Proc. Natl. Acad. Sci. USAVol. 84, pp. 4210-4214, June 1987Immunology

Molecular cloning of Lyt-3, a membrane glycoprotein marking asubset of mouse T lymphocytes: Molecular homology toimmunoglobulin and T-cell receptor variable and joining regions

(lymphocyte differentiation antigen/immunoglobulin super gene family/CD8)

HIROMITSu NAKAUCHI*, YO-ICHI SHINKAI, AND Ko OKUMURADepartment of Immunology, Juntendo University School of Medicine, Tokyo 113, Japan

Communicated by Leonard A. Herzenberg, February 17, 1987 (received for review December 30, 1986)

ABSTRACT Lyt-3 is a membrane glycoprotein expressedon thymocytes and class I major histocompatibility complex-restricted cytotoxic T cells. Lyt-3 is expressed as a heterodimerwith Lyt-2, and this complex is considered to be a homologueof the human Leu-2/T8 (CD8) that has been postulated to bea receptor for the class I major histocompatibility complex. Wehave determined the complete primary structure of Lyt-3 fromthe nucleotide sequence of its cDNA clones. Analysis of thepredicted amino acid sequence indicates that the Lyt-3 poly-peptide has a 21-amino acid leader peptide, and the matureprotein consists of an NH2-terminal region of 146 amino acids,a transmembrane region of 27 residues, and a C-terminalregion of 19 amino acids, The NH2-terminal 110 residues showclear homology to the T-cell receptor and immunoglobulinvariable region sequences. In addition, Lyt-3 has 11 residuesthat have strong homology to the joining region sequences ofthe T-cell receptor and the immunoglobulin heavy and lightchains. The presence of immunoglobulin variable- as well asjoining-region-related sequences in Lyt-3 further supports theidea that these molecules may be recognition molecules belong-ing to the immunoglobulin super gene family.

The murine cell surface molecules Lyt-2 and Lyt-3 (CD8 inhumans) have been used as a marker delineating cytotoxic Tlymphocytes from helper/inducer T cells expressing thealternative marker L3T4 (CD4 in humans) (1-4). However,there is a more strict association between the expression ofthese molecules by T cells and their restriction pattern; Lyt-2and Lyt-3 are expressed on T cells restricted by class I majorhistocompatibility complex (MHC), whereas L3T4-express-ing T cells are restricted to recognizing class II MHC (5, 6).

Lyt-2 and Lyt-3 molecules are believed to contribute to theoverall avidity of T-cell interactions by binding to thenonpolymorphic portions of the class I MHC molecules,since antibodies to Lyt-2 or Lyt-3 will block the lysis of targetcells by many cytotoxic T lymphocytes (7-9). The Lyt-2 andLyt-3 are coexpressed except on fetal thymocytes for a briefperiod of time when Lyt-2 alone may be expressed (10). Thestructural genes for Lyt-2 and Lyt-3 are tightly linked to eachother and to the K light chain locus on murine chromosome6 (11).

In the mouse, the Lyt-2 antigenic determinant is found ontwo related polypeptides, 38-kDa (a) and 34-kDa (a') poly-peptides resulting from the alternative splicing of mRNAtranscribed from a single Lyt-2 gene (12, 13). Each of thesepeptides is disulfide bond-linked to a Lyt-3 polypeptide (f3)for cell surface expression (14, 15). It has been shown thatLyt-2 is the homologue of human Leu-2 (CD8) (12, 16).

Similarly, in the rat, CD8 consists of 32-kDa and 37-kDapolypeptides, and the 32-kDa chain has been shown to be the

rat homologue of the human CD8 and the mouse Lyt-2molecules (17). Molecular cloning of the gene correspondingto the 37-kDa chain of the rat CD8, a putative rat homologueof mouse Lyt-3 (18), produced a probe that we have used toclone a gene encoding the mouse Lyt-3.We describe here the isolation of Lyt-3 cDNAs, present its

predicted amino acid sequence, describe the general struc-ture of the Lyt-3 polypeptide, and compare the Lyt-3 se-quence with those of Lyt-2 and other members of theimmunoglobulin super gene family.

MATERIALS AND METHODSEnzymes and Cells. Restriction enzymes, exonuclease III,

and mung bean nuclease were obtained from Takara (Tokyo).The AKR thymic lymphoma cell lines were established by H.Yagita in our laboratory.

Screening of Mouse cDNA Library and Hybridization Con-ditions. A cDNA library made from the alloreactive cytotoxicT lymphocytes clone 2C, derived from BALB/B mice, in thephage vector Xgtl0 (19) was plated on Escherichia coliC600/Hfl and probed with a nick-translated rat 37-kDa CD8cDNA clone (pX9.15; kindly provided by P. Johnson and A.Williams, Oxford) (18). Plaque hybridization was performedas described (20) with low-stringency washing in 6x SSPEwith 0.1% NaDodSO4 then in 4x SSPE/0.1% NaDodSO4 at650C. (lx SSPE = 180 mM NaCl/10 mM sodium phosphate,pH 7.7/1 mM EDTA.)

Nucleotide Sequencing. The insert of Xgtl0 phage clonesscreened as described above were subcloned into M13mp18phage vectors. Clones that contained series of unidirectionaldeletions were made by digestion with exonuclease III andmung bean exonuclease from either end following the methoddescribed by Yanisch-Perron et al. (21). The nucleotidesequence was determined by the dideoxynucleotide termi-nation method of Sanger et al. (22).DNA and RNA Hybridization. Digested DNA was analyzed

by the Southern blot technique (23). Total RNAs wereextracted from cell lines and tissue as described (24) andanalyzed by RNA blot hybridization (25).Computer Analysis. Nucleotide and amino acid sequence

analysis were done by using the Beckman MicroGenieprograms. The National Biomedical Research FoundationProtein Data Bankt was searched by the program describedby Beckman MicroGenie.

RESULTSIsolation and Sequence of Lyt-3 cDNA. We screened a

cytotoxic T-lymphocyte clone 2C cDNA library at low

Abbreviations: V, immunoglobulin variable; J, joining; MCH, majorhistocompatibility complex.*To whom reprint requests should be addressed.tProtein Identification Resource (1986) Protein Sequence Database(Natl. Biomed. Res. Found., Washington, DC), Release 8.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Immunology: Nakauchi et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4211

stringency with a full-length cDNA probe, pX9. 15, encoding (XLY3C-23 and XLY3C-24) contained the NH2-terminal ami-the rat 37-kDa CD8 polypeptide. Of 180,000 plaques no acid sequence. Fig. 1 shows the nucleotide and deducedscreened, 46 positive phage clones were obtained. Five amino acid sequence obtained from these cDNA clones.clones were further examined in detail. Two of those clones Comparison of the amino acids predicted from the cDNA

AAGAGGAGTGAGGGGAAGAGTGTGCGGAAAAGCGGCAA(; 38

-21ATG CAG CCA TGG CTC TGG CTG GTC TTC AGT ATG AAG CTG GCA GCT CTC TGG AGC AGC TCTMet Gin Pro Trp Leu Trp Leu Val Phe Ser Met Lys Leu Al Ala Leu Trp Ser Ser Ser

-_ I 10GCC CTC ATT CAG ACC CCT TCG TCC CTG CTG GTT CAA ACC AAC CAT ACG GCA AAG ATG TCCAla Leu lie Gin Thr Pro Ser Ser Leu Leu Val Gin Thr Asn His Thr Ala Lys Met Ser

20 30TGT GAG GTT AAA AGC ATC TCT AAG TTA ACA AGC ATC TAC TGG CTG CGG GAG CGC CAG GACCys Glu Val Lys Ser lie Ser Lys Leu Thr Ser lie Tyr Trp Leu Arg (;lu Arg Gin Asp

40 50CCC AAG GAC AAG TAC TTT GAG TTC CTG GCC TCC TGG AGT TCT TCC AAA GGA GTT TTG TATPro Lys Asp Lys Tyr Phe GIu Phe Leu Ala Ser Trp Ser Ser Ser Lys GIv Vat Leu Tyr

60

GGT GAA AGTGlt (tlu Ser

70GTG GAC AAG AAA AGA AAT ATA ATT CTT GAG TCT TCA GAC TCA AGA CGG CCCVal G;I Lys Lys Arg Asn lie lle Leu Glu Ser Ser Asp Ser Arg Arg Pro

9 () 4

TTT CTC AGT ATC ATG AAT GTG AAG CCA GAG GAC AGT (GAC TTC TAC TTC TGC GCG ACG (;TTPhe Leu Ser lIe Met Asn Vat Lvs Pro GInu Asp Ser Asp Phe Tv r P he Cys Ala Thr Vat

1 00

GGGGI y

AGC CCC AAG AT(; GTC TTT GG(; ACA G(;G ACG AAG CTG ACT GTG GTT GAT (;TC CTT CCTSer Pro Lys Met Vat Phe Gte Thr Gly Thr Lys Lenu Ther Vat Vat Asp Vat Lenu Pro

120 1 30ACA ACT GCC CCA ACC AAG AAG ACT ACC CTG AAG ATG AAG AA(; AAG AAG CAA TGC CCG TTCThr Thr Ala Pro Thr Lys Lys Thr Thr LenL Lys Met Lys Lys Lys Lvs GIn Cvs Pro Phe

140 151)CCC CAC CCA GAG ACC CA(; AAG GGC CT(; ACA TGC AGC CTT ACC ACC CTC AGC CTG CTG (;TAPro His Pro Glu Thr Gin Lys Gly Leu Thr Cys Ser LetLi Thr Thr Leu Ser Leu Leu Vat

160 170GTC TGC ATC CTG CTT CTG CTG GCA TTC CTC GGA GTG GCC GTC TAC TTT TAC TGT GTG CGGVal Cys lie Leu Leu Len Leu Ala Phe Leu (GIy Val Ala Val Tyr Phe Tvr Cvs Val Arg

180 19(1AGG AGA GCC CGA ATT CAC TTC ATG AAA CAG TTT CAC AAA TGA TCAGCGCCCACAACACTGATAGCArg Arg Ala Arg lie His Phe Met Lys GIn Phe His Lys End

CTGCTAGCAATAAAA(;GACTGTAACTCAATCAAAGCAA(;ATTT(;AACTAGTG;A(;AGACAGC(;CATTCAA(;ACTG;(;A(;A(;

GCCCTTGCCCTTGCTCAAGCTGCCT(;CTTT(;AACTGCT(;CAAC(;TCTGTGTGT(;(;TCTGG(;GTCTCACA(;GGAA(;(;CCC

CAGAACCCAAGCTCACTCACA(;AGT(;CCTGAAGAACTAAGAACTCCCACACCACCATGTACTCTTCTT(;(;ACTGGGCCT

TGGCAGTGGCCTTCCAGCCACTGT'CTTTGTCA(;TTGCTT(;GTAT(;ACAGCCTTATT(;ACTCTTT(;(;CCTTTCAT(GAAA

AGAGCTAGCCGGGACCCTGGAGAAGACGT(;CAAATCCT'CACACA(;AGAAGCCACCTCATGAATAAAGTTTTCT(;CAAACA

GTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCCT

B sco4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ipoly (A)

-01

.Z~~~~~~~~~~~~~~~~~~~P

100 bp

ALY3C 23

A LY3C 241 1

458

518

5 78

6 38

7 00

7 811

81)0

940

1020 FIG. 1. (A) Nucleotide and aminoacid sequences derived from cDNA

1101( clones encoding Lyt-3. Cysteine resi-dues believed to participate in intra-

1144 chain disuffide bonding are marked byarrows. The possible N-linked glycosyl-ation site is marked by asterisks. TheJ-related segment is indicated by thedashed line. The transmembrane regionis underlined. The 21-amino acid leaderpeptide is residues -21 to -1. Themature peptide is residues 1 to 192. Theputative polyadenylylation signal is in-dicated by a wavy line. (B) Restrictionendonuclease map of Lyt-3 cDNA cod-ing for Lyt-3. The protein coding regionis boxed. The shaded region is themature protein after cleavage of leaderpeptide. The DNA sequence strategyutilizing M13-deletion mutants is pre-sented below the map. Below the se-quencing strategy are the two cDNAclones used in this sequencing; theregions that were sequenced are indi-cated by the bold lines.

98

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4212 Immunology: Nakauchi et al.

sequence with the reported amino acid sequence of purifiedLyt-3 protein (26) show<s correspondence of all but threeamino acids out of 13 residues.Mouse Lyt-3 sequenceProtein microsequencing L I Q A P S S L L V Q K KPredicted from cDNA L I Q T P S S L L V Q T N

NH2-terminal microsequencing identifies residue 4 as ala-nine, and residues 12 and 13 as lysine, whereas the cDNAsequence predicts threonine, threonine, and asparagine,respectively. Since residues 4, 12, and 13 ofLyt-2 are alanine,lysine, and lysine, the discrepancy between the two seemsmost likely to be due to the contamination of Lyt-3 materialby Lyt-2. However, this could also be due to the allotypicdifferences between BALB/B (Lyt-3.2) and B6/PL (Lyt-3.1)strains.Based on DNA and protein sequence data as well as the

hydrophobicity plot analysis of the predicted protein (datanot shown), we conclude that the mature peptide is 192 aminoacids long (predicted molecular size, 21.9 kDa) with a leaderpeptide of 21 residues. Analysis of the hydrophobicity plotalso reveals a strongly hydrophobic area near the carboxylterminus (residues 147-173) followed by a hydrophilic se-quence (residues 174-192). These regions may be the trans-membrane and cytoplasmic regions. There is one possible

-2 1 -ILYT-3 M Q P W W L V F S M K A A W S -- - -[ -LYT-2 MASPLPTRFLSLNT- L G ES I I L GSG[E

1 10 20

WI Q T P S SL L Q T N H T- - - - - - C E V K[f I SKK P Q A P E L R I F P K K M DAE L G Q V D C E V- L GSVI T Q S P R YWl I L - G R A N K - - - - S - - L E C Q H L G H -

V T Q P D A R V TW1S E G A S L - - - - Q - - L R C K Y S Y

V T Q E S A L T T - S P G E WV- - - - T - -L T C R S T

W]T Q SW A I M A A S L G Q K V - - - T - C - S A S S SQ L Q Q S GA Q RAGS S V --- - MS C K AS G Y T F

Proc. Natl. Acad. Sci. USA 84 (1987)

N-linked glycosylation site at position 13. The discrepancy ofthe observed size of Lyt-3 as 30 kDa and that of predictedprimary protein sequence may be due to O-linked and/orN-linked glycosylation.

Lyt-3 Is a Member of the Immunoglobulin Super GeneFamily. A computer search of protein sequence bankt forsequences similar to Lyt-3 indicated that the NH2-terminal110 residues of Lyt-3 are homologous to the immunoglobulinand the T-cell receptor variable (V) domain sequence with thegreatest similarity to T-cell receptor p-chain V-region se-quences (Fig. 2). Like other members of the immunoglobulinsuper gene family, this domain ofLyt-3 has two cysteines thatcan form the intrachain disulfide bond as well as a trypto-phan, 13 residues downstream from the first cysteine of thedisulfide loop. In addition, Lyt-3 has 11 residues almostidentical to the joining (J) region sequences of the T-cellreceptor and immunoglobulins. The highest homology toLyt-3 was found in J segments of the immunoglobulin Xlight-chain gene and the T-cell receptor a-chain gene, where9 out of 11 residues matched (Fig. 3). Schematic diagrams ofthe murine CD8 molecules, Lyt-2 (a), Lyt-3 (a'), and Lyt-3(B) predicted from the cDNA sequences are shown in Fig. 4.A Single Gene Without DNA Rearrangement Is Responsible

for the Expression of Lyt-3. Southern blots containing DNAsfrom AKR liver, an Lyt-3-expressing thymoma line, and

L - - - N - -S - - - Q - -

- - N - -S A - TP - -GA V TWS NV - S SS - -- T S - Y - -

t

30 40 50 60

S W L R E RQWD P K DW Y F F LWA S W [ST SSG V L Y G E S V G K N

G C W L F Q N S S SJL P Q PTFV V Y M AIS sH N K I T W D E LN S S K L

A MYW Y K Q - --S A EE]P P - L M F L Y N L E3Q L I R N E T P -

Y L F W Y V Q Y P - R Q G L Q L - - L L K Y YsG - D PWV Q V- -Y A N W V Q Q K P FDH L - - -WIT G - L I G G T N - - - N R - - A P G V P A R FY L H W Y Q Q --- K S G A S P K P -LI H R TW- - - N r- A ( V P AR F

GW]N W V K QFRIP G 0 G L E W I G Y - I N - - P G N G Y T K{N F --F- G K

7 0I1IL SSD S|RF S A M R D T N NFWPf IC ] SE K ARPF KNS G S L I G NS G S - -G S G TT TWLT V D K[]S

80 990 1 00

R P r80 - L S WlM NWEGK P ED S D F Y F C T - - - - - p

K Y V -L T L N K F S EIN E G Y Y F C- S - - - - TLN SK L L -L H S A V E DStA V Y F CAS S - - H G Q G V SS S nF H L R K A S H W S D S A V Y F C A V S - - G F A S

K A A - L T T G A Q T E D E A I Y F C A L W Y - - - - - - S

S Y S -L T S SW1E A E D D A T YMC Q W - - - s P

S T A Y M Q L R S L T S E D S|A V Y F C AR S V Y Y GG S|Y Y

LYT-3LYT-2TCR flTCR a

V A

VH

I I z0

K - M v G T K L T V VID L P A -- -PT K

V V -FIS S V V P V L Q KN SI l- T K V L

G N T LYF GIE IIEA - - L TF GS GTKV I L

N H - W F G|G|G T K L L- - - - |F G|SG T K L K

F DYWGQ jTj-jS

(

K T L K M K K K K Q C PR P S P V H P T G T SQ

1 4 I 5 6 I 7 (LYT-3 F PT H E- T Q K G L T C S LHTH- S L L V V CIL L LFF L G. V Y F Y

LYT2 P Q R P D C R1C R G S V K G G D F A C 1) I YLW A PI A G I C VJL L L S

1 80 1 0

LYT-3 C V R R R A R I - F M Q FH[ *LYT-2 L I I T L I C Y R S R R V C C P R P L V R Q E (; K P K 11 S E K I V *

FIG. 2. Amino acid sequence align-ment of Lyt-3, Lyt-2, and the variableregion of a mouse T-cell receptor chain(TCR 83) (86T1) (27), T-cell receptor a

chain (TCR a) (pHDS58) (19), immuno-globulin X light chain (VX) (MOPC104E)(28), immunoglobulin K light chain (VK)(S107) (29), and immunoglobulin heavychain (VH) (M36-65) (30) are shown. Thedashes indicate gaps introduced to maxi-mize the alignments. Homologies of theseproteins to Lyt-3 are boxed. The number-ing on top of the sequence is that of Lyt-3.The cysteines that could make an immu-noglobulin-like intrachain disulfide bondare marked by arrows.

LYT-3LYT-2TCR fiTCR aV AVaVH

LYT 3LYT-2TCR 6

TCR aV Av a

VH

LYT-3LYT-2TCR fTCR aV AvHVH

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Proc. Natl. Acad. Sci. USA 84 (1987) 4213

MOUSE LYT-3 ! VX U G I- I. I IJA. (Consensus) \\\ F F-L (I UI V-

JTa (TA39) 1) N[F G1 l - T IKLJT,8 (TM86) 1) F F- 1 I) I NX . 1. \ IL

FJT y (2C) Va- A iL N,JKc (Consensus) I I| i 1

JH (Consensus) V)1; I l; 1

L3T4 J' VI1i I_

L3T4 J \\ 1NI -

FIG. 3. Alignments of residues 104-115 of Lyt-3 with J-regionsequences from immunoglobulin X light chain (JX), T-cell receptor a(JTa) (TA39) (31), ,3 (JT/3) (TM86) (32), and y (JTy) (2C) (33) chains,immunoglobulin K light chain (JK), heavy chain (JH), and L3T4 (J andJ') (34). Residues that are identical to any of Lyt-3 residues areboxed, and dashes indicate gaps introduced to maximize the simi-larities. The J-region consensus sequences for immunoglobulinchains were determined from ref. 28.

AKR thymocytes digested with BamHI, EcoRI, HindIII, orXho I, and hybridized to the insert of XLY3C-23 showed nodifferences (Fig. 5A). Since the probe contains the entirecoding sequence, we conclude that major rearrangements arenot required for expression of Lyt-3. This is also supportedby our preliminary sequence data of the Lyt-3 gene in whichno intervening sequences were found between the immuno-globulin V and J regions (unpublished data).The data shown in Fig. 5A also suggest that only a single

Lyt-3 gene is present in the mouse genome. However,expressed mRNA inRNA gel blot analysis showed two bands(at 1.2 and 2.2 kb) (Fig. 5B). The 1.2-kb band corresponds tothe isolated cDNA, whereas the 2.2-kb band may be partiallyprocessed precursor RNA. Similar bands have been reportedin RNA gel blot analyses with mouse Lyt-2 cDNA and ratCD8 37-kDa chain cDNA (12, 16).

aLyt-2) (Lyt-2)(L-a13(Lyt-2) (Lyt-2) (Lyt-3)

NH2 NH2

26 26

S S

102 102

s

NH2

1

20

S

96

s S

2 ; 157 St t 15 g CVvv ~ ~~~~~~~~~~~~~~~~~~~~~ _ _

s

220COOH

1195(C0OH A 192

C (H1

FIG. 4. Schematic diagram showing three chains of the murineCD8 on the cell surface membrane. The a and a' chains of Lyt-2 are38 kDa and 34 kDa, respectively, and produced by alternativesplicing ofa primary mRNA transcribed from a single Lyt-2 gene (12,13). The murine CD8 structure is found as a heterodimer ofLyt-3 andeither the a or the a' chain of Lyt-2 (14). Cysteine residues are

indicated with an S, and possible N-linked glycosylation sites areindicated by black dots. A circle represents a predicted V-domain-like structure with a postulated intrachain disulfide bond.

A

1 2 3 4 5 6 7 8 9 10kb-12-10

B U) -.zD ha +

> Jl>- jI 0 or qrF U3 m :

-28S-8-76

-5

-4

-3

-18S

FIG. 5. (A) Autoradiogram of Southern blot analysis using a32P-labeled insert of XLY3C-23 as a probe. High molecular weightDNA isolated from AKR liver (lanes 1, 4, 6, and 9), AKR thymocytes(lanes 2, 5, 7, and 10), and Lyt-3-expressing AKR thymic lymphoma(lanes 3 and 8) were digested with restriction enzymes EcoRI (lanes6, 7, and 8), BamHI (lanes 4 and 5), HindIII (lanes 9 and 10), andXhoI (lanes 1, 2, and 3) and electrophoresed in 0.75% agarose. The sizesof comigrated 1-kilobase (kb) ladder markers are indicated in theright margin. (B) Autoradiogram of RNA gel blot of RNA fromdifferent sources hybridized to a nick-translated insert of XLY3C-23.Ten micrograms of cytoplasmic RNA from AKR thymocytes, spleencells, Lyt-3-negative R4L2(-) and -positive R4L2(+) AKR thymomacell lines.

DISCUSSIONThe murine CD8 is composed of two subunits, Lyt-2 andLyt-3, that are each encoded by two lightly linked genes. Thiscomplex in turn is tightly linked to the immunoglobulin Klocus. We (16) and others (12) have determined the primaryprotein structure of Lyt-2 by molecular cloning of the cDNAencoding Lyt-2. Considering their similarities in expression,presumed function, and genetic linkage, we assumed thatLyt-3 was also structurally similar to Lyt-2. However, wecould not observe any cross-hybridizing bands in Southernhybridization at low stringency when Lyt-2 cDNA was usedas a probe in our attempts to clone Lyt-3 (H.N., L. A.Herzenberg, unpublished results). In fact, comparison of theDNA and the deduced amino acid sequence of Lyt-2 andLyt-3 reveals surprisingly low homology between the twomolecules in spite of their biological similarities.The primary structure of Lyt-3 deduced from cDNA

nucleotide sequence shows that Lyt-3, as with Lyt-2, is atypical cell surface protein composed of a 21-residue hydro-phobic leader peptide, two extracellular domains, a trans-membrane hydrophobic domain, and a cytoplasmic domain.The membrane-proximal region of Lyt-3 is distinct in that

it contains a cluster of lysine residues (8 out of 31 residues).Although its biological significance is not clear, this lysinecluster may well be a suitable site for a proteolytic cleavage.This is in accord with the report that Lyt-3 antigens arecleaved more readily than Lyt-2 by trypsin treatment (14).This region is also rich in proline (5 out of 31 residues) similarto the immunoglobulin hinge region. On an assumption thatLyt-3 is a receptor molecule, this region might help orient theimmunoglobulin V-like domain away from cell surface mem-brane.From the biochemical study using endo-,8-N-acetylglucos-

aminidases, Lyt-3 was predicted to have only one asparagine-linked glycan unit (35). The deduced amino acid sequence ofthe Lyt-3 cDNA reveals one such unit (asparagine-13), andconfirms their data. This glycosylation site matches one ofthe three units in rat 37-kDa chain CD8 (asparagine-13). Therat 32-kDa chain CD8 has one N-linked glycosylation site,whereas its mouse counterpart Lyt-2 has three N-linked sites.There is no N-linked glycosylation in Leu-2, human CD8 (36).In contrast to Thy-1 or class II MHC in which all N-linked

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4214 Immunology: Nakauchi et al.

glycosylation sites are conserved, in CD8 molecules, N-linked sites are less conserved among species.The first external domain of Lyt-3 has homology to the

T-cell receptor and the immunoglobulin V-region sequences.Important structures to make the immunoglobulin domainstructure, such as two cysteines and a tryptophan, are foundappropriately located in this domain. In addition, Lyt-3 hasa short sequence that shows strong homology to J-regionsequences of immunoglobulin and T-cell receptor molecules.These data further support the idea that Lyt-3, immunoglob-ulin, and T-cell receptor genes have evolved from a commonancestral gene. Lack of somatic rearrangement for expres-sion of Lyt-3 and an absence of intervening sequence be-tween V and J sequences may suggest that the V-Jjoining canbe considered to be a reversal of an ancient, accidentalinsertion of a transposon-like DNA fragment carrying theinverted repeat sequences at the margins into a putativeancestral gene of immunoglobulin super gene family asproposed by Sakano et al. (37).Between mouse and rat, the Lyt-3 chain is more conserved

(75%) than is the Lyt-2 chain (64%) (17). In addition, Lyt-3has J-like sequences following immunoglobulin V-like do-main. These data may indicate the relative importance ofLyt-3 over Lyt-2. On the other hand, there is no knownequivalent of Lyt-3 in humans. It will be of particular interestto see whether there is a human equivalent of Lyt-3 at DNAlevel.

The authors are grateful to Drs. P. Johnson and A. Williams forproviding us with the rat OX8 cDNA clone (pX9.15) prior topublication. We also thank Dr. H. Saito for the cDNA library, Dr. Y.Arata for helpful discussions in the analysis of the protein structure,Dr. H. Yagita for providing AKR thymoma lines, and Dr. D. Loh forcritically reviewing the manuscript. This work was supported in partby grant-in-aid from the Mochida Memorial Foundation for Medicaland Pharmaceutical Research.

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