Proc. Natl. Acad. Sci. USAVol. 88, pp. 1746-1750, March 1991Immunology

A serum heterodimer from hagfish (Eptatretus stoutii) exhibitsstructural similarity and partial sequence identitywith immunoglobulin

(superfamily/immunological crossreactivity/amino acid sequence)

JUDITH VARNER*t, PETER NEAMEt, AND GARY W. LITMAN*t*Department of Pediatrics, University of South Florida, All Children's Hospital, 801 Sixth Street South, St. Petersburg, FL 33701; tLaboratory of MolecularGenetics, Tampa Bay Research Institute, St. Petersburg, FL 33716; and tShriners Hospital, Tampa, FL 33612

Communicated by Lewis Thomas, November 2, 1990

ABSTRACT We have isolated, characterized, and par-tially sequenced immunoglobulin from the most primitiveextant nonvertebrate craniate, the hagfish, a jawless fish thatmay have diverged from the vertebrate lineage more than 500million years ago. The 160-kDa protein, which is a minor serumcomponent, is composed oftwo different heavy chains of69 and74 kDa and a light chain of 29 kDa and resembles knownimmunoglobulin on the basis ofan equimolar ratio ofheavy andlight chains, N-linked glycosylation of heavy chains, presenceof intra- and interchain disulfide bonds, and polydispersity ofeach peptide chain. High molecular mass (polymeric) as well aslow molecular mass (monomeric) forms were isolated fromserum. The hagfish immunoglobulin is unique in that eachheterodimer is composed of two different heavy chains and twolight chains. The partial peptide maps and amino acid com-positions of the two heavy chains differ; the chains do notcrossreact immunologically. Slight crossreactivity of the 74-kDa heavy chain with antisera against purified shark immu-noglobulin and some conservation of amino acid sequences,including those surrounding a cysteine, suggest that the isolatedprotein is an immunoglobulin.

A fundamental issue in developmental immunology ad-dresses the origins of the immunoglobulin/T-cell antigenreceptor (TCR) recognition molecule. Whereas TCRs have sofar been identified only in mammals (1) and in birds (refs. 2,28), typical immunoglobulins have been isolated from ani-mals as phylogenetically primitive as the ratfish (3) and theshark (4-6). For all species characterized to date, a basicheterodimeric structure consisting of two identical heavychains and two identical light chainsjoined by disulfide bondshas been preserved, but the structures of polymeric immu-noglobulin forms, number of antibody classes, and the orga-nization of immunoglobulin genes vary significantly (7).Whereas the nature of immunoglobulin molecules in the

modem descendants of primitive jawed vertebrates is wellestablished, considerably less is known about the nature ofimmunoglobulin in primitive jawless fish (Agnathans). Im-mune organs are not well-defined (8), and it has been difficultto isolate and characterize molecules of the immune system.Heterodimeric molecules have been isolated from the sera oftwo species of hagfish, Eptatretus stoutii (9, 10) and Epta-tretus burgerii (11), but structural characterization of thesemolecules has been limited (9-11) as has that of immunoglo-bulin-like serum proteins in Petromyzon marinus (sea lam-prey), a member of the other lineage of extant jawless fish.

In a continuing effort to identify the prototype antigenrecognition molecules and to characterize the nature of theevolution of the genomic organization and rearrangement

mechanism(s) of the immunoglobulin multigene family (7),we have isolated and characterized an immunoglobulin fromthe Pacific hagfish, E. stoutii. Evidence is presented that thehagfish heterodimer is an immunoglobulin on the basis ofsimilar physical properties, slight immunological crossreac-tivity with antisera raised against horned shark (Heterodon-tus francisci) immunoglobulin, and localized amino acidsequence identity with both immunoglobulins, TCRs, andother members of the immunoglobulin superfamily, but it isunique in its low serum concentration and the apparentpresence of two different heavy chains in each molecule.

MATERIALS AND METHODSIsolation of Serum. E. stoutii were obtained from Pacific

Biomarine (Marina del Rey, CA). Animals were maintainedin seawater over a 2-day period at 40C. They were anesthe-tized in MS222 (100 mg/liter) and bled from the tail sinus.Pooled blood was centrifuged at 40C at 700 x g for 10 min andthen at 10,000 x g for 30 min to remove cells and cellulardebris. Serum was delipidated by centrifugation at 50,000 xg for 2 hr in a 75Ti rotor at 40C and stored at -200C.

Iodination ofImmunoglobulin. Hagfish immunoglobulin (10jig) in 100 dul of 0.15 M NaCl/0.05 M Tris, pH 8.0 wasiodinated by the addition of 0.5 mCi (1 Ci = 37 GBq) of Na125Iand one Iodo-Bead (Pierce) for 30 min. Free iodine wasremoved by gel-filtration chromatography, and the iodinatedimmunoglobulin was stored at -20°C.

Preparation of Antisera. Two milligrams of purified hagfishimmunoglobulin (see Results) was precipitated by the addi-tion of 5 volumes of acetone to 1 volume of protein at -20°Cfor 1 hr and centrifuged at 10,000 x g for 10 min at 4°C.Precipitated immunoglobulin was dried, boiled in SDS sam-ple buffer containing 5% 2-mercaptoethanol, and then appliedto preparative SDS/7.5% polyacrylamide gels. The gels werelightly stained with Coomassie brilliant blue, and bandscontaining heavy and light chains were excised and pulver-ized. Two rabbits each were immunized with the bandscontaining both heavy chains, the 74-kDa heavy chain (H1),or the light chain.

Protein Sequencing. Reduced and alkylated immunoglob-ulin chains were separated on a Superose 12 FPLC column(Pharmacia) at a flow rate of 0.5 ml/min. Separated chainswere digested with sequencing grade endoprotease Lys-C(Boehringer Mannheim) in 50 mM Tris Cl at pH 8.0. Peptideswere separated on a Brownlee RP300 reverse-phase HPLCcolumn (2.1 x 30 mm) at a flow rate of 200 ,ul/min (monitoredat 220 nm), eluted with a gradient of 0-70%o acetonitrile in0.1% trifluoroacetic acid over 45 min, and sequenced on anApplied Biosystems 477A gas-phase sequencer using the

Abbreviations: TCR, T-cell antigen receptor; TBST, Tris-bufferedsaline containing Tween 20.

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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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program FAST-1 as described in the manufacturer's literature.Phenylthiohydantoin amino acids were analyzed on an Ap-plied Biosystems 120A on-line HPLC using a 2.1-mm diam-eter column as supplied by the manufacturer.

RESULTSPurification of Putative Immunoglobulin. Delipidated hag-

fish serum was fractionated by successive precipitations with30%, 45%, and 60% (wt/vol) saturated ammonium sulfate,and precipitated proteins were analyzed by comparing re-ducing and nonreducing SDS/polyacrylamide gel profiles(data not shown). Nonreduced molecules of 150-180 kDa inthe 30% precipitate yielded bands of -72 and 29 kDa uponreduction that were identified as candidate immunoglobulins.This resuspended precipitate was applied to a Pharmacia

FPLC Mono Q (anion-exchange) column. Approximately 1%of the protein was eluted before application of a linear saltgradient (Fig. 1A) and consisted primarily of an -160-kDaband on nonreducing gels (Fig. 1B) and 72- and 29-kDa bandson reducing gels (Fig. 1C). Application of a linear salt gra-dient removed the remainder of the serum protein but onlysmall additional amounts of the reduction-sensitive 160-kDaheterodimer. The immunoglobulin heterodimer (0.3% of thetotal serum protein) eluted from a Pharmacia FPLC Superose12 column at a position corresponding to a native molecularmass of -160 kDa (data not shown). Some polymeric het-erodimer detected in the void volume of the Superose 12column chromatographed on an FPLC Superose 6 columnprimarily as a tetramer, thus resembling the polymeric immu-noglobulin of bony fishes (7).

A

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VOLUME (ml)

7 9 11 13 15 17 19 21

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FIG. 1. Anion-exchange chromatography of hagfish serum fractions. Delipidated hagfish serum was adjusted to 30o with saturatedammonium sulfate, incubated 2 hr on ice, and centrifuged at 10,000 x g for 30 min at 40C. The precipitate was resuspended in and dialyzed against0.15 M NaCl/0.05 M Tris HCI, pH 8.0 and applied to a Pharmacia FPLC Mono Q HR 5/5 column equilibrated in the same buffer. The columnwas developed in buffer followed by a 20-ml linear gradient of 0.15-1 M NaCl in 0.05 M Tris at pH 8.0 and analyzed by absorbance at 280 nm(A). One hundred microliters of each 1-ml fraction was analyzed by SDS/PAGE under nonreducing (B) and reducing (C) conditions. Molecularmass markers (in kDa) are indicated at left.

BFraction no. 3

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On an SDS/5-7.5% polyacrylamide gel (see Fig. 2B, lane1), the purified hagfish immunoglobulin resolved into onemajor band of -160 kDa and minor bands of 180, 170, and 140kDa. Upon reduction (see Fig. 2B, lane 2), these yielded onlythe two heavy-chain bands of SDS/PAGE molecular massesof 74 (H1) and 69 (H2) kDa and a single light chain of 29 kDa.An 87-kDa band was a minor contaminant. To determine ifthe several nonreducing gel forms represented different com-

AS |65 NR

R210

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29--

B1 2 3 4 5 6

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FIG. 2. Two-dimensional nonreducing/reducing gel electropho-resis of hagfish immunoglobulin. Ten-microgram aliquots of purifiedhagfish immunoglobulin were acetone precipitated, boiled 3 min inreducing (R) or nonreducing (NR) SDS sample buffer, and electro-phoresed in individual lanes on an SDS/5-7.5% PAGE gradient gel.One lane containing nonreduced immunoglobulin was excised, in-cubated in reducing SDS sample buffer, and electrophoresed per-pendicular to the original direction of electrophoresis on a 5-7.5%gradient gel. (B) Immunoblotting and immunoprecipitation of hagfishimmunoglobulin with heavy-chain-specific antisera. Ten-microgramaliquots of purified hagfish immunoglobulin were electrophoresed ona 7.5% polyacrylamide gel under nonreducing (lane 1) or reducing(lane 2) conditions and stained with Coomassie brilliant blue. Tenmicrograms of reduced immunoglobulin was electrophoreticallyblotted from a 7.5% gel to nitrocellulose in 25 mM Tris/192 mMglycine at 400 mA for 4 hr (12), blocked for 1 hr at room temperaturein 1% bovine serum albumin in Tris-buffered saline containing Tween20 (TBST), incubated 4 hr in a 1:200 dilution of a polyclonal antiseraraised against hagfish immunoglobulin subunit H1, washed in TBST,incubated with iodinated protein A (New England Nuclear), washedin TBST, and autoradiographed (lane 3). Two 100-,ul aliquots ofiodinated hagfish immunoglobulin were adjusted to 0.5% NonidetP-40 (lanes 4 and 5), precleared with 10 Al of rabbit nonimmune sera,and immunoprecipitated with 10 ,ul of anti-H1 specific antisera andprotein A-Sepharose as described (13). A similar aliquot of iodinatedimmunoglobulin was denatured by boiling 3 min in 0.5% SDS/2 mMdithiothreitol, adjusted to a final concentration of0.5% Nonidet P-40,and similarly immunoprecipitated (lane 6). The immunoprecipitateswere boiled 3 min in 50 Al of nonreducing (lane 4) or reducing (lanes5 and 6) SDS sample buffer, electrophoresed on SDS/7.5% PAGEgels, and autoradiographed. Prestained molecular mass markers (inkDa) are indicated by arrowheads.

binations of heavy and light chains- (H1L)2 or (H2L)2-ormixed heterodimers- (H1H2)L2-of differing mobilities, asingle gel lane of nonreduced immunoglobulin was incubatedin reducing SDS sample buffer and electrophoresed perpen-dicular to the original direction of electrophoresis into asecond 5-7.5% polyacrylamide gel. Each of the high molec-ular mass bands reduced to yield both ofthe heavy chains andthe light chain (Fig. 2A), suggesting that only (H1H2)L2 formsexist. If any nonreduced forms (including the major compo-nent) had consisted of either one or possibly both homodimerforms, discrete H1 or H2 bands or diagonal bands would havebeen observed. The presence of several molecular mass var-iants composed ofthe same number and apparent proportionsof subunits may be accounted for by slightly different con-formational states (perhaps imparted by different SDS bind-ing constants) arising from sequence microheterogeneity.The remainder of the bands on the two-dimensional gelappear to be minor nonimmunoglobulin contaminants.The nature of the hagfish immunoglobulin heterodimer was

examined further by Western blotting and immunoprecipita-tion experiments. Rabbit antisera specific for H1, based onanalytical Western blotting analyses, were obtained (Fig. 2B,lanes 2 and 3). When purified immunoglobulin was iodinated,denatured, and immunoprecipitated with H1-specific anti-sera, only H1 was precipitated (Fig. 2B, lane 6). In contrast,when immunoglobulin was iodinated and immunoprecipi-tated without denaturation with the same antisera, one nonre-ducing band of 160 kDa, which yielded H1, H2, and lightchains in equal proportions upon reduction, was precipitated(Fig. 2B, lanes 4 and 5). These results are consistent with thetwo-dimensional gel analyses described above.The failure of the two chains to crossreact immunologically

suggests that they are distinct peptide chains. Striking dif-ferences are observed when H1 and H2 are digested withchymotrypsin in Cleveland (14) peptide map analyses (datanot shown). In addition, the amino acid compositions of thetwo heavy chains differ; H2 contains significantly moreproline (2.3-fold) and glycine (1.5-fold) and less tyrosine(0.3-fold) and lysine (0.6-fold) than H1 (values not shown).The distribution of amino acids, particularly for H1, are inaccordance with the average amino acid compositions ofimmunoglobulins (15).

Hagfish Immunoglobulin Structurally Resembles OtherImmunoglobulins. Immunoglobulins electrophoresed onSDS/5-7.5% polyacrylamide gels under nonreducing condi-tions (Fig. 3A) were compared with immunoglobulins thatwere mildly reduced or were completely reduced and alky-lated. Mildly reduced immunoglobulin from horned shark,Heterodontus francisci, which electrophoresed as one bandat 180 kDa, yielded a heavy chain of 72 kDa and a light chainof 25 kDa. When completely reduced and alkylated, theseheavy and light chains migrated with reduced mobility at 79kDa and 30 kDa, consistent with cleavage of intrachaindisulfide bonds. Mildly reduced hagfish immunoglobulinyields two heavy-chain bands and one light-chain band, eachof which migrates with lesser mobility, at 76, 72, and 38 kDa,when more extensively reduced and alkylated. Upon reduc-tion and alkylation, the shift in mobility of the light chain wasconsiderably greater than the shift in mobility of the Heter-odontus light chain. This may be due to the presence of largerdomains and/or additional disulfide bonds in the hagfish lightchain. The heavy-chain band shifts, while significant, werenot as marked as those of either the light chain or the controlhorned shark heavy chains, perhaps due to the presence ofsmaller domain sizes or fewer disulfide bonds.When either hagfish or horned shark immunoglobulin was

treated with endoglycosidase F (16) under denaturing condi-tions (Fig. 3B) and electrophoresed on SDS/5-7.5% poly-acrylamide gels, horned shark heavy chains shifted from anapparent molecular mass of 72 kDa to 62 kDa, hagfish H1

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AE. stout ii H. f rancisci

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FIG. 3. (A) Reduction and alkylation. Three 10-I.tg aliquots eachof hagfish (E. stoutii) or homed shark (H. francisci) immunoglobulinwere acetone precipitated and resuspended in 25 Al of 0.15 MNaCI/0.05 Tris HCl, pH 8.0. One aliquot was not treated (NR), onewas mildly reduced with 1 mM dithiothreitol (R), and one wascompletely reduced and alkylated by addition of20mM dithiothreitoland 0.5% SDS prior to boiling for 3 min, incubation at roomtemperature 1 hr and addition of 1 mg of iodoacetic acid and 7.5 ,lof 2 M Tris at pH 6.8 (R,A). All samples were adjusted to 0.5% SDSand boiled 3 min prior to electrophoresis on SDS/5-7.5% PAGE gels.S, standards. (B) Deglycosylation. Twenty micrograms of purifiedhagfish (E. stoutii) or homed shark (H. francisci) immunoglobulinwas acetone precipitated and resuspended in 60 ul of 0.5% SDS/1%2-mercaptoethanol/0.1 M Tris-HCI, pH 8.8/0.01 M EDTA, boiled 3min, and then cooled on ice. Six microliters of l1o Nonidet P-40 wasadded to each sample. The samples were divided into two aliquots;one (+) received 5 Al of endoglycosidase F (endo F) (Calbiochem, 20unit/pl), while the other (-) remained untreated. Both were incu-bated overnight at 37°C. Twelve microliters of a solution of 5%SDS/25% glycerol/20%o 2-mercaptoethanol/0.05% bromphenol bluewas added to each sample; these were boiled 3 min, electrophoresedon an SDS/5-7.5% PAGE gel, and stained with Coomassie blue.Molecular mass markers (in kDa) are indicated by arrowheads.

shifted from 74 kDa to 69 kDa, and hagfish H2 shifted from 69kDa to 66 kDa. Treatment with endoglycosidase F had noapparent effect on the mobility of either hagfish or homedshark light chains, although minor molecular mass shifts maynot be discernible in an SDS gel at this percentage of acryla-mide. The magnitude of the shift in mobility of hagfish heavychains is consistent with one to two glycans per heavy chain,based on the average size of biantennary N-linked glycans.By quantitation ofchromatography separated, reduced and

alkylated, guanidine hydrochloride-denatured hagfish immu-noglobulin heavy and light chains (data not shown), heavyand light chains are in equal molar proportions (-1:1).Densitometric scanning of SDS/polyacrylamide gel-sep-arated, Coomassie blue-stained heavy and light chains indi-cates that 1.5 moles of heavy chains are present for every

FIG. 4. Immunological crossreactivity. Two micrograms of pu-rified hagfish or homed shark immunoglobulin was boiled in reducingSDS sample buffer, electrophoresed on SDS/7.5% polyacrylamidegels, and transferred electrophoretically to nitrocellulose in 25 mMTris/192 mM glycine for 4 hr at 400 mA. Blots were blocked for 1 hrin 1% bovine serum albumin in TBST, incubated 4 hr in a 1:200dilution of rabbit antisera against hagfish immunoglobulin (Left) orhomed shark immunoglobulin (Right), washed in TBST, incubated2 hr in iodinated protein A, washed in TBST, and autoradiographed.

mole of light chains (data not shown). Immunoglobulin lightchains are known, however, to bind disproportionately lessCoomassie blue than their molecular masses would warrant.

Isoelectric focusing of separated subunits (data not shown)indicates that all three hagfish immunoglobulin chains arepolydisperse (as would be expected of immunoglobulin sub-units). When reduced immunoglobulin was subjected totwo-dimensional O'Farrell gel analysis (17), H1 separatedinto at least 10 different isoforms, 7 ofwhich fell into a narrowpH range; H2 and light chains were dispersed into at least asmany isoforms as H1. All of these data suggest that the hag-fish heterodimer is an immunoglobulin-like molecule.

Hagfish Immunoglobulin Is Related to Horned Shark Immu-noglobulin. Rabbit antisera raised to SDS/polyacrylamidegel-purified hagfish immunoglobulin heavy chains crossreactslightly with homed shark immunoglobulin chains on West-ern blots (Fig. 4 Left). Rabbit antisera raised to intact homedshark low molecular mass immunoglobulin crossreactslightly with Western blotted hagfish immunoglobulin H1heavy chains (Fig. 4 Right). The sequences of several hagfishpeptides further suggest a relationship between the hagfishheterodimeric protein and other immunoglobulin superfamilymolecules. Although much of the peptide sequence obtainedcould not be aligned with that ofany known immunoglobulin,limited sequence identities, such as those between a hagfishlight-chain peptide, Heterodontus light chain (18), and humanA light chains (19) in a region surrounding a cysteine, areapparent (Fig. SA). One heavy-chain peptide (derived frommixed heavy chains) could be aligned with a mouse heavy-chain variable region (20) (Fig. 5B); another heavy-chain

AHagfish light chain [[ES I NLPSPPASWCOV S|

Human lambda light chain [!TPEIWK S HR S Y SC Q VT

Heterodontus light chain V PATAWN K G S S Y jS1D

BHagfish heavy chain IS T A Y|G NPA AMQOPE E

Mouse heavy chain (VH 1)

CHagfish heavy chain

Human heavy chain (CH 1)

Heterodontus heavy chain (CH 1)

Human T-cell receptor

Is T MUS SLT[IT

MN P [I S S P _ V V

[dY S LS S VI VTI VI

TYTR|SSIQILIT

S Y C RR RR

FIG. 5. Comparison of hagfish light- and heavy-chain sequenceswith corresponding human, shark, and mouse sequences.

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peptide could be aligned equally well with human (21) andshark (22) heavy-chain constant region exon 1 and humanTCR f3 chain (23) (Fig. 5C) or with shark light-chain frame-work 1 of the variable region (data not shown); these latterassignments are consistent with a significant degree of inter-as well as intradomain relatedness within immunoglobulins.

DISCUSSIONAttempts to identify hagfish immunoglobulin genes fromgenomic or cDNA libraries using a number of different sharkor mammalian nucleic acid probes specifying highly con-served segments of the variable (24), constant, and trans-membrane (22) regions ofheavy chains as well as variable andconstant regions of light chains (18) have been unsuccessful(J.V. and G.W.L., unpublished observations). While nega-tive, these observations led us to postulate that the sequencesof hagfish and higher vertebrate immunoglobulin genes differby more than the 55-60% nucleotide sequence identityrequired to support specific hybridization. The alternativeapproach that was pursued and is reported here involved theinitial isolation of the hagfish immunoglobulin protein. Themolecule that has been identified in this study resembles aserum heterodimer that had been described previously (9-11); however, absolute identification of the molecule as animmunoglobulin in these studies was not possible based onthe limited amount of structural information that was avail-able. In the studies reported here, we isolated and charac-terized a minor hagfish serum component and suggest on thebasis of similar physical properties as well as both serologicalcrossreactivity and partial peptide sequence identities that itis hagfish immunoglobulin. This serum heterodimer resem-bles a molecule that can be elicited in slightly elevatedquantities upon immunization (9).The hagfish serum heterodimer is unique compared with all

other immunoglobulins studied to date in that two differentheavy chains combine with two (presumably) identical lightchains in each molecule. The two heavy chains are notbiosynthetic intermediates of each other; deglycosylation byendoglycosidase F reduces the molecular mass of each chainindependently of the other, and peptide mapping and compar-ison of amino acid compositions reveal that the sequences ofthe chains differ.

Hagfish are unusual in that they have been shown toefficiently reject skin grafts (25) while producing serumantibody titers only 2-fold higher than resting titers (9, 10), inthat their immunoglobulin is only 0.3% of serum protein(compared with 50% for sharks), and in that immunoglobulin-surface-positive lymphocytes react in a mixed lymphocyteresponse assay (26, 27). It has been suggested that T and Bcells are not separate in this species (26). The structure ofhagfish immunoglobulin is unique in that different heavychains are associated within a single heterodimer. The pres-ence of dissimilar polypeptide chains in a single immuno-globulin family heterodimer is a feature of both a//3 and y/STCRs, although these are not associated with light chains.The limited amount of peptide sequence information avail-able to date suggests that hagfish immunoglobulin is asrelated to TCRs as to immunoglobulin, but considerably moreprotein data will have to be obtained before the full extent ofthese relationships can be realized. While it is premature tospeculate that the hagfish heterodimer may share functionalproperties with TCRs, these results establish a precedent forthis type of molecular configuration in an immunoglobulinmolecule. Similarities between the immunoglobulin genesfound in horned shark and TCR genes identified in highervertebrates also have been established (18), but the overallstructure of the horned shark immunoglobulin clearly resem-bles that of mammalian antibodies rather than TCRs.

Phylogenetic relationships have to be considered whenassessing the similarities between proteins found in primitivespecies and in more recent forms. Hagfish represent a phylo-genetic lineage distinct from that shared by the modem highervertebrates. Given the enormous separation in phylogenetictime between the divergence of the craniate lineage from thatof jawed vertebrates, atypical molecules could be expected.Continued characterization of the hagfish immunoglobulin,cellular immune system components, and immunoglobulingenes will provide insight into the origins of progenitors of themodern immune recognition molecules and the evolutionarychanges that have altered these during phylogenetic time.We thank Michael Shamblott for initial protein studies, preparation

ofuseful antisera, and helpful discussions; Drs. James Kaufman, ChrisAmemiya, and Guy Bradley for helpful discussions; and Mrs. BarbaraPryor for excellent editorial assistance. This work was supported byNational Institutes of Health Grant A123338. G.W.L. is the HinesProfessor of Pediatric Research, University of South Florida.1. Kronenberg, M., Siu, G., Hood, L. E. & Shastri, N. (1986) Annu.

Rev. Immunol. 4, 529-591.2. Sowder, J. T., Chen, C.-L. H., Ager, L. L., Chan, M. M. &

Cooper, M. D. (1988) J. Exp. Med. 167, 315-322.3. Sanchez, G. A., Gajardo, M. K. & De loannes, A. E. (1980) Dev.

Comp. Immunol. 4, 667-678.4. Marchalonis, J. & Edelman, G. M. (1966) Science 154, 1567-1568.5. Frommel, D. W., Litman, G. W., Finstad, J. & Good, R. A. (1971)

J. Immunol. 106, 1234.6. Clem, L. W. & Small, P. A. (1967) J. Exp. Med. 125, 893-920.7. Litman, G. W., Shamblott, M. J., Haire, R., Amemiya, C.,

Nishikata, H., Hinds, K., Harding, F., Litman, R. & Varner, J.(1989) in Progress in Immunology VII, ed. Melchers, F. (Springer,Berlin), pp. 361-368.

8. Good, R. A., Finstad, J., Pollard, B. & Gabrielsen, A. E. (1966) inPhylogeny ofImmunity (Univ. Florida Press, Gainesville, FL), pp.149-169.

9. Raison, R. L., Hull, C. J. & Hildemann, W. H. (1978) Proc. Natl.Acad. Sci. USA 75, 5679-5682.

10. Raison, R. L., Hull, C. J. & Hildemann, W. H. (1978) Dev. Comp.Immunol. 2, 253-262.

11. Kobayashi, K., Tomonaga, S. & Hagiwara, K. (1985) Mol. Immu-nol. 22, 1091-1097.

12. Towbin, H., Staehlin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci.USA 76, 4350-4354.

13. Kaufman, J. F., Skjoedt, K., Salomonsen, J., Simonsen, M., DuPasquier, L., Parisot, R. & Riegert, P. (1990) J. Immunol. 144,2258-2272.

14. Cleveland, D. W., Fischer, S. G., Kirschner, M. W. & Laemmli,U. K. (1977) J. Biol. Chem. 252, 1102-1106.

15. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M. & Gottes-man, K. (1987) Sequences of Proteins of Immunological Interest(U.S. Dept. Health and Human Services, Washington).

16. Edler, J. & Alexander S. (1982) Proc. Natl. Acad. Sci. USA 79,4540-4544.

17. O'Farrell, P. (1975) J. Biol. Chem. 250, 4007-4021.18. Shamblott, M. J. & Litman, G. W. (1989) Proc. Nat!. Acad. Sci.

USA 86, 4684-4688.19. Langer, B., Steinmetz-Kayne, M. & Hilschmann, N. (1968) Z.

Physiol. Chem. 349, 945-951.20. Gilliam, A. C., Shen, A., Richards, J. E., Blattner, F. R., Mushin-

ski, J. F. & Tucker, P. W. (1984) Proc. Natl. Acad. Sci. USA 81,4164-4168.

21. Ellison, J. & Hood, L. (1982) Proc. Natl. Acad. Sci. USA 72,1984-1988.

22. Kokubu, F., Hinds, K., Litman, R., Shamblott, M. J. & Litman,G. W. (1988) EMBO J. 7, 3413-3422.

23. Morinaga, R., Fotedar, A., Sengh, B., Wegmann, T. G. & Taamaki,T. (1985) Proc. Natl. Acad. Sci. USA 82, 8163-8167.

24. Litman, G. W., Berger, L., Murphy, K., Litman, R. T., Hinds,K. R., Jahn, C. L. & Erickson, B. W. (1985) Proc. Natl. Acad. Sci.USA 82, 2082-2086.

25. Hildemann, W. & Thoenes, G. H. (1969) Transplantation 7,506-521.26. Raison, R. L., Seppelt, I., Hanley, P. & Trent, R. (1989) Dev.

Comp. Immunol. 13, 345.27. Gilbertson, P., Wotherspoon, J. & Raison, R. L. (1986) Dev. Comp.

Immunol. 10, 1-10.28. Tjoelker, L. W., Carlson, L. M., Lee, K., Lahti, J., McCormack,

W. T., Leiden, J. M., Chen, C.-I. H., Cooper, M. D. & Thompson,C. B. (1990) Proc. Natl. Acad. Sci. USA 87, 7856-7860.

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embe

r 24

, 202

0