Proc. Natl. Acad. Sci. USAVol. 85, pp. 208-212, January 1988Immunology

Isolation and structure of a cDNA encoding the B1 (CD20)cell-surface antigen of human B lymphocytes

(lymphocyte differentiation antigen/gene expression/in vitro translation)

THOMAS F. TEDDER*t, MICHEL STREULI*t, STUART F. SCHLOSSMAN*4, AND HARUO SAITO*§*Division of Tumor Immunology, Dana-Farber Cancer Institute, and Departments of tPathology, tMedicine, and §Biological Chemistry, Harvard MedicalSchool, 44 Binney Street, Boston, MA 02115

Communicated by Barul Benacerraf, September 14, 1987

ABSTRACT The Bi (CD20) molecule is a Mr 33,000phosphoprotein on the surface of human B lymphocytes thatmay serve a central role in the humoral immune response byregulating B-cell proliferation and differentiation. In thisreport, a cDNA clone that encodes the Bi molecule was isolatedand the amino acid sequence of Bi was determined. B-cell-specific cDNA clones were selected from a human tonsillarcDNA library by differential hybridization with labeled cDNAderived from either size-fractionated B-cell mRNA or size-fractionated T-cell mRNA. Of the 261 cDNA clones isolated, 3cross-hybridizing cDNA clones were chosen as potential can-didates for encoding Bi based on their selective hybridizationto RNA from Bl-positive cell lines. The longest clone, pBl-21,contained a 2.8-kilobase insert with an 891-base-pair openreading frame that encodes a protein of 33 kDa. mRNAsynthesized from the pBl-21 cDNA clone in vitro was translatedinto a protein of the same apparent molecular weight as Bi.Limited proteinase digestion of the pBl-21 translation productand Bi generated peptides ofthe same sizes, indicating that thepBl-21 cDNA encodes the Bi molecule. Gel blot analysisindicated that pBl-21 hybridized with twomRNA species of 2.8and 3.4 kilobases only in Bl-positive cell lines. The amino acidsequence deduced from the pBl-21 nucleotide sequence appar-ently lacks a signal sequence and contains three extensivehydrophobic regions. The deduced Bi amino acid sequenceshows no significant homology with other known proteins.

B1 (CD20), a human B-lymphocyte surface structure, iswidely expressed during B-lymphocyte ontogeny, from earlypre-B-cell developmental stages until final differentiation intoplasma cells (1-3). Although the exact role of B1 in vivo isunknown, functional studies using monoclonal antibodieshave shown that antibody binding to B1 inhibits B-cellproliferation caused by mitogens, Epstein-Barr virus, oractivated T cells by inhibiting progression through the S/G2andM stages ofthe cell cycle (4, 5). The binding of antibodiesto B1 also inhibits B-lymphocyte differentiation (4-6). Oneanti-Bi monoclonal antibody, 1F7, has been reported toaugment B lymphocyte function (6-8). These results suggestthat the B1 molecule regulates a step of the B-cell activationprocess that is required for cell cycle progression anddifferentiation (9).

Relatively little is known about the structure of B1. It is ahighly hydrophobic phosphoprotein (10). B lymphocytesexpress three isoforms of this molecule with a predominantform of Mr 33,000 and two less prevalent forms of Mr 34,500and 36,000 that may result from differential phosphorylationof a Mr 33,000 species of B1 (11). In this report, we describethe primary structure of B1 deduced from a cloned cDNA.1

MATERIALS AND METHODSSize Fractionation of Poly(A)+ RNA. Isolation of the

microsomal fraction from cell lysates of the human B-lymphoblastoid cell line Raji and the human T-lymphoblas-toid cell line HSB-2 was as described (12). The isolation ofpoly(A)+ RNA was according to Maniatis et al. (13). ThisRNA (150 pug) was size-fractionated by density gradientcentrifugation as described (14) except that the gradient wascentrifuged in a Beckman SW41 rotor at 35,000 rpm for 14.5hr. mRNA in each of 33 fractions was isolated and translatedin vitro as described below.

In Vitro Translation of mRNA. Poly(A)+ RNA was trans-lated in a nuclease-treated rabbit reticulocyte lysate systemaccording to the procedure recommended by the supplier(New England Nuclear/DuPont, Boston, MA) except thatadditional hemin, essential amino acids, phosphocreatine,creatine kinase, and RNasin (Promega Biotec, Madison, WI)were added as described (15). Each reaction mixture (120 ,l)contained 50 t4 of reticulocyte lysate, 1 ug ofmRNA, and 25tLCi of [35S]methionine (1123 Ci/mmol; 1 Ci = 37 GBq) andwas incubated for 1 hr at 370C.

Immunoprecipitation and PAGE Analysis. In vitro transla-tion products were suspended in 0.5 ml of a buffer containing1% (vol/vol) Triton X-100 (Sigma) and proteinase inhibitorsas described (10). Cultured Raji cells were metabolicallylabeled with [35S]methionine and lysed using this buffer (10).Detergent-insoluble material and nuclei were removed bycentrifugation at 12,000 x g for 15 min in a microcentrifugeat 40C. Cell lysates and translation products were pretreatedto remove materials that bind to protein A and immunoglob-ulin by the addition of 50 Al of a 50% (vol/vol) suspension ofprotein A-Sepharose CL-4B (Pharmacia) per ml of solutionwith rotation at 40C for 4 hr. The lysates were furtherprecleared by the addition of a nonreactive antibody of theIgG2a isotype and protein A-Sepharose as above. B1 wasimmunoprecipitated using the anti-Bla monoclonal antibody(1) provided by Coulter Immunology. HLA class I antigenswere precipitated using the W6/32 antibody (16). Eachantibody was used at a concentration of 50 pug/ml of lysateand was precipitated using protein A-Sepharose beads.Following overnight incubations at 40C the beads werewashed as described (10) and analyzed by NaDodSO4/PAGEaccording to Laemmli (17).

One-dimensional peptide analysis of [35S]methionine-la-beled proteins was carried out as described by Cleveland(18).

Isolation of B-Lymphocyte-Specific cDNA Clones. Con-struction of the cDNA library derived from poly(A)+ RNAisolated from pooled human tonsils (approximately 50% B

IThe sequence reported in this paper is being deposited in theEMBL/GenBank data base (Bolt, Beranek, and Newman Labora-tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg)(accession no. J03574).

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lymphocytes) has been described (19). About 40,000 plaqueswere subjected to differential screening as described (20).32P-labeled cDNA probes were made from size-fractionatedpoly(A)+ RNA isolated from the microsomes of either theRaji or the HSB-2 cell line. Duplicate sets of filters of thetonsil cDNA library were separately hybridized with thecDNA probes. Plaques that hybridized with the B-cell (Raji)cDNA probe but not with the T-cell (HSB-2) cDNA probewere isolated and subjected to a second round of screening inthe same manner.RNA Blot Analysis. Dot blot hybridization using total

cytoplasmic RNA of lymphoblastoid cell lines was carriedout as described (21). For gel blot analysis, approximately 2,ug of poly(A)+ RNA isolated from B- and T-lymphoblastoidcell lines was denatured with glyoxal, fractionated by elec-trophoresis in a 0.8% agarose gel, and transferred to nitro-cellulose (22). The phage cDNA inserts were isolated, radio-labeled by nick-translation (23), and hybridized with thefilters according to Wahl et al. (24).DNA Sequencing. cDNA fragments were subcloned into

the plasmid vector pSP64 (25). Restriction maps were gen-erated as described by Maniatis et al. (13), and nucleotidesequences were determined according to the method ofMaxam and Gilbert (26).

In Vitro RNA Synthesis and Translation. RNA transcriptswere synthesized in a reaction mixture (0.1 ml) containing 10,ug of template pBl-21 DNA (linearized with the restrictionenzyme Pvu II), 40 mM Tris-HCI (pH 7.5), 6 mM MgCl2, 2mM spermidine, 10 mM dithiothreitol, 100 ,ug of acetylatedbovine serum albumin, 100 units of RNasin, 0.5 mM eachrNTP, 0.5 mM G(5')ppp5'G (Pharmacia), and 40 units of SP6RNA polymerase (Promega Biotec) for 2 hr at 40°C. Thefull-length RNA products were purified by agarose gelelectrophoresis, extracted four times with phenol, precipitat-

21 28S 18S

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FIG. 1. Sedimentation analysis of B1 mRNA. Poly(A)+ RNA wasisolated from the microsomal fraction of a lysate from the B-lymphoblastoid cell line Raji and separated by sucrose densitygradient centrifugation into 33 fractions. Each mRNA fraction wassubjected to in vitro translation and immunoprecipitation withanti-Bla and W6/32 (anti-HLA class I) antibodies. Immunoprecipi-tated materials were analyzed by NaDodSO4/PAGE and B1 (o) andclass I (u) molecules were immunoprecipitated only from translatedmRNA fractions 2-3 and 3-5, respectively. Relative amounts ofimmunoprecipitated B1 and class I molecules were determined byquantitative scanning densitometry of autoradiographs and areexpressed in arbitrary units. The average length of mRNA in eachfraction was determined by electrophoresis in a 1% agarose gel with18S and 28S ribosomal RNA and DNA fragments of known length asstandards. The migration of the 28S and 18S ribosomal RNAsfractionated in a companion centrifuge tube is also shown (arrows).

Proc. Natl. Acad. Sci. USA 85 (1988) 209

Table 1. B-lymphocyte-specific clones isolated from a tonsillarcDNA library

No. of clonescDNA group mRNA length, kb isolated

A * 35Invariant chain 1.4 21B 2.0 11,i heavy chain 1.8 11HLA-DP,DQ,DRB chain 1.2 10K light chain 1.1 8HLA-DP,DQ,DR a chain 1.2 8C 1.2 4D (Bi) 2.8 and 3.4 3E * 3F 2.2 3G 2.5 2H 12.0and 8.7 2I 2.2 2cDNA clones were isolated that hybridized with size-fractionated

cDNA of the B-lymphoblastoid cell line Raji but that did nothybridize with size-fractionated cDNA of the T-lymphoblastoid cellline HSB-2. Thirty groups that contained only single clones are notshown.*Multiple mRNA species.

ed with ethanol, and dissolved in water. In vitro synthesizedmRNA was translated in a nuclease-treated rabbit reticulo-cyte lysate according to the procedure recommended by thesupplier (Promega Biotec). The translation mixture (0.1 ml)contained 70 1.d of reticulocyte lysate, 1 AuM [35S]methionine(1150 Ci/mmol, New England Nuclear/DuPont), and 2 gg ofin vitro synthesized mRNA and was incubated for 60 min at300C.

RESULTS

Characterization of B1 mRNA Length. Poly(A)+ RNAisolated from the microsomes of the Bi-positive lymphoblas-toid cell line Raji was size-fractionated through a sucrosedensity gradient. Individual fractions were translated in vitroand assayed by immunoprecipitation for the ability to pro-duce B1 protein (Fig. 1). The peak of B1 mRNA activity waslocated within fractions containing mRNA with an estimatedlength of2200-3000 nucleotides [2.2-3 kilobases (kb)]. There-fore, B1 mRNA is about 2- to 3-fold longer than would berequired to encode a protein of 33 kDa. In contrast to B1mRNA, mRNA that encoded class I molecules (45-kDaprotein) was about 2000 nucleotides long.

Isolation and Characterization of B-Cell-Specific cDNAClones. The fraction of Raji RNA enriched for B1 mRNA(Fig. 1, fractions 2-3) and a corresponding size fraction ofmRNA from the T-cell line HSB-2 were used to synthesize32P-labeled cDNA probes. These probes were used to screenfor B-lymphocyte-specific clones within a cDNA library

_Z 7 < :. * X YCX

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FIG. 2. Restriction map of the pBl-21 cDNA clone and thestrategy for determining the nucleotide sequence. The map wasconstructed by the standard single, double, or triple digestions ofpBl-21. The putative coding region is shown in black, and the 3'untranslated region is stippled. Arrows indicate the direction andextent of nucleotide sequence determination. Open and closedcircles indicate 5'- and 3'-end labeling, respectively. bp, Base pairs.

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gaattccCCT CAATGACACT CATGGAGGAA ATGCTGAGAG AAGCATTCAG ATGCATGACA CAAGGTAAGA CTGCCAAAAA TCTTGTTCTT GCTCTCCTCA TTTTGTTATT1 10 20M T T P R N S V N 0 T F P A E P M K G P I

TGTTTTATTT TTAGGAGTTT TCACACCAAA ATC ACA ACA CCC AGA AAT TCA GTA AAT GCG ACT TTC CCG GCA GAG CCA ATG AAA GCC CCT ATT30 40 50

A M Q S G P K P L F R R M S S L V G P T Q S F F M R E S K TGCT ATG CAA TCT GGT CCA AAA CCA CTC TTC AGG AGG ATG TCT TCA CTG GTG GGC CCC ACG CAA AGC TTC TTC ATG AGG GAA TCT AAG ACT

60 70 80L G A V Q I M N G L F H I A L G 0 L L M I P A 0 I Y A P I C

TTG GGG OCT CTC CAG ATT ATC AAT 000 CTC TTC CAC ATT CCC CTG 0GG GGT CTT CTG ATG ATC CCA GCA GGG ATC TAT GCA CCC ATC TOT90 100 110

V T V W Y P L W 0 G I M Y I I S G S L L A A T E K N S R K CGTG ACT OTO TGO TAC CCT CTC TGG OGA GGC ATT ATO TAT ATT ATT TCC GGA TCA CTC TTG GCA GCA ACG GAG AAA AAC TCT AGG AAO TOT

120 130 140L V K G K M I M N S L S L F A A I S G M I L S I M D I L N I

TTO OTC AAA GGA AAA ATG ATA ATG AAT TCA TTG AGC CTC TTT GCT GCC ATT TCT GGA ATG ATT CTT TCA ATC ATG GAC ATA CTT AAT ATT150 160 170

K I S H F L K M E S L N F I R A H T P Y I N I Y N C E P A NAAA ATT TCC CAT TTT TTA AAA ATG GAG AGT CTG AAT TTT ATT AGA GCT CAC ACA CCA TAT ATT AAC ATA TAC AAC TGT GAA CCA OCT AAT

180 190 200P S E K N S P S T Q Y C Y S I Q S L F L 0 I L S V M L I F A

CCC TCT GAG AAA AAC TCC CCA TCT ACC CAA TAC TGT TAC AGC ATA CAA TCT CTG TTC TTG GOC ATT TTO TCA OTO ATO CTG ATC TTT 0CC210 220 230

F F Q E L V I A 0 I V E N E W K R T C S R P K S N I V L L STTC TTC CAG GAA CTT GTA ATA GCT GGC ATC GTT GAG AAT OAA TOO-AAA AGA ACG TGC TCC AGA CCC AAA TCT AAC ATA OTT CTC CTG TCA

240 250 260A E E K K E Q T I E I K E E V V 0 L T E T S S Q P K N E E D

OCA OAA OAA AAA AAA OAA CAG ACT ATT OAA ATA AAA OAA OAA OTO OTT 0G0 CTA ACT OAA ACA TCT TCC CAA CCA AAO AAT OAA GAA GAC270 280 290

I E I I P I Q E E E E E E T E T N F P E P P Q D Q E S S P IATT GAA ATT ATT CCA ATC CAA GAA GAO OAA OAA OAA OAA ACA GAO ACO AAC TTT CCA GAA CCT CCC CAA GAT CAG OAA TCC TCA CCA ATA

297E N D S S P '

OAA AAT GAC AOC TCT CCT TAA OTOATTTCTT CTGTTTTCTG TTTCCTTTTT TAAACATTAO TGTTCATAOC TTCCAAGAOA CATOCTGACT TTCATTTCTT

OAOOTACTCT OCACATACGC ACCACATCTC TATCTGGCC .............

FIG. 3. Nucleotide sequence and predicted amino acid sequence ofcDNA clone pBl-21. The numbers shown above the amino acid sequencedesignate amino acid residue positions. The amino acid regions with hydrophobic character are underlined. Amino acids are designated by thesingle-letter code, and an asterisk indicates the termination codon.

made from human tonsillar lymphocytes (19). Approximately40,000 plaques were screened, and 261 plaques that hybrid-ized with Raji cDNA but not with HSB-2 cDNA wereidentified and isolated. Fifty-eight of these clones hybridizedwith DNA encoding known B-lymphocyte proteins (Table 1).The cDNA inserts were isolated from 60 of the remaining 203clones, nick-translated, and used to establish groups ofcross-hybridizing clones. Although not all clones have beengrouped, nine groups that contained more than one clonewere identified (Table 1, groups A-I).These nine groups ofcDNAs were examined further by dot

blot hybridization using cytoplasmic RNA isolated from celllines that are either positive or negative for cell-surfaceexpression of B1 (21). Group D clones hybridized to RNAfrom the Bi-positive cell lines Raji, Bjab, and Daudi, but notto RNA from the Bi-negative cell lines, Nalm-6, PB697,HSB-2, HuT-78, CEM, Jurkat, K-562, and U937 (data notshown). The hybridization patterns of the remaining groupsdid not correlate with B1 expression. Since this pattern ofexpression is unique among known B-lymphocyte surface-antigens, group D was further characterized. The longestcDNA insert (2.8 kb) from one of the group D clones wassubcloned into pSP64, and the resultant plasmid was calledpBl-21.

Nucleotide and Deduced Amino Acid Sequence of pBl-21. Arestriction map was generated for pB1-21, and the nucleotidesequence was determined according to the method ofMaxamand Gilbert (26), using the strategy shown in Fig. 2. Thenucleotide sequence of 1153 base pairs at the 5' end of pB1-21was determined and is shown in Fig. 3. The extreme 3' endof the B1-21 cDNA clone, which was also sequenced (datanot shown), contained a potential poly(A)-attachment signalsequence (AATAAA) and ended with a short stretch of Aresidues (27). The 5' cDNA sequence contained an openreading frame of 891 nucleotides, shown in Fig. 3. The firstATG is most likely the initiation codon for translation, sincethis is the firstATG that conforms to the proposed translationinitiation consensus sequence, ANNATG (28).The pBl-21 cDNA insert can encode a protein of297 amino

acids with a molecular mass of 33 kDa. Hydropathy analysisas described by Kyte and Doolittle (29) identified threestretches of strongly hydrophobic amino acids (underlined in

Fig. 3). The pBl-21 cDNA does not encode a typical signalpeptide at the 5' end of the protein. Three potential N-linkedglycosylation sites (N X S or N X T) were found at amino acidpositions 9, 171, and 293. A computer search of proteinsequences using the Protein Identification Resource databasell indicated that no known proteins share significantsequence homology with the putative pBl-21 protein prod-uct.

Demonstration That pBl-21 Encodes the Bl Molecule. ThepBl-21 cDNA insert, subcloned in the appropriate orienta-tion into the pSP64 expression vector, was used to synthesizemRNA in vitro (25). This RNA was translated in an in vitrotranslation system in the presence of [35S]methionine and theprotein product was compared with the B1 protein im-munoprecipitated from metabolically labeled Raji cells. Boththe pBl-21 protein product and the B1 molecule from Rajicells migrated with an approximate Mr of 33,000 whenanalyzed by NaDodSO4/PAGE (Fig. 4). The anti-B1 anti-body failed to specifically immunoprecipitate the pBl-21protein product due to a possible reduction in antibodyaffinity for the protein produced in vitro from syntheticmRNA and increased background binding of the singularlabeled protein product from in vitro translation. However,peptide mapping by limited proteolysis with gel electropho-resis provides highly reproducible peptide fingerprints thatare characteristic of individual proteins (18). Limited pro-teinase digestion of the B1 protein and the pBl-21 geneproduct demonstrated that identical peptide fragments weregenerated from the cleavage of those two proteins by twoproteinases with different substrate specificities (Fig. 4).These results indicate that the pBl-21 cDNA insert encodesthe same protein immunoprecipitated by the anti-B1 anti-body.

Expression of Bi mRNA. Poly(A)+ RNA was isolated from11 lymphoblastoid cell lines and examined by gel blot analysisusing the pBl-21 as a probe (Fig. 5). pBl-21 hybridized withmRNA of 2.8 and 3.4 kb in Raji, SB, GK5, and Bjab cells. Inall cases the 2.8-kb species was expressed at about 10-fold

UProtein Identification Resource (1987) Protein Sequence Database(Natl. Biomed. Res. Found., Washington, DC), Release 11.

210 Immunology: Tedder et al.

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FIG. 4. Biochemical comparison of the protein product of thepBl-21 cDNA clone with the B1 molecule. (A) mRNA produced invitro from pBl-21 was translated in vitro in the presence of[35S]methionine (pBl-21). The labeled proteins produced in thetranslation reaction without RNA added is shown (no RNA). Rajicells were metabolically labeled with [35S]methionine, lysed withdetergent, and immunoprecipitated with a nonreactive IgG2a anti-body (control). This lysate was then immunoprecipitated with theanti-Bla antibody (B1). The immunoprecipitated materials and invitro synthesized materials were analyzed by NaDodSO4/10%6PAGE with subsequent autoradiography. (B) Peptide mapping of B1and the pBl-21 protein product by limited proteolysis. The Mr 33,000proteins, pBl-21 and B1 from A, were excised from the gel andone-dimensional peptide mapping was carried out by the method ofCleveland (18). Protein fragments generated by digestion withStaphylococcus aureus V8 proteinase (150 ng) or chymotrypsin (10j.g) were analyzed by NaDodSO4/15% PAGE followed by auto-radiography.

higher levels as determined by quantitative scanning densi-tometry. mRNA from the microsomal fraction of Raji cellswas enriched 2.5-fold for B1 mRNA as compared to totalcellular mRNA from Raji (Fig. 5). The Bi-negative pre-B-celllines Nalm-6 and PB697 did not express B1 mRNA. How-ever, the PB697 cell line is unique in that exposure to tumorpromoters induces the selective expression of B1 on the cellsurface (30), whereas this treatment does not induce expres-sion of B1 by Nalm-6 cells. Consistent with the surfaceexpression ofB1, phorbol ester treatment induced B1 mRNAexpression by PB697 cells (both the 2.8- and 3.4-kb forms),while treatment of Nalm-6 cells did not induce B1 mRNAexpression (Fig. 5). pBl-21 did not hybridize with mRNAfrom the T-cell lines CEM, MOLT-3, HuT-78, and HSB-2 orthe erythroleukemia cell line K-562 (data not shown).

DISCUSSIONA cDNA clone has been isolated and shown to encode theB-lymphocyte-specific molecule B1 (Fig. 4). The deducedprotein structure of B1 (Fig. 3) is unique among lymphocytesurface structures and shares no significant homology withany known proteins. Hydropathicity analysis indicated thatthere were three extensive hydrophobic regions. Two ofthese regions, 25 and 27 amino acids long, are sufficient inlength for the protein to transverse the membrane. The otherextensive stretch of hydrophobicity, 55 amino acids long, is

FIG. 5. Gel blot analysis of B- and T-lymphocyte mRNAshybridized with labeled pBl-21 and pB70 probes. Poly(A)+ RNA wasisolated from the B-cell lines Raji, SB, GK5, and Bjab; the pre-B-celllines Nalm-6 and PB697; and the T-cell line CEM. RNA was isolatedfrom the two pre-B-cell lines, PB697 and Nalm-6, before and after a24-hr exposure to phorbol 12-myristate 13-acetate (PMA; 1 ng/ml).RNA was also isolated from the microsomal (memb.) fraction of Rajicells. Two micrograms of each RNA was used per lane. RibosomalRNA (28S and 18S) was run in parallel as an indicator of size.Autoradiography was with an intensifying screen for 5 days. Thefilter was first hybridized with the pB70 probe (group C, Table 1),which identifies a 1.2-kb mRNA species expressed by all B- andpre-B-cell lines, and was then hybridized with the pBl-21 probe,which identifies the 2.8- and 3.4-kb mRNA species.

sufficient in length to transverse the membranes at leasttwice. The putative amino-terminal end of the protein doesnot have a hydrophobic region typical of signal sequences.However, other membrane-embedded proteins, such asrhodopsin, and the f3-adrenergic receptor, also lack signalsequences (31, 32). These data suggest that the B1 moleculemay also traverse the membrane more than once, as dorhodopsin and the j-adrenergic receptor (31, 32).Although three potential N-linked glycosylation sites exist

on B1, previous studies indicate that the native B1 moleculeis not glycosylated (10, 11). The finding that the in vitrosynthesized B1 protein was identical in apparent molecularweight with B1 immunoprecipitated from Raji cells (Fig. 4)further demonstrates that posttranslational glycosylationdoes not occur with B1. Although it is not possible todetermine the membrane orientation ofthe amino terminus ofB1, the region between residues 142 and 185 is most likelyexposed on the cell surface, since it is the only hydrophilicportion ofthe molecule containing tyrosine residues availablefor iodination, and cell-surface B1 is readily iodinated (10,11). This would indicate that amino acid residues locatedbetween position 213 and the carboxyl terminus of themolecule are most likely located within the cytoplasm. Thereare 44 serine and threonine residues in B1 providing numer-ous candidate sites for phosphorylation (10, 11). Proteinkinase C phosphorylates serine and threonine residues thatare on the carboxyl-terminal side (one or two residues away)of a number of basic residues, and cyclic GMP-dependentprotein kinase requires multiple basic residues amino-termi-nal to the phosphorylation site (33). Thus, possible phospho-

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pBl -2'E

PB70

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rylation sites for these enzymes may include B1 amino acidpositions 35, 36, 49, 108, 144, 219, 221, and 225. Therefore,the deduced structure of the B1 protein is consistent withprevious results of B1 being a generally hydrophobic phos-phoprotein.Gel blot hybridization analysis showed that two distinct

mRNA species of 2.8 and 3.4 kb hybridized with the pBl-21cDNA insert (Fig. 5). The 3.4-kb B1 mRNA species repre-sented less than 10% of the total B1 mRNA. Three differentmolecular weight forms of B1 protein are expressed by Blymphocytes, with the higher molecular weight forms beingless than 20% of the total B1 protein (11). The structural basisfor the different forms of B1 protein is unknown. To deter-mine whether or not the different mRNA species producedifferent B1 structures, the isolation of a cDNA correspond-ing to the larger form of B1 mRNA will be required. B1mRNA was expressed by Bi-positive B-cell lines but not byT-cell lines, the erythroleukemia cell line K-562, or themyelomonocytic cell line U937 (Fig. 5 and data not shown).In addition, the pre-B-cell line PB697 does not express B1 onthe cell surface but selectively expresses B1 following expo-sure to tumor-promoting agents (30). PB697 expressed B1mRNA only after exposure to phorbol ester (Fig. 5). There-fore, expression of the B1-21 gene is consistent with cell-surface expression of B1.The 85 amino acid carboxyl-terminal region of the B1

protein is most likely located within the cytoplasm (Fig. 3).The length of this region contrasts with that of other B-cell-specific surface structures such as IgM, IgD, and IgG heavychains or histocompatibility antigen class II a or, chains,which have relatively short intracytoplasmic regions (3, 3,28,15, and 16 amino acids, respectively; refs. 34 and 35).Another unique feature of this protein is that of the last 61carboxyl-terminal amino acids, 21 are acidic residues, where-as only 2 are basic, indicating that this region may have astrong net negative charge (Fig. 3). The overall structure ofB1 suggests that the function of B1 may involve the gener-ation of transmembrane signals resulting in further interac-tions with cytoplasmic structures. Functional studies indi-cate that antibody binding to B1 does not trigger an increasein intracellular Ca2l concentration (5), but B1 may serve aregulatory role through interactions with other molecules.The hydropathicity profile and overall structure of B1 aresimilar to those of the rhodopsins and,B-adrenergic receptors(31, 32). Both rhodopsin and the 3-adrenergic receptor arephosphoproteins and are involved in signal-transductionmechanisms through interactions with guanine nucleotide-binding regulatory proteins (36, 37). The structure andfunctional properties of B1 suggest that it may also be asignal-transducing protein.

We thank Laurie Hall, Gabriel Kleijman, Dr. Michael Seckl, andAnn Penta for assistance with these experiments; Drs. L. Klicksteinand D. T. Fearon for the human tonsillar cDNA library and the CR1probe; Dr. C. F. Webb for the IgM heavy chain probe; Dr. J. Weisfor the CR2 probe; Dr. J. Strominger for the DP, DQ, and DR probes;Dr. E. 0. Long for the invariant chain probe; and Drs. J. Lambert,J. Breitmeyer, and J. Mole for helpful discussions. This work wassupported by grants from the National Institutes of Health (CA34183)and the Whitaker Health Sciences Fund and by an InvestigatorAward from the Cancer Research Institute (H.S.). T.F.T. is aResearch Fellow of the Damon Runyon-Walter Winchell CancerFund (DRG-864). M.S. is a recipient of a fellowship from the SwissNational Science Foundation.

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