248

Biochimica et Biophysica Acta, 577 (1979) 248--252 © Elsevier/North-Holland Biomedical Press

BBA 38146

LACTOPEROXIDASE-CATALYZED IODINATION OF HUMAN IgM

DIFFERENCES BETWEEN 7 S IgM AND 19 S IgM AND BETWEEN CELL SURFACE 7 S IgM A N b SERUM 7 S IgM

BEN K. SEON and DAVID PRESSMAN

Department of Immunology Research, Roswell Park Memorial Institute *, Buffalo, NY. 14263 ,(U.S.A.)

(Received August 30th, 1978)

Key words: Lactoperoxidase-catalyzed iodination; Immunoglobulin M; Serum immunoglobulin; Cell surface immunoglobulin

Summary

We have studied the lactoperoxidase-catalyzed iodination of human IgM and have measured the ratio of radioactivity incorporated into the ~ chain to that incorporated into the L chain (i.e. the p/L ratio). Both 7 S and 19 S IgM were examined. The ratio of radioactivity was found to be larger for 7 S IgM than for 19 S IgM for all four of the monoclonal IgM proteins examined. The data suggest that some tyrosines of the p chain which are buried and not available for iodination in 19 S IgM become exposed on conversion of 19 S IgM to 7 S IgM.

The p/L ratio for the IgM found on the cell surface of RPMI 8392 cells was significantly smaller than the ratios for all of the five 7 S IgM proteins studied in solution. It appears, therefore, that a portion of the p chain of the cell sur- face IgM of the RPMI 8392 cells is buried in the membrane.

Introduct ion

In a previous s tudy of immunoglobulins on human lymphoid cells of estab- lished cultured cell lines, we identified cell surface IgM by use of lactoperoxi- dase-catalyzed iodination with carrier-free l~sI, specific immunoprecipitat ion and SDS polyacrylamide gel electrophoresis [1]. As a reference protein during the immunoprecipitat ion and the gel electrophoresis, we included a monoclonal

* A'unit o f t h e N e w Y o r k S ta te Department of Health. A b b r e v i a t i o n : SDS, sodium dodecyl sulfate.

249

serum IgM protein, CARD, which was iodinated with [131I]iodine by lactoper- oxidase-catalysis.

When the ratio of radioactivity in the g chain to that in the L chain (g/L ratio) was calculated for the cell surface IgM on RPMI 8392 cells and the carrier IgM CARD using the results shown in Fig. 1 of the above report, the p/L ratio for the cell surface IgM was only 1.2 whereas the ratio for IgM CARD was 1.8. This difference of the p/L ratio between the cell surface IgM and serum IgM is of interest in view of the fact that such ratios have been used by other investigators as a measure of the degree to which cell surface IgM mole- cules are buried in the cell membrane [2,3].

The problems involved in utilizing the/~/L ratio to determine the exposure are that the ratio may vary widely among individual monoclonal IgM molecules and that even for the same immunoglobulin molecule, the ratio may vary according to the extent of iodination as was shown for several human IgG pro- teins [4]. Differences in the p/L ratio among different IgM proteins would be due to the fact that different monoclonal IgM proteins derived from different patients have variations in the amino acid sequence in the variable region of the IgM molecule although they have essentially the same sequence in the constant region of the molecule [5--7]. In comparing the p/L radioactivity ratio for serum IgM with that for cell surface IgM it should be considered that serum IgM is of pentameric (19 S) form while cell-surface IgM is monomeric (7 S) [2]. We have now determined the p/L ratios for both monomeric and penta- meric forms of several human monoclonal IgM proteins after both forms of each IgM protein were iodinated at several different iodination levels and have found that in all IgM proteins studied, the/~/L ratios for 7-S IgM proteins are larger than those for 19-S IgM proteins although the ratios observed vary at dif- ferent levels of iodination. Moreover the ratios for 7-S serum IgM proteins are significantly larger than that for cell surface 7 S IgM on RPMI 8392 cells.

Materials and Methods

The purified monoclonal serum IgM proteins CARD, KUNZ, MILL and WIDR are those used in the previous s tudy [8]. The 7-S IgM proteins of the serum IgM proteins were obtained by reducing the proteins with 20 mM mer- captoethylamine and alkylating the resulting SH groups as described previ- ously [8]. The 7- and 19-S IgM proteins were radioiodinated with lactoper- oxidase-catalyzed procedure and the amount of iodine incorporated into the protein was determined as before [4]. The iodinated proteins were fully reduced with dithiothreitol in the presence of 1% SDS and 9 M urea and anal- yzed by electrophoresis on polyacrylamide gels.

The molecular weights of the substances corresponding to radioactive peaks in polyacrylamide gels were determined as before [4].

Results

Examples of analyses of radioiodinated 19-S and 7-S IgM proteins are shown in Fig. 1 where the SDS polyacrylamide gel electrophoresis patterns are illus- trated. The /~/L was determined from the ratio of the iodine content of the

2 5 0

CARD BS p

~3 0291/IoMs

8

2

~ ;~ 6

x

CARD 7S

L

2O 40 6O

2O

15

5

2O

~411 Zglds

/a.

L

40 60

FRACTION NO.

Fig. 1. SDS p o l y a e r y l a m i d e gel e lee t rophores i s of i od ina t ed and r e d u c e d IgM. Io d in a t ed 19- and 7-S IgM pro te ins C A R D con ta ined 0 .29 and 3.4 iodine a t o m s pe r IgMs (7 S subuni t ) mo lecu le , respect ive ly . The iod ina ted 19- and 7-S IgM pro te ins were fully r e d u c e d wi th d i th io th re i to l and subjec ted to gel e lect ro- phoresis in t he p resence of 1% SDS. The gel was sliced by an au toge l d iv ider and the 125 i . rad ioac t iv i ty in each gel-slice f rac t ion was d e t e r m i n e d by use of a Packard 9"-ray s p e c t r o m e t e r . Th e mo lecu l a r we igh t of each p e a k and t he re la t ive a m o u n t s of r ad io iod ine in # and L chains were d e t e r m i n e d as b e f o r e [4 ] .

g-chain peak to the iodine content of the Llchain peak. In Fig. 2, the ~/L ratio for several IgM proteins which were iodinated at two or more different iodina- tion levels are summarized. The filled symbols and open symbols represent 19 S IgM and 7 S IgM, respectively. It should be noted that in all the IgM proteins studied, the p/L ratio for 7 S IgM is larger than that for 19 S IgM, although the ratio varies among different IgM proteins. For each level of iodination for each protein the p/L ratio for the 7 S IgM was greater than that for the 19 S IgM for

- I 4

2

=L .¢

o) CARD .,IgS, o?S

6

I~ KUNZ •IgSj A?S

"7

61 c)Mlt.Lvl9S~ v7S

6~°'.. a d)WIDR zlg~ aTS

: t 5 IO 15

~I-LABELED IO01NE A~S/ /MOLECULE OF 19Ms

Fig. 2. Ra t ios of r ad io iod ine i n c o r p o r a t e d in the p chains to t h a t in the L chains of 19- and 7-S IgM pro- reins. Each 19 S IgM p ro t e in was iod ina ted at several d i f fe ren t iod ina t ion levels and each 7 S IgM p ro t e in was i od ina t ed a t t w o d i f f e r en t i od ina t i on levels. Each of the iod ina ted p ro te ins were ana lyzed as illus- tza ted in Fig. 1. The resul ts fo r 19 S IgM and 7 S IgM were r ep r e sen t ed b y filled s y m b o l s and o p e n sym- bols , respec t ive ly . Each e x p e r i m e n t a l po in t r ep resen t s an average value of dup l ica te e x p e r i m e n t s in all cases for 7-S IgM pro te ins and in m o s t cases for 19-S IgM pro te ins . On the abscissa, the a m o u n t of iodine i n c o r p o r a t e d in to IgM was r e p r e s e n t e d pe r 7 S subun i t (19 • 10 4 tool. wt . ) for b o t h 19 S IgM and 7 S IgM.

251

each experiment performed. The g/L radioactivity ratio varies at different iodination levels as we observed for IgG [4]. The variation of the p/L ratio, however, was not significant at the iodine levels below one iodine atom per molecule of 7 S IgM (Fig. 2) as expected on theoretical bases. The iodination of cell surface IgM with carrier-free 12sI results in the iodination at such a low iodination level.

The p/L ratio for 7-S IgM proteins CARD, KUNZ, MILL and WIDR ranged between 1.7 and 7.5 and were all larger than the ratio (1.2) of cell surface IgM on RPMI 8392 cells. The light chains of four IgM proteins of Fig. 2 were of type. IgM WAI, whose light chain was of k type was also studied. The p/L ratio of iodinated 7 S IgM WAI were 3.9 and 2.3 at iodination levels of 0.86 and 32 iodine atoms per molecule of 7 S IgM, respectively.

Discussion

The present results of the higher p/L radioactivity ratio for 7 S IgM than for 19 S IgM (Fig. 2) is compatible with the three-dimensional model of IgM mole- cule [9--13]. According to this model, the C-terminal ends of p chains of each 7 S subunit IgM form a central core in 19 S IgM with the N-terminal ends of

chain and L chains protruding outwards. It is likely that some tyrosines * in the C-terminal region of p chains in 19 S IgM are buried and that some of the buried tyrosines became exposed in 7 S IgM. Such exposures of tyrosines of chains in 7 S IgM would result in a higher p/L radioactivity ratio for 7 S IgM as we have found in the present s tudy (Fig. 2).

The 7 S IgM used in the present s tudy was obtained from 19 S serum IgM by mild reduct ion with mercaptoethylamine as described before [8]. The 7 S IgM obtained in this way was a mixture of g2L2, a covalent 7 S molecule, and (pL)2, a noncovalent dimer of a half molecule. Upon ultracentrifugal analysis in a non- dissociating neutral buffer, p2L2 and (~L)2 co-sedimented. The relative ratio p2L2/(pL)2 was 1.9, 1.3, 1.5 and 1.4 for CARD, KUNZ, MILL and WIDR, respec- tively. No significant correlation was observed between the p2L2/(pL)2 ratio and the ratio of radioactivity incorporated into the ~ chain to that incorpora- ted into the L chain (the p/L ratio). The fact that the p/L ratio for cell surface 7 S IgM on RPMI 8392 cells is smaller than the ratios of any of the five 7-S IgM proteins including IgM WAI studied here suggests that a port ion of the p chain of the cell surface IgM of the RPMI 8392 cells is buried in the membrane. Fu and Kunkel [17] reported that the C-terminal port ion of the heavy chain of cell surface IgM on human B lymphocytes is buried in the membrane. Their conclusion was based upon the observation that specific antigenic sites of cell surface IgM is not available for reaction with antibody. Vitet ta and Uhr [18] compared the efficiency of ant ibody binding of cell surface IgM by anti-~ and anti-~ antisera and found that anti-~ was far less effective in binding cell surface IgM than anti-~. Their result suggests that a port ion of p chain of IgM is buried in cell membrane. Our results are consistent with theirs.

* A l t h o u g h his t id ine o f pro te ins can be i od in a te d by l a c t o p e r o x i d a s e - c a t a l y z e d i o d i n a t i o n [ 1 4 , 1 5 ] , t y ro - sine is the p r i m a r y a m i n o acid res idue o f p r o te in that is i od ina ted [ 1 4 , 1 6 ] .

252

Acknowledgments

This investigation was supported in part by Grant No. CA-17276 and Grant No. CA-17609 awarded by the National Cancer Institute, DHEW. We wish to thank Mr. S. Grimm, Mr. M.W. Kim and Miss Marie Kampshoff for their excel- lent technical assistance. Thanks also go to Mrs. C. Zuber and Miss J. Stevens for their help in the preparation of the manuscript.

References

1 Seon, B.K., Minowada, J. and Pressman, D. (1975) ImmunoL Commun. 4, 149--158 2 Vitetta, E.S., Baur, S. and~3hr, J.W. (1971) J. Exp. Med. 134, 242--264 3 Marchalonis, J.J. and Cone, R.E. (1973) Transplant. Rev. 14, 3--49 4 Seon, B.K. and Pressman, D. (1976a) Immunochemistxy 13,407--415 5 Putnam, F.W., Florent, G., Paul, C., Shinoda, T. and Shimizu, A. (1973) Science 182, 287--291 6 Watanabe, S., Barnikol, H.U., Horn, J., Bertram, J. and Hflschmann, N. (1973) Hoppe-Seyler's Z.

Physiol. Chem. 354, 1505--1509 7 Florent, G., Lehman, D. and Putnam, F.W. (1974) Biochemistry 13, 2482--2498 8 Seon, B.K. and Pressman, D, (1976b) Immunochemistry 13,895--900 9 Svehag, S.E., Bloth, B. and Seligmann, M. (1969) J. Exp. Mcd. 130, 691--705

10 Feinstein, A. and Munn, E.A. (1969) Nature 224, 1307--1309 11 Shelton, E. and McIntire, K.R. (1970) J. Mol. Biol. 47,595--597 12 Pa~khouse, R.M.E., Askonas, B.A. and Dourmashkin, R.R. (1970) Immunology 18, 575--584 13 Metzger, H. (1970) Adv. Immunol. 12, 57--116 14 Morrison, M. (1968) Gunma Syrup. EndocrLnoL 5, 239--243 15 Krohn, K.A. and Welch, M.J. (1974) Int. J. Appl. Rad. Isotopes 25, 315---323 16 Marchalonis, J.J. (1969) Biochem. J. 113,299--305 17 Fu, S.M. and Kunkel, H.G. (1974) J. Exp. Med. 140, 895--903 18 Vitetta, E.S. and D-hr, J.W. (1974) J. Exp. Med. 139, 1599--1620