Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1886-1891, March 1996 Immunology

The joining (J) chain is present in invertebrates that do not express immunoglobulins TOMIHISA TAKAHASHI*, TAKASHI IWASE*, NOBUKO TAKENOUCHI*, MANABU SAITO*, KUNIHIKO KOBAYASHIt, ZINA MOLDOVEANUt, JIRI MESTECKYt, AND ITARU MORO*§ *Department of Pathology, School of Dentistry, Nihon University, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo 101, Japan; tDepartment of Pediatrics, Hokkaido University, School of Medicine, Kita 15 Jyo-Nishi 7, Kita-ku, Sapporo 060, Japan; and tDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 Communicated by Marion E. Koshland, University of California, Berkeley, CA, November 22, 1995 (received for review August 19, 1995)

ABSTRACT Joining (J) chain is a component of poly- These findings indicate that the may possess functions meric, but not monomeric, immunoglobulin (Ig) molecules in addition to Ig polymerization. and may play a role in their polymerization and transport In human ontogeny, the J chain has been detected in fetal across epithelial cells. To date, study of the J chain has been liver at the sixth gestational week (18), and its expression was confined to vertebrates that produce Ig and in which the J found to precede synthesis of the ,u chain at least by 1 week. chain displays a considerable degree of structural homology. Phylogenetically, the J chain has been detected in many The role of the J chain in Ig polymerization has been ques- vertebrates including fish, amphibia, reptiles, birds, and mam- tioned and, since the J chain can be expressed in lymphoid mals, all ofwhich produce Ig (1-3, 19). In mammals, nucleotide cells that do not produce Ig, it is possible that the J chain may sequences encoding human (4), mouse (20), and bovine (21) have other functions. To explore this possibility, we have J chains, as well as the sequences of rabbit (22) and surveyed J-chain , mRNA, and protein expression by bullfrog (23) J chains have been reported. However, expression using reverse transcriptase-coupled PCR, Northern blot anal- of the J chain has not been investigated in invertebrate species and in invertebrate species that do in which the defense systems against foreign antigens are ysis, immunoblot analysis considerably different from those in vertebrates. Therefore, we not produce Ig. We report that the J-chain gene is expressed have examined the phylogeny of J chains in various animal in invertebrates (Mollusca, Annelida, Arthropoda, Echino- species, especially in invertebrates that do not produce Ig. dermata, and Holothuroidea), as well as in representative We detected the J-chain gene in various invertebrates and vertebrates (Mammalia, Teleostei, Amphibia). Furthermore, compared the earthworm J-chain DNA sequence with those of J-chain cDNA from the earthworm has a high degree of mammals. The J-chain gene was detected in 12 invertebrate homology (68-76%) to human, mouse, and bovine J chains. species but not in the sea anemone or amoeba. Furthermore, Immunohistochemical studies reveal that the J chain is local- the J-chain cDNA sequence determined from earthworm ized in the mucous cells of body surfaces, intestinal epithelial RNA by reverse transcriptase (RT)-coupled PCR displayed a cells, and macrophage-like cells of the earthworm and slug. high degree of homology to J chains from mammals, which are This study suggests that the J chain is a primitive polypeptide known to produce Ig. These results indicate that the J chain is that arose before the evolution of Ig molecules and remains a primitive protein that is highly conserved in invertebrates and highly conserved in extant invertebrates and vertebrates. vertebrates.

The joining (J) chain was first identified as an additional MATERIALS AND METHODS component of human colostral secretory IgA (1-3). An acidic polypeptide with a molecular mass of 15 kDa, it is covalently Invertebrate and Vertebrate Species and Human Cell Lines. linked to the penultimate Cys residues of the a and ,i chains The following invertebrate and vertebrate species were used in of dimeric IgA and pentameric IgM molecules (1-3). The J this study: amoeba (Acanthamoeba castellani), sea anemone chain has been detected by a variety of techniques in mam- (Metridium senile), sea cucumber (Stichopus japonica), slug malian stem cells, pro-B, pre-B, and B cells, plasma cells, and (Incilaria bilineata), snail (Gastropoda littorinidae), oyster thymic T cells (4-13). Furthermore, recombinant technologies (Crassostrea gigas), clam (Meretrix lusoria), earthworm (Eisenia using insects (14) and plants (15) have demonstrated that the foetida), sea squirt (Ascidia japonica OKA), shrimp (Penaeus chain an role in the formation of secreted japonicus), crab (Portunus trituberculatus), silkworm (Bombyx J plays important mori), spider (Nepila clavata), butterfly (Artogenia rapae- dimeric IgA. Accordingly, the J chain is associated with crucivora), lamprey (Lampetra japonica), sardine (Sardinops extracellularly secreted polymeric immunoglobulin (Ig) and is melanostictus), and lizard (Eumeces latiscutatus). The Epstein- thought to function in the polymerization of Ig molecules and Barr virus-transformed human B-cell line Daudi was used as in their ability to bind the polymeric Ig receptor (pIg-R) on a J-chain-positive control (24), and the erythroleukemia cell epithelial cells (1-3). line K562 was a negative control. However, it has been reported that IgG-secreting cells and RT-PCR Procedure. Oligonucleotide primers were synthe- B-cell precursors in which Ig gene rearrangement did not occur sized by using a DNA synthesizer (Applied 13iosystems) from are able to produce the J chain (4-7, 10-12). Other studies the regions of the published DNA and amino acid sequences revealed that polymeric IgM is secreted by a glioma cell line common to human (4), mouse (20), rabbit (22), and bullfrog transfected with IgM heavy and light chain cDNA, even though (23) J chains. Primer sites were determined by using GenBank this cell line does not produce J chains (16). In IgM, substi- and EMBL data bases (GENETYX-MAC Software Develop- tution of a Ser for the Cys involved in J-chain binding results ment, Tokyo). Primer sequences designed for RT-PCR were in the formation of polymeric IgM without a J chain (17). as follows: pJ-1, 5'-GAGGACATTGTGGAGAGAAA-3';

The publication costs of this article were defrayed in part by page charge Abbreviations: J chain, joining chain; RT-PCR, reverse transcriptase- payment. This article must therefore be hereby marked "advertisement" in coupled PCR; Ig, immunoglobulin. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1886 Downloaded by guest on October 1, 2021 Immunology: Takahashi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1887 pJ-2, 5'-TAGCACTTGTTTCTGTCATA-3'; pJ-3, 5'- CTGTAAAAAATGTGATCC-3'; pJ-4, 5'-GTCAGGATG- GCAGGCAT-3'; ,B-actin, 5'-CCTTCCTGGGCATG- GAGTCCTG-3' and 5'-GGAGCAATGATCTTGATCTTC- 3'. To test the ability of each primer to detect the J-chain gene in earthworm RNA, several sets of synthesized primers were used in RT-PCRs. Total RNA was extracted from -1.0 g of earthworm tissue or 1 x 106 Daudi cells by the acid guani- dinium thiocyanate/phenol/chloroform method (25). RT- B 1 1) I A 7 Q 110)11 111 1A 1 PCR was performed as follows: random-hexamer-primed sin- gle-stranded cDNA was synthesized from 1.0 j,g of total RNA in a final volume of 10 ,ul with 2 units of Rous sarcoma- 310 bp -- associated virus 2 reverse transcriptase (Takara Shuzo, Kyoto) at 42°C for 60 min. An aliquot (1.0 t,l) of the reaction mixture was diluted with 50 ,ul of PCR buffer containing the various primer sets described above. The PCR was then performed by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 using 2.5 units of Thermus aquaticus DNA polymerase (Strat- agene). The procedure for PCR was as follows: denaturing, 1 min at 95°C; annealing, 2 min at 55°C; extension, 3 min at 72°C. 310 bp -O PS a This cycle was repeated 35 times. The amplified DNA was electrophoresed on a 1.7% agarose gel, stained with ethidium bromide, and visualized and then photographed on a UV illuminator. After electrophoresis, the blot was transferred to C 1 2 3 4 5 6 7 8 9 10 a Hybond nylon membrane (Amersham) under standard con- ditions (26). Hybridization was performed in hybridization 1.4 Kb- buffer containing salmon sperm DNA (100 ,tg/ml), 6x SSC, 0.5% SDS, and 5x Denhardt's solution at 55°C for 16 h by using a human J-chain cDNA (kindly provided by E. E. Max, Laboratory of Cell and Viral Regulation, Food and Drug D 1 2 3 4 5 6 7 Administration Center for Biologics Evaluation and Research, Bethesda) encoding exons 1-4 labeled with [a-32P]dCTP by a 1.4Kb * random priming kit (Takara Shuzo). After hybridization, the membrane was washed three times at a high stringency (0.1 x SSC/0.2% SDS) at 55°C for 15 min and then exposed to Kodak FIG. 1. J-chain in earthworm by RT-PCR. (A) X-Omat film for 16 h at -80°C. 4X174/Hae III DNA as size marker (lanes 1 and 16); PCR products DNA Sequencing Analysis of the Earthworm J Chain. The from earthworm (lanes 2, 5, 8, and 11), from Daudi cells as a earthworm J chain was sequenced by using the TA cloning kit J-chain-positive control (lanes 3, 6, 9, 12, and 14), and from K562 cells (Invitrogen). Briefly, the RT-PCR product amplified by as a J-chain-negative control (lanes 4, 7, 10, 13, and 15). Combinations primer set pJ-1 and pJ-4 (Fig. IA, lane 5) was purified of primer sets used for PCR were pJ-1 and pJ-2 (lanes 2-4), pJ-1 and (Ultrafree centrifuge tube, Millipore) and subcloned into the pJ-4 (lanes 5-7), pJ-3 and pJ-2 (lanes 8-10), and pJ-3 and pJ-4 (lanes 11-13); /3-actin primers were used as positive control (lanes 14 and 15). plasmid vector pCR1000 (Invitrogen). After restriction en- (B) Analysis of J-chain gene expression in invertebrate and vertebrate zyme analysis with BamHI and HindlIl (Toyobo, Osaka), three species by RT-PCR. Animals used were sardine (lane 2), silkworm positive clones with '312-bp fragments were selected. DNA (lane 3), shrimp (lane 4), crab (lane 5), spider (lane 6), oyster (lane 7), sequencing was performed by the dideoxynucleotide chain- sea squirt (lane 8), earthworm (lane 9), slug (lane 10), sea cucumber termination method using Sequenase Version 2.0 (United (lane 11), sea anemone (lane 12), and amoeba (lane 13). K562 cells States Biochemical). The nucleotide sequences from these (lane 14) and Daudi cells (lane 15) were used as controls. 4X174/Hae three clones that encode the earthworm J chain between III DNA size markers are in lanes 1 and 16. (Upper) Ethidium primers pJ-1 and pJ-4 were identical. Furthermore, to deter- bromide-stained PCR products. (Lower) DNA blot analysis of the PCR products using a human J-chain cDNA probe. (C) Northern blot mine the upstream sequence of earthworm J chain, a primer, analysis with the human J-chain probe of J-chain mRNAs from various pJ-5 (5 '-AACAAATGCATGTGTACCCG-3'), that corre- animals. Silkworm (lane 1), shrimp (lane 2), sea squirt (lane 3), sponds to the region of the mouse J chain described by earthworm (lane 4), sea cucumber (lane 5), slug (lane 6), sea anemone Matsuuchi et al. (20) was synthesized. The same PCR and (lane 7), amoeba (lane 8), K562 cell (lane 9), and Daudi cell (lane 10) cDNA sequencing procedures were repeated with primers pJ-5 RNAs were used. (D) Northern blot analysis with the earthworm and pJ-2. J-chain PCR product in earthworm (lane 1), silkworm (lane 2), slug Northern Blot Analysis. Ten micrograms of RNA from (lane 3), sea squirt (lane 4), snail (lane 5), and K562 (lane 6) and Daudi various invertebrate species and two human cell lines, K562 (lane 7) cells. and Daudi, were used for Northern blot analysis. RNA was denatured and electrophoresed on a 1.0% agarose/ Tris-HCl, pH 7.5/0.5 M NaCl/1% Nonidet P-40/1 mM formaldehyde gel, stained with ethidium bromide, transferred EDTA) containing aprotinin (Sigma; 10 Ag/ml) and 1 mM to a Hybond membrane by standard procedures (26), and then phenylmethylsulfonyl fluoride (Sigma). The solubilized earth- hybridized with the human J-chain cDNA and earthworm worm extract was centrifuged at 25,000 x g for 30 min at 4°C J-chain PCR products (DNA sequence between pJ-1 and pJ-4) to remove debris. The total protein concentration was deter- in salmon sperm DNA (100 ,ug/ml)/50% (vol/vol) form- mined by a protein assay kit (Bio-Rad). An aliquot of extract amide/0.65 M NaCI/5 mM EDTA/0.1% SDS/0.1 M (10 uId) was analyzed by SDS/PAGE on 12.5% gels (27). As Pipes-NaOH, 5x Denhardt's solution/10% (wt/vol) dextran controls, monomeric and polymeric IgA and J chains purified sulfate at 42°C for 16 h. After hybridization, the membrane was from polymeric IgM (6, 7) were used. After SDS/PAGE, the washed for three 15-min periods in 2x SSC/0.1% SDS at 55°C separated protein bands were transferred onto a poly(vinyli- and then exposed to Kodak X-Omat film for 16 h at -80°C. dene difluoride) membrane (Immobilon-P, Millipore) by the Protein Analysis of the Earthworm J Chain. An earthworm procedure of Towbin et al. (28). The membrane was allowed to (1.0 g) was homogenized with cell lysis buffer (50 mM react with a rabbit polyclonal anti-human J-chain (19) Downloaded by guest on October 1, 2021 1888 Immunology: Takahashi et al. Proc. Natl. Acad. Sci. USA 93 (1996)

and then with horseradish peroxidase-conjugated anti-rabbit chain. Briefly, the sections were immersed in absolute meth- IgG (heavy and light chain) antibody (Cappel). Substrate anol containing 0.3% H202 to block endogeneous peroxidase reaction was performed in a diaminobenzidine solution con- activity. After washing with phosphate-buffered saline (PBS), taining 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sig- the sections were incubated with 1% bovine serum albumin ma), 0.005% H202, and 0.05 M Tris HCl for 10 min. and then with a rabbit polyclonal anti-human J-chain antibody Immunohistochemical Analysis. Frozen earthworm and (19). After washing with PBS, peroxidase-conjugated anti- slug sections were fixed in 10% buffered formalin and sub- rabbit IgG antibody (Cappel) was applied to the sections, and jected to immunoperoxidase staining for the detection of J then the substrate reaction described above was performed.

CAA GLA OLT GAL AGO ATT GTT CTT GTT GAC AAC AAA TGT AAG TGT GCC CGG ATT ACT TCC AH *** *** ** * **** ** ** * ** * EA AAC AAA TGC ATG TOT ACC CGA GTG ACG GCG *** * ******** * pJ-5 NO GOT GAC GAC GAA GCG ACC ATT CTT GCT GAC AAC AAA TGC ATG TGT ACC CGA GTT ACC TCT

EU AGG ATC ATC CGT TCT TCC GAA GAT CCT AAT GAG GAC ATT GTG GAG AGA AAC ATC CGA ATT *** *** * ** * ** *** *** *** *** *** *** *** *** *** ** *** *** *** AGG ATC AGO GOT ACC AGG GAG GAT CCT AAT OG GAC ATT GTG GAG AGA TAC ATC CGA ATT *** ***** ** * *** *** *** *** *a**** *** *** *** *** * *** *** *** pJ-1 AGO ATC ATC CCT TCC ACC GAG GAT CCT AAT GAG GAC ATT GTG GAG AGA AAT ATC CGA ATT EL EU ATT GTT CCT CTO AAC ARC AGG GAG AAT ATC TCT GAT CCC ACC TCA CC- ATT GAG AAC CAG ** *** *** *** ** *** *** * * *** *** *** *** *** *** ** ** *** * *** *** EL AAT GTT CCT CTG AAL AAC AGO 000 AAT ATC TCT GAT CCC ACC TCC CCA ATT AGG AAC CAG * ** *** ** ** *** *** * * *** *** *** *** *** *** *** *** * ** * * NO OTT GTC CCT TTG AAC AAC AGO GAG AAT ATC TCT GAT CCC ACC TCC CCA CTG AGA AGG AAC

EU ATT TOT GTA CCA TTT GTC TGA CCT CTG TAA AAA ATG TGA TCC TAC AGA A------** *** ** *** *** *** *** *** *** *** *** *** * ** * EA -TT TOT ATA CCA TTT GTC ACC TTC CTG TAA AAA ATG TGA TCC TTA CGA A------** *** *** *** *** *** * * ** *** *** * ** *** **i* * ** * no -TT TOT ATA CCA TTT GTC AGA CCT CTO TAA GAA ATG CGA TCC TGT GGA AGT GGA GCT GGA

EU -G- TGO AGC TG- --- -GA TAA TCA GA- TAG TTA CTG CTA CCC AGA GCA ATA TCT GTG ATG * *** ** ** ** * ** ** *** *** *** *** ** * ** EL -GA TGO AGT TOT TAC TGC CAC CGA GAC TAA CAT CTO CTA CCC AGA CCA A-- --G GTG --T ** * ** *** *** *** *** ** *** ** *** *** ** *** ** * *** * MD AGA TCA GGT TOT TAC TGC CAC CCA GAG CAA CAT CTG, CAA TGA AGA CGA T-- --O GTG --T Hu AAL ACA GTG CTA CAG AGA C-- CTG CTA CAC TTA TGA CAG~~~~~~~~~~~~pJ-3AAA CAA GTG CTA CAC AGC TGT ** *** ** *** *** * ** * ** *** *** *** *** *** ** * * TCC TCA GTC CTG CAG AGA CTA TTG CCC GOA ACT AGA CAG AAA CAA GTO CTA TAC COT OCT *** * * * ** * * * * ** ** ** ** ** ** ** * *p12 NOHa TCC TGA GAC CTG C------T ACA TGT A-- TGA CAG AAA CAA GTG CTA TAC CAC TAT EL Eu GGT CCC ACT CGT ATA TGO TGG TGA GAC CAA AAT GOT GGA AAC AGC CTT AAC CCC AGA TGC ** *** ** * ** * *** *** *** *** *** *** ** ** ** *** ** *** ** * Er AGT CCC ACC TGG GTA TAC TGG TGA GAC CAA AAT GGT GCA AAA TGC CTT GAC CCC CG4 TAA ** *** *** *** *** * *** *** *** *** *** *** *** * ** *** *** ** *** GOT CCC ACC TGG GTA TCA TGG TGA GAC CAA AAT GOT GCA AGC AGC CTT GAC CCC CGA TTC EU CTO CTA TCC TGA C pJ-4 ***CTG ***CTA CCC** ****TGOAC ** *** *** *** * MO TTG CTA CCC TGA C

CHO B 20 40 + 50 HU QEDERIVLVDKCARITRIRSSEDPNEDIVERNIRIIVPINNRENIS DO -DENERIV------P-A---SQ------V------S----- RA D--ATI-A----M-T-V-----P-T------V------NO ---ST------Q-V------DPDN-S------T----- BU EQEYI-AN-----VK-S--FVP-T-R-G-E-L- ---Q-TI-TSS-MX-- FIG. 2. Nucleotide (A) and deduced amino EA ---N-T-V-A--ROTR------acid (B) sequence of cDNA synthesized by RT- 60 80 100 PCR from RNA in earthworm. (A) Alignment of HU DPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSAT ETC the nucleotide sequences of earthworm (EA),

DO ---- M--K-- -T-E----D-V --- S------S-A --- human (HU), and mouse (MO) J chains. Aster- isks indicate identical nucleotides; double- RA ------RN ------V------V-- --ED-V ------N--DGVP --- headed arrows indicate the primer sites. (B) M ------E-K-N-AN------I-----VF--S-----PD-DYS --- Alignment of deduced amino acid sequences of EU --Y-----Q---N-W-I -Q ----VQL -IGGIP-L-S-PXXSKP-dE earthworm (EA), human (HU), bovine (BO), EL ------NQ------PS ------YEDOV----ET---YP-QOVPQS- rabbit (RA), mouse (MO), and bullfrog (BU) J chains. Amino acids identical to the human se- 120 140 HU YTY DRNKCYRLVVPLVYGGETKVETALTPDACYPD quence are indicated by a dash. Arrows indicate the location of Asn-linked glycosylation sites. BO ------TNRVK-S-R-Q-5------Nine Cys residues are present in the earthworm RA -N------I ---R-H--Q--A--OA-----S---- J chain; of these, seven are conserved in all five MD ------TL-- ITER-V-R- -KAT----S---- animal species at identical positions (asterisks), are BU ---TE -NFK KKVP---- S--EYSE and six conserved in four species (excluding the EA RD- CPEL------VL--PG-T------QN------bullfrog-residue 100 of the bullfrog J chain has not been determined). Downloaded by guest on October 1, 2021 Immunology: Takahashi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1889 The sections were finally washed, counterstained with Meyer's hematoxylin, and mounted. As negative control, normal rabbit IgG was used. .< 3 .5 X E s. 0 s cj~~~E °_ RESULTS Detection of the J-Chain Gene in Earthworm. To detect J-chain DNA in invertebrates, we used an RT-PCR employing several sets of oligonucleotide PCR primers (deduced from published DNA and amino acid sequences) that are conserved in human (4), mouse (20), bovine (21), rabbit (22), and bullfrog 28KD - _MMfm (23) J-chain . RNA was extracted from the whole body of earthworms and from human J-chain-positive B-lymphoma cells (Daudi) (24) and amplified by RT-PCR using various sets of primers to detect the J-chain gene. The J-chain message could be detected in earthworm and Daudi cells by RT-PCR FIG. 3. Immunoblot analysis of the crude extracts from earthworm with different sets of primers (Fig. 1A). Based on these (whole bodies). As controls, purified human monomeric and polymeric experiments, a primer set pJ-1 and pJ-4, which amplified a IgA and J chain were used. The polyclonal anti-human J-chain J-chain gene transcript of -312 bp, was chosen for the antibody detected the earthworm J chain at 28 kDa. detection of J-chain expression in various animal species. Presence of the J-Chain Gene in Various Invertebrate and Protein Analysis of the Earthworm J Chain by SDS/PAGE Vertebrate Species. As shown in Fig. 1B, J-chain transcripts and Immunoblot. The presence of the J-chain protein in were detected in various animals including slug, oyster, sea invertebrates was verified by SDS/PAGE analysis of cell cucumber, earthworm, shrimp, crab, silkworm, spider, sea lysates of the earthworm. Multiple bands seen on a Coomassie squirt, and sardine. In other experiments, the J-chain mRNA blue-stained gel corresponded mainly to low molecular weight was also detected in clam, butterfly, lamprey, and lizard (data proteins (data not shown); one protein migrated at a position not shown). However, J-chain mRNA could not be detected in similar to that of purified human J chain (30, 31). Immunoblot sea anemone or amoeba. The authenticity of the presumptive analysis of the earthworm extract and of purified human J PCR-amplified J-chain products was confirmed by blotting and chain and polymeric IgA developed with a polyclonal anti- hybridization to a cDNA probe encoding exons 1-4 of human human J-chain antibody revealed a single band corresponding J chain (Fig. 1B). to J chain; no analogous band could be detected in a mono- Northern Blot Analysis of the J-Chain Gene in Inverte- meric IgA preparation (Fig. 3). These results indicate that the brates. The presence of J-chain gene transcripts in inverte- earthworm J-chain protein can be detected by a reagent brates was also detected by Northern blot analysis using the specific for the human J chain. The apparent difference in the human J-chain cDNA probe; J-chain transcripts ('1.4 kb) molecular mass of the J chains determined by SDS/PAGE were detected in slug, sea cucumber, earthworm, shrimp, (25-28 kDa) and ultracentrifugation (15-15.6 kDa) has been silkworm, and sea squirt (Fig. 1C). The same results were explained by aberrant behavior of reduced and denatured J obtained when the earthworm J-chain cDNA probe (using pJ-1 chains during SDS/PAGE (3, 30, 31). and pJ-4 primers and amplified by RT-PCR) was used on RNA Immunohistological Localization of the J Chain in Earth- extracted from earthworm, silkworm, snail, slug, and sea worm and Slug. The J-chain protein was localized in frozen squirt; Daudi cells and K562 cells were used as positive and sections of earthworm and slug stained by the immunoperox- negative controls, respectively (Fig. 1D). idase method using anti-human J-chain antibody. J chain was Sequence Analysis of the Earthworm J Chain and Compar- localized in mucous cells in the dorsal and ventral surfaces of ison of its DNA and Amino Acid Sequences to those of the body, the intestinal epithelial cells, and infiltrating cells Mammalian Species. Sequence analysis of the J-chain gene called macrophage-like cells (32) (Fig. 4). As positive controls, was performed to define the cDNA sequence of product (Fig. we used human tissues stained with this anti-J-chain reagent 1A, lane 5) amplified by RT-PCR from the earthworm. After (6-8, 33). In negative controls, anti-J-chain IgG was replaced RNA extraction, single-stranded cDNA was synthesized and with IgG from an unimmunized rabbit (data not shown). amplified by PCR with the same pJ-1 and pJ-4 primers. An additional primer pair, pJ-2 and pJ-5, was used to confirm the sequence and to obtain the sequence on the 5' side of the DISCUSSION coding region sequence. This allowed us to determine the In vertebrates, the J chain is found exclusively in association earthworm J-chain cDNA sequence of 380 nt (Fig. 2A). As with polymeric Ig and participates in Ig polymerization (1-3, shown in Fig. 2A, the earthworm J-chain cDNA sequence has 14). Randoll et al. (34) demonstrated that transfection of a a high degree of homology to human and mouse J-chain DNA J-chain cDNA into antibody-secreting B cells lacking J-chain sequences (75%, 76%, and 68%, respectively). The deduced expression resulted predominantly in the secretion of the amino acid sequence of earthworm J chain revealed that it has pentameric form of IgM. Furthermore, inclusion of the J-chain a high degree of homology to human, mouse, bovine, and gene into baculovirus-infected epithelial cells producing mo- rabbit J chains (64%, 69%, 61%, and 56%, respectively) (Fig. nomeric IgA resulted in the synthesis of IgA polymers (14). 2B). Analysis of the deduced amino acid sequence from the Because antibody to the J chain blocks binding of the poly- earthworm J chain by Hopp and Wood's hydropathy plot (29) meric Ig receptor (pIg-R) to polymeric IgA and pentameric indicates that the arrangement of hydrophilic segments was IgM, the J chain not only may be required for Ig polymeriza- similar to human (4) and mouse (20) J chains. The locations of tion but also may play an important role in the epithelial Cys and Asn residues are also similar to their respective transport of polymeric Ig into exocrine secretions (2, 3, 35). positions in human (4), mouse (20), and rabbit (22) J chains. Several studies have demonstrated that J-chain synthesis In human and mouse J chains, the Asn residue is linked to a appears to be regulated independently of heavy and light chain single oligosaccharide chain. On the other hand, Cys-Pro-Glu- production (1-3, 5-7). These reports imply that the J chain may Leu (CPEL) is found in the earthworm J chain at positions have functions other than Ig polymerization. In this report, we 104-107. Although this Cys residue may form a disulfide bond, examined the presence of the J chain in invertebrate species it is difficult to explain its function at present. from primitive to highly developed animals, as well as in Downloaded by guest on October 1, 2021 1890 Immunology: Takahashi et al. Proc. Nati. Acad. Sci. USA 93 (1996)

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FIG. 4. Immunohistochemical analysis of J-chain protein in earthworm and slug. (A and C) The J chain was localized in mucous cells of the dorsal surface of epithelium in earthworm and slug. (Approximately x 160.) (B and D) The J chain was detected in the intestinal epithelial cells and macrophage-like cells (arrows). (Approximately X95.) vertebrate species, and detected the J chain in 14 invertebrate highly homologous between different mammalian species as and 3 vertebrate species. Although the function of the J chain assessed by DNA and amino acid sequence analysis (4, 21-23). in invertebrates is not known, our data indicate that the J chain Because it has been shown that to the human J chain is expressed in invertebrates even though they do not produce cross-react with J chains from various vertebrate species (19), Ig. we used such antibodies for detection of the J chain in Based on the DNA and deduced amino acid sequence data earthworm and demonstrated its presence by immunoblot and immunochemical cross-reactivity with polyclonal antibod- analysis and immunohistochemistry. Thus, our results reveal ies, it is clear that the J chain is a primitive and remarkably that the earthworm J chain has high homology to human, conserved protein that has retained the same properties, mouse, and rabbit J chains and suggest that the J chain, which including molecular mass, the distribution of Cys residues, and is conserved in invertebrate and vertebrate, is a very primitive primary structure in both vertebrates and invertebrates. In- protein that appeared at an early stage of coelomate evolution. terestingly, secondary structure analyses have suggested that Invertebrates that expressed J chains have three germ layers human and rabbit J chains display single Ig-like domain and a deuterocoel, whereas invertebrates that did not, a structures (22, 31, 36). Other proteins that are not involved in protozoan (amoeba) and a sea anemone, have only two germ the recognition of foreign antigens or in defense mechanisms layers. This may suggest that the J chain occurs in invertebrates also display such Ig-like domain structures (37-46). It has been that possess a mesoderm and a digestive canal. Indeed, im- reported that highly developed invertebrates, such as insects munohistochemical studies demonstrated that in the earth- and tunicates, produce proteins that belong to the Ig super- worm and slug, the J chain is found in macrophage-like cells family (37). These include amalgam (38) and fasciclin II (39) and in epithelial cells covering the external (dorsal cuticle) and in the neurosystem of insects, the Thy-1-like molecule (40, 41, internal (digestive tract) surfaces. Presence of the J chain in 42) and Lyt-1 homolog (43) found in Mollusca, Annelida, and epithelial cells does not necessarily indicate that it is synthe- Protochordata, and a 132-microglobulin-like molecule (44, 45) sized in these cell; epithelial cells in the intestine and secretory in various invertebrates. A hemolin molecule (46), which is an glands of vertebrates acquire this polypeptide during trans- antibacterial protein, has also been identified in insects. endocytosis of J-chain-containing polymeric IgA or IgM, The J chain occurs in various vertebrates including mam- which is produced by adjacent plasma cells (2, 3). Immuno- mals, birds, reptiles, amphibians, and fishes (1-3, 19) and is histochemical studies of tissues from invertebrate species Downloaded by guest on October 1, 2021 Immunology: Takahashi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1891 indicate that the J-chain protein is also found in cells adjacent 19. Kobayashi, K., Vaerman, J.-P., Bazin, H., Lebacq-Verheyden, to the skin and intestinal surface, which are in direct contact A.-M. & Heremans, J. F. (1973) J. Immunol. 111, 1590-1594. with foreign environmental substances. This suggests that the 20. Matsuuchi, L., Cann, G. M. & Koshland, M. E. (1986) Proc. Natl. Acad. Sci. USA 83, 456-460. J chain may be involved in the defense system of invertebrate. 21. Kulseth, M. A. & Rogne, S. (1994) DNA Cell Biol. 13, 37-42. We conclude that the J-chain gene and protein are expressed 22. Hughes, G. J., Frutiger, S., Paquet, N. & Jaton, J.-C. (1990) in invertebrate and vertebrate species and that the J chain is a Biochem. J. 271, 641-647. primitive polypeptide that arose at an early stage of evolution 23. Mikoryak, C. A., Margolies, M. N. & Steiner, L. A. (1988) J. and has been highly conserved from lower to higher animal Immunol. 140, 4279-4285. species. These findings suggest the possibility that the Ig- 24. Kutteh, W. H., Moldoveanu, Z., Prince, S. J., Kulhavy, R., independent functions of J chain, as observed in invertebrates, Alonso, F. & Mestecky, J. (1983) Mol. Immunol. 20, 967-976. 25. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156- may be retained as additional functions in Ig-producing ver- 159. tebrates. 26. Maniatis, T., Fritsch, E. F. & Sambrook, J. 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