether any of these possibilities account for our observations.

Acknowledgments. We acknowledge Drs. Brian Seed and Alejandro Aruffo (Massachusetts General Hospital, Boston, Massachusetts) and Dr. David Jackson (Molecular Medicine Unit, Oxford University, Oxford, UK) for their advice on the method of cloning (Brian Seed). Grant

Aruffo, A. and Seed, B.: Molecular cloning of a CD28 cDNA by a highefficiency COS cell expression system. Proc Natl Acad Sci USA 84: 8573-8577, 1987 Fisher, R. C., Lebeau, M. M., Laurence, J.B., and Thorley-Lawson, D. A.: Genomic organization and chromosomal localization of the human leukocyte activation antigen BLAST-1. In W. Knapp (ed.): "Leukocyte Typing IV", Oxford University Press, Oxford, 1989 Gilman, M.: Preparation and analysis of RNA. In F. M. Ausubel (ed.): Current Protocols in Molecular Biology, pp. 4.1.1-4.1.6, Wiley, New York, 1989 Goding, J.W.: Immunoprecipitations and eleetrophoretic analysis of membrane proteins. In J.C. Venter and L.C. Harrison (eds.): Membrane, Receptor Purification and Characterisation Techniques, pp. 31-60, Luiss, New York, 1980 Henniker, A. J., Bradstock, K. F., Atldnson, K., Barclay, S., Kabral, A., and Grimsley, P.: Expression of a novel human pan leukocyte differentiation antigen on leukaemic cells. Leuk Res 13: 689-697, 1989 Henniker, A. J., Bradstock, K. F., Grimsley, P., and Atkinson, K. : A novel human leukocyte surface membrane antigen defined by murine monoclonal antibody. Tissue Antigens, in press, 1990 Killeen, N., Moessner, R., Arvieux, J., Willis, A., and Williams, A. F.: The MRC OX-45 antigen of rat lenkocytes and endothelium is in a subset of the immunoglobulin snperfamily with CD2, LFA-3 and carcinoembryonic antigens. EMBO J 7: 3087-3091, 1988 Knapp, W. (ed.): Leukocyte Typing/V, Oxford University Press, Oxford, 1989 McMichael, A. J. (ed.): Leukocyte Typing III, Oxford University Press, Oxford, 1987 Parish, C. R. and McKenzie, I. F. C.: A sensitive rosetting method for detecting subpopulations of lymphocytes which react with alloantisera. J Immunol Methods 20: 173-183, 1978 Seed, B. and Aruffo, A.: Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor by a rapid immuno selection procedure. Proc Natl Acad Sci USA 84: 3365-3369, 1987 Sparrow, R. L. : Human antigen defined by monoclonal antibodies. Ph.D. thesis, University of Melbourne, Parkville, Australia, 1983 Sparrow, R. L. and McKenzie, I. F. C. : A function for human T200 in natural killer cytolysis. Transplantation 36: 166-171, 1983 Starling, G. C., Davidson, S. E., McKenzie, J. C., and Hart, D. N. J.: Inhibition of natural killer cell mediated cytolysis with monoclonal antibodies to restricted and non restricted epitopes of the leukocyte common antigen. Immunology 61: 351-356, 1987 Staunton, D. E. and Thorley-Lawson, D. A.: Molecular cloning of the lymphocyte activation marker Blast 1. EMBO J 6: 3695-3701, 1987 Staunton, D. E., Fisher, R. C., LeBeau, M. M., Lawrence, J. B., Barton, D. E., Francke, U., Dustin, M., and Thorley-Lawson, D. A.: Blast-1 possesses a glycosyl-phosphatidylinositol (GPI) membrane anchor, is related to LFA-3 and OX45, and maps to chromosome 1@1-23. J Exp Med 169: 1087-1099, 1989 Thorley-Lawson, D.A., Schooley, R.T., Bhan, A.K., and Nadier, L.M.: Epstein-Barr virus superinduces a new human B cell differentiation antigen (BLast 1) expressed on transformed lymphoblasts. Cell 30: 415-425, 1982 Thurlnw, P. J., Lovering, K. E., and McKenzie, I. F. C.: A monoclonal antibody detecting a new human T cell antigen, HnLy-m2. Transplantation 38: 143-147, 1984 Vaughan, H.A., Thompson, C.H., Sparrow, R.L., and MeKenzie, I. F. C. : H u L y - m 3 - a human leukocyte antigen. Transplantation 36: 446-450, 1983