Scand. J. hnmttnol. 9, 141-149, 1979

Characterization of IgD 1. Isolation of Two Molecular Forms from Human Serum

G. CORTE, P. T O N D A , E. C O S U L I C H . CELIA P. M I L S T E I N , A, BARGELLESI & M. F E R R A R I N I Institute of Biological Chemistry and Chair of Clinical Immunology. University of Genoa, Genoa. Italy; and Department of Immunology. A.R.C. Instiltiie of Animal Physiology Babraham, Cambridge, UK

Cone. G..Tonda. P.. Cosulich. t., Milstein. C. P.. Uargelksi, A. & f-"errarini. M. Characieri/aiion of IgD. I. Isolation ofTwo Molecular Forms from Human Scrum. Scand. J. Im/nmiol. 9, 141 149, 1979. Human IgD present in ihc scrum of nornial individuals or of palienis «iih Hadakin's disease (having high IgD tonccntriUions) was characicri/cd ami compared with live IgD myeloma proteins. IgD wus isolated using a highly specilic anii-S insniuhic inimunoabsorbcnl from which [lie bounti maicriul v.as cluicd with sodiuin Jodccjl sulphaic (SDS) or urea. Ihc latter rtagciii could be removed by exlensivc t!ial>sis. thus makiriB possible llie renaiuration of the eluled molecules. The purii> of the IgD ihus isolated was confirmed h> aniigcnic analysis. Boih xand >. lighl chain dcierminanis were prt'seni on serum IgD. although >, light chain was prcUominani wiih il ratio over Ihe K chain of 2:L SnS-polyacr\lami.) and one with an l g D ( \ i monoclonal protein. The scrum concentration of IgD in normal individuals ranged from 2 lo .12 ixg ml, as (JcterminciJ by radial mimunodilFusion ^^ radioimmunoassay [2|. The IgD level in H D patients was I.TO-450 iig/mK whereas that of myeloma patients

1979 Blackwell Scientific Publications

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•was oT 8-20 mg/ml. In the case of liealthy individuals freshly drawn sera were generally used, allhougli identical results could be obtained with stored sera. All sera were stored at -.10 C following ihe addition of e-aminocaproLC acid at the final concentration of ]OniM (to inhibii proteolysis by plasmin). Attlisera. Anti-human FCab').^, IgD, IgM, IgA. IgG, K and >. ligiii chain antisera. raised in rabbiis or goats, were made specific for the appropriate Ig class or light chain type by absorplion with proteins coupled to Sepliarosc-4B and tested for specificity as detailed previously [1 4|, IgG fraciions ofthe antisera were made insoluble with Sepharose-4B (Pharmacia, Uppsala. Sweden*. Generally 1 g of dried CnBr-acti\ated Sepharose bound 10-15 mg of IgG. Preparation of tgD. IgD was prepared from myeloma sera by chromalography on DEAE-cellulose followed by gel tillralion on Ultrogel AcA 34 12]. Normal serum IgD was prepared by inimunoabsorption. Aliquots of serum (diluteii, when required, to a final maximum concentration of 50 ;.ig/ml) were rotated with anii-S bound lo Sepharose (Seph-aS) in the presence of 10 mM EDTA and 10 niM s-aminocaproic acid for Ifi h at 4 C. To avoid nonspecific absorption of serum componenis or of 'nainral" antibody to the Sepharose immunoabsorbent, incubalion with Seph-aS was generally preceded by a short incubalion of ihe serum (l-.l h) wilh purified rabbii IgG anii-phytohaemaggluiinin (PHA) coupled to Sepharose. The amoiini of Seph-a^ used was of 100 [j.\ of a 50",, (v/v) suspension in PBS per ml of scrum. The immunoabsorbent was washed twice wilh 0.5",, NtinlDel P40 (NP40, Shell Italiana, Genoa) in phosphate-buffered saline (PBS) and ihree times with 0 . 5 \ NP40 in 0.5 M NaCl, 10 niM phosphate buffer, pH 7.4. Elution of the bound material was carried out by boiling the Seph-aS wilh an equal volume of 2",, sodium dodecyl sulphate (SDS). The Seph-aS was then centrifuged at low speed and rinsed with a further aliquot of 2",, SDS. When irreversible denaturation of the eluaie was tindesirable, Seph-aS was treaied with 10 M urea for 30 min at 37 C. The Scph-aS was separated from the supernatant and rinsed with an equal volume of 10 M urea. Renaturation o'^ Ihe eiuted material was obtained by extensive dialysis againsi PBS at 4 C. lodinaiion. Proteins were labelled with '-•'! using Ihe chloramine-T method according to Greenwood ei at. [1\. In order to ensure maximum and uniform iodinalion of lyrosines. all the proieins lo be processed for pepiide mapping were labelled in the presence of 2",, SDS (Sequanal Grade, Pierce, USA). SDS-polyacrylamicte get electrophiiresis (PAGE) and motecidar wcigttt determinaiion. SDS-PAGE was carried oui in slab gels using either ihe Tris or ihe phosphate bulTer system [9). The gels were stained wilh Coomassie blue or auloradiographed, or both [22]. Proteins were recovered by cutting the relevant hands out of the dried slab gel and eluting lor 24 h w ith 0.1" „ SDS in PBS al room temperature. Molecular weights were determined by using the following markers: mouse lgG2b (MPC II) (I50,0(H) daltons), monomeric IgM (MOPC 104E) (190,000 dallons), monoclonal human IgA and IgE (160,000 and 184.000 daltons respectively), phosphorylase a (95.000 daltons), bovine transferrin (75,000 daltons) and ovai-

btimin (43,000 dallons) and human y chain (50,000 dallons). Pfpiiitc tiicipping. Fingerprims of potypeptide chains were obtained according to the method of Mole [121 as modified by Richardson . lighi chain tlcicrminanis on IgD extracted from normal sera Percent* radioaciiviiy+:f precipitated b>: Exp. 1 2

Ami-^•

Anti-/.

'/JK ratio

.11

6ti fi8

2,0 :,:

*80-90% of radiolabelled lyD was preeipitabic by anti-A- ami-/, or ami-S, This value was laken as IOO",, of immunnprecipilable counts. •I- KXUKHIcpmofradiolabelled IgD were u.sed in each experinieni, * The non-specilic values of ihe immunoprecipitaies (obtained with an aiitiPitA antiserum coupled to Sepharose) were 5(KX)-lO.OOO cpm.

SDS-PAGE. As can be seen in Table 1. >- light ehain was predominant over the «• chain with a ratio of about 2:1. In evaluating these results one should consider that, although light chains contribute to a minor fraction of the total radioactivity of an Ig molecule, they could, however, be differently labelled and therefore Induce a small shift from the real values. It is noteworthy that neither >. nor K ehains were preferentially associated with SI or ^2 chains as shown by SDS-PAGE analysis.

Is lgD2 a molecular fortn originated tiy conversion of IgDl in the serum? To investigate whether IgDl could be converted into IgD2 by "factors' present in human serum, several approaches were used. Two normal sera and one HD serum were incubated for 24 h at 37 C. immunoprecipitated with Seph-a^, and analysed in SDS-PAGE. The rationale behind this experiment was that, if IgDl was converted into lgD2 in the serum. then prolonged ineubation should result in a change of the lgDI/IgD2 ratio. However. SDS-PAGE analysis followed by Coomassie blue staining eonsistently failed to show any obvious change in the relative proportions of Ihe two molecules. In order to investigate whether monoelonal IgD could be converted

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into lgD2, '"'I-labellcd IgD purified from two different myeloma sera Vias incubated separately with three different normal human sera at 37 C for 24 h. The radiolabelled material was recovered by immunoprecipitation with Seph-aS and analysed by SDS-PAGE followed by autoradiography. In no case was conversion into an IgD2 moleeular form observed. To rule out the possibility that radiolabelled and unlabelled IgD might be processed differently by serum "factors", 50 ijg of purified, nonlabelled monoclonal IgD v-as added to I ml aliquots of two normal sera containing a very low amount of IgD (2 |ig/ml). The IgD was recovered by immunoabsorption with Seph-aS either immediately or after different periods of incubation at 37 C. The bound material was eluted and analysed by SDS-PAGE followed by Coomassie blue staining. No band corresponding to lgD2 was ever deteeted. It is noteworthy that no major loss of monoclonal IgD was detected in the course of the above procedure as judged by the intensity of the IgD bands siained by Coomassie blue.

Peptide mapping analysis of M and S2 heavy ehain.s To compare the peptide structure of the different chains, a miero-technique was employed with radiolabelled moleeules. Isolated ^1 and S2 chains from two normal sera were prepared for peptide analysis as described in Materials and Metliods. The peptide maps were compared with those of the S chain ofa inonoclona! IgD protein. A comparison between monoclonal and normal SI chain is shown in Fig. 4(a). The two ehains have a high degree of homology. all peptides present in the normal HI being also present in the myeloma S chain. However, the myeloma heavy chain shows four additional peptides. It is possible, although not proven, that these additional peptides are derived from the variable region of the monoclonal prolein. In heavy ehains from normal sera, the variable-region heterogeneity would obviously result in an apparent absence of peptides from that region of the molecule. The peptide pattern displayed by S2 was identical to that of SI (Fig. 4b) with the exception of three additional spots. Tbese peptides were, bowever, different from the extra peptides found in the monoclonal S ehain (Fig. 4c).

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G. Corte ct al.

C H R O M A T O G R A P H Y FIG. 4. Autoradiograph of pcpiidc maps of '"l-iabcllcd normal and myeloma S chains, (a) Sl chain from normai serum IgD (left) anil S chain from Ha myeloma proicin (right), (b) Sl (lefi) and S2 (righ;) chain from normal scrum IgD. (c)S2 chain from normal serum_lgD(lert) and H chain from Ha myeloma protein (righi). The arrows indicate the ihreo characteristic extra spots of the S2 chain.

Characterization of Httttian Serttm fffD

DISCUSSION In the present study IgD was isolated from human sera by using a highly specific anti-i^ insoluble immiinoabsorbcnt. Previous attempts lo isolate IgD from human and mouse serum have been carried out with soluble-phase immunoprecipitation [23]. In those experiments, in order to identify the molecules present in (he immunoprecipitate, the sera were labelled with '•-^1 prior to addition of the antiserum. Insoluble immunoabsorption offers several major advantages, in particular that the IgD can be eluted in a high degree of purity suitable for further immunochemical characterization. For the same reason, radioactive labelling of the isolated molecule Is not strictly necessary for SDS-PAGE analysis, provided a sufficient qtiantity of starting material is used. Finally, when required, a more efTective and controlled iodinalion of IgD can be obtained by using purified material rather ihan unfractionated serum. The method for the elution was critical. For example, elution with SDS was the method of choice in terms of yield of material recovered, but had the disadvantage of concomitant irreversible denaturation. Reagents such as KCI, ammonia and KSCN eluted only a small fraction of the bound material. Urea represented a good compromise, since its use combined a reasonably good recovery with the possibility of renaturation to the native structure. The IgD recovered from Seph-anti-S was resolved into two distinct bands in SDS-PAGE which, for convenience, we have named IgD I and IgD2. Upon reduction, both igDI and lgD2 dissociated in heavy and light chains and the difference in their SDS-PAGE mobility was due to the smaller size of the ^i™ chain. The apparent molecular weight of IgDI and of its constituent ^^1 chain was 188,000 and 72,000 daltons, respectively. These values are identical to those obtained for all the IgD myeloma proteins (seven cases) that we have studied and are in agreement with the results obtained by others on different myeloma proteins with the same technique [15], The lower values recenily reported by Goyert et al. [6] are possibly explained by their different SDS-PAGE technique. While the presence of L chains, detected hy SDS-PAGF: and confirmed by serological analysis, clearly demonstrated the immuno-

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globulin nature of lgD2, the apparent molecular weight (160,000) of this molecule raised the possibility that lgD2 represented another Ig class contaminating the preparations. This hypothesis was excluded by antigenic analysis of the IgD preparations, by the unique mobility of the n chain on SDS-PAGE, i.e. different from that of all the other heavy chain classes, and by the high degree of homology between ^I and S2 chains indicated by the peptide maps. By immunochemical analysis A chain was found to be [he predominant light chain type on both IgDI and IgD2 isolated from normal sera, with a ratio over the K chain of approximatively 2 to I. This particular ratio confirms the remarkable predominance of >, light chain detected by previous studies on IgD myeloma proteins [20] and normal IgD cont;'.ining plasma cells [14] and excludes further a contamination by other Ig classes. The finding of lgD2 raises several problems concerning its origin and relationship with IgDI. It is unlikely that IgD2 results from degradation of IgD I in the serum, since 'factors' capable of converting IgDI into IgD2 were never detected in normal :^era. In addition alihough 'spontaneous" degradation of the heavy chain of monoclonal IgD has been observed, this never gave rise to molecules resembling a S2 chain. Rather, H chains, rtmning just in front of the Si chain were observed and their altered mobility was related to the loss of a few residues from the Cicrminal end [6, 20]. Finally, no spots were missing from the map of the 82 chain as compared lo that of the Hi chain. The interpretation of the peptide map of radiolabelled proteins is not as straightforward as that of a conventional map. First of all, with this technique only peptides containing tyrosine are visualized, and hence any difference due to nonlabelled peptides would go undetected. Second, even though iodination is carried out in SDS, sonic tyrosines may escape iodination depending upon the conformation of the molecule. Third, in the one case of IgD myeloma studied (which, incidentally, is identical by peptide analysis to the one used for our mapping experiments) no tyrosines were found in the first twenty-seven residues of the S chain [11]. The comparison between the maps of the SI and the S2 chains excluded the possibility that the absence of a peptide at either end of the 82

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C. Corte ct cl.

chain could be per se a sufficienl explanation for the difference in molecular weight. The lack of a certain amino acid sequence at the O terminal end would have resulted in the absence, or, possibly, in the ditTerent migration of a peptide, whereas, for the reason explained above, the absence of the first twenty-seven amino acids would have gone undetected. In neither case would the appearance of three extra spots have been expected. Consequently, if the reason for the different molecular weight resides exclusively in the peptide portion of the two chains, one is then left with the possibility that they diifer in their amino acid composition and sequence and therefore represent the H chains of two distinct IgD subclasses. The finding that all of the monoclonal IgD so far studied migrate in SDS-PAGE as IgDl. together with the difficulties encountered in demonstrating convincingly antigenic heterogeneity and the absence of structural differences amongst IgD myeloma proteins [10, 17] make this possibility rather unlikely. In addition, a large difference in molecular weight would represent a rather unusual finding for two subclasses of Ig molecules. Differences in the carbohydrate portion of IgDl and lgD2 could also explain the observed differences in their SDS-PAGE mobility and peptide map. Previous studies on myeloma proteins have shown that the ^ chain is a carbohydrate-rich molecule with an average content of about \5% [20]. In view of the similarities between IgD myeloma proteins and IgDl, it is likely that the SI chain is alsoa carbohydrate-rich molecule. A lower carbohydrate content of the S2 chain would thus contribute to explain its lower apparent molecular weight. In addition, differences in the quantity and possibly in the composition of the carbohydrate portion may also account for conformational changes, which may influence the availability of certain tyrosine residues for iodination, causing the appearance of three extra spots in the map. In this connection it is perhaps worth recalling that all carbohydrate is attached to the S chain of myeloma proteins at three defined sites [21]. Recent studies have demonstrated that in man and mice surface and intracytoplasmic IgD displays two molecular forms whose SDSPAGE mobility closely resembles that of IgDl and IgD2, respectively [Bargellesi et al.. in

preparation; 13. 22]. It has also been suggested that in the mouse intraccllular "factors" are capable of converting IgDl into lgD2 [13]. The same process could occur for hitman IgD and may take place at the cell level by modification of either the peptide or the carbohydrate moieties.

ACKNOWLEDGMENTS We thank Dr A. Fcinstein and Dr R. M. E. Parkhouse for helpful discussions and for critically reading the manuscript and Dr A. Carbonara, Dr F. Dammacco and Dr L, Scolari for the generous gift of IgD myeloma sera. Collaboration between the two laboratories was made possible by an E.M.B.O. fellowship to G. Corte. Supported by grants CNR 42/76 and NATO 1464.

REFERENCES 1 Boxel. J.A. van. PJUI. W.E.. Terry. W.D. & Green. !. IgD bearing liiiniiin lymphocyies. J. Itnmtiitol. 109, 648. 19722 Corle. G.. Ferrarini, M.. Tonda. P. & Biirgellesi, A. Increased serum IgD concentrations in patients with Hodgkin's disease. Clin. e.\p. liutnutwi. 28. 359. 1977. .' Ferrarini. M., Bargellesi. A., Corte. G.. Viale, G. & Pernis. B. Comparative studies of membrane anil cytoplasmic immunoglobulin classei in tiuman lymphoid cells. Antt. N, Y. Acad. Sci. 254, 243. 1975. 4 Ferrarini. M.. Corle. G.. Viale. G.. Duranie. M.L. & Bargellesi. A. Membrane Igon human lymphocytes; rale of turnover of IgD and IgM on the surface of human tonsil cells. Ettr, J, Imtitiittnl. 6, 372, 1976. 5 Fii. S.M.. Winchester. R.J. & Kunkel. H.G. Similar idiotypic specilicily Tor the membrane IgD and IgM of human B lymphocyies. J. Immttnol. 144, 250. 1975. 6 Goyert. S.M.. Hugli, T.E. & Spiegelbcrg. H.L. Sites of spontaneous degradation of IgD. J, Imttiiutol. Its, 2138. 1977. 7 Greenwood. F.C. Hunier. W.M. & Glover. J.S. The preparation of '^'1 labelled human growth hormone ai higli specific activity. Bioihcm, J. 89, 114. 1963. S Jetieris, R. & Miiuhews. J.B, Structural studies of human IgD paraproteins. tmmiinid. Rcw 37, 25. 1977. 9 Mai/el. J.V. Polyacrylamide gel-electrophoresis o( viral proteins, p. 179 in Maramorosh. K. & Koprowski. H. (eds.) Methods in Virnloffv. Vol. 5. Academic Press. New York. 1971. II) Mendez, [I.. Frangione. B. & Kochna. S. Chemical typing of human immunoglobulins L and D. FEBS Lett, 33,4. 1973.

Characterization of Htiman Serum IgD 11 Milstein. C P . & Noihrop, F. N-termina! aminoacid sequence of a human delta chain myeloma protein. Eur. J. Immunol. 6, 222. 1976. 12 Mole, L.E. A gcneiic marker in the variable region of rabbit imtnuiioglobulin heavy chain. Biochem. J. 151, 351. 1475. L^ Parkhouse, R.M.E. & Cooper. M.D. A model for ihe dilferentiation of B lymphocytes with implications for the biological role of IgD. Imtmmol. Rev, 37, 105, 1977. 14 Pernis. B. Relationship between ihe heterogeneity ot" immunoglobulins and the differentiation of plasma cells. Cold Spring llarbcr Svmp. qttanr. Biol. 32. 333. 1967. 15 Perry. M.B. & Milstein. C. Interchuin bridges of IgD. Nature. 228, y.M. 1970. 16 Richardson. N.. Mclllhinney. R.A.J. & Feinstein, A. An association of immunoglobulin M with actin. Submitted for publication. I? Rivat, C . Roparli^. C. & Rowe. D.S. Antigenic heierogeneity of human IgD Immunoglobtilins. Nature: New Biol. 231, 279. 1971.

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18 Rowe. D.S.. Hug, K.. Forni. L. & Pernis, B. Immunoglobulin D as a lymphocyte recepior. J. c.\p. Mcd. 138, 965, 1973. 19 Salsano, F., Froland, S.S., Natvig. J.B. & Michaelsen. T.E. Same idiotype of B lymphocyte membrane IgD and IgM. Evidence for monoclonality of chronic lymphocyiic leukemia cells. Scand, J. Immunol. 3, 841, 1974. 20 Spiegelberg. H.L. The structure and biology of human IgD. Immtimil. Rev. 37, 3. 1977. 21 Spiegelberg. H.L.. Prahl. J.W. & Grey, H.M. Structural studies of a human IgD myeloma protein. Bi>Hhemi.stry, 9, 2115. 1970. 22 Sitia. R.. Corte. G.. Ferrariui. M. & Bargellesi. A. Lymphocyte membrane Immunoglobulins: similarities between human IgD and mouse IgD-like molecules. Eur. J. Immunol. 7, 503, 1977. 23 Vitetta. E.S. & Uhr. J.W. IgD and B cell differentiation. Imtmmol- Rev. 37, 50. 1977. Received 21 June 1978 Received in revised form 16 Ociober 1978

Characterization of IgD. I. Isolation of two molecular forms from human serum.

Scand. J. hnmttnol. 9, 141-149, 1979 Characterization of IgD 1. Isolation of Two Molecular Forms from Human Serum G. CORTE, P. T O N D A , E. C O S...
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