Tryptic and plasmic cleavage of a rat myeloma IgD

0161-5890/81/030249~7 102.00/O

Mokcular Immunology.Vol. 18,pp. 249 to 255. Pergamon PressLtd. 1981. Printedin Great Brilain.

TRYPTIC

GISfiLE

AND PLASMIC CLEAVAGE MYELOMA IgD

ALCARAZ, ALLAIN JOSBPHINE SIRE,

Centre d’Immunologie

COLLE$ HERVE

INSERM-CNRS

ANNIE BAZIN*

OF A RAT

BONED, BRIGITTE KAHN-PERLES, AND ALAIN BOURGOIS

de Marseille-Luminy, 1200 Brussels, Belgium

France

and *University

of Louvain,

(Received 23 October 1980) Abstract-Rat cell surface immunoglobulins were analysed in sodium dodecyl sulfate polyacrylamide gels. By specific immunoprecipitation a 200,000 dalton IgM and a 165,000 dalton IgD were demonstrated. A molecule similar to the mouse and rabbit Fc receptors was non-specifically precipitated. The digestion of a rat myeloma IgD with trypsin or plasmin was studied. Although the IgD molecule was rapidly converted in Fab and Fc fragments by trypsin (1 min., 0°C) it was relatively resistant to plasmin. (Several hours at 37°C were necessary to obtain a complete conversion.) During the plasmin digestion a Facb fragment was observed. These results are discussed in regard to a possible physiological cleavage of cell surface IgD.

MATERIALS

INTRODUCTION

Preparation

IgD is now well characterized as one of the two main cell surface immunoglobulins in all species studied (Van Boxel et al., 1972; Rowe et al., 1973; Abney & Parkhouse, 1974; Melcher et al., 1974; Martin et al., 1976; Fiebig & Ambrosius, 1976; Ruddick & Leslie, 1977; Yurin et al., 1977; Bazin et al., 1978; Sire et al., 1979). In contrast, it is poorly represented in the serum (Rowe & Fahey, 1965b; Bargellesi et al., 1979; Pauwels et al., 1979). The occurrence of IgD myeloma in man (Rowe & Fahey, 1965a) and the rat (Bazin et al., 1978) is then of particular interest for structural studies of this class of immunoglobulins. Biochemical studies have revealed a striking feature of IgD: the extreme susceptibility of its hinge region to proteolysis (Spiegelberg et al., 1970; Jefferis & Matthews, 1977; Bourgois et al., 1977a; Sire et al., 1979). This property led some authors (Vitetta & Uhr, 1975; Bourgois et al., 1977a) to propose that the splitting of IgD complexed with the antigen would be an essential event of the immune response. In this work presented here, antisera directed against a rat myeloma IgD were used to study the rat IgD receptor on the lymphocyte membrane. The splitting of this rat myeloma IgD by trypsin and plasmin was analysed in regard to a possible physiological cleavage of this molecule.

AND METHODS

of iodinated membrane

lysate

Spleen cell suspensions from 3 month old LOU/c/WSl rats were labelled by the lactoperoxydase-catalysed procedure (Marchalonis et al., 1971), washed once in phosphate-buffered saline (PBS) containing potassium iodide (lo- 3 M), and then lysed for 10 min at 0°C in 1% Nonidet-P 40 (NP 40, Shell Chemical Co.) in PBS containing 1 mA4 phenylmethylsulfonyl fluoride and 100 mM recrystallized iodoacetamide. This lysate was centrifuged (10,000 g x 30 min) and then passed over Sephadex G25 equilibrated with the same solution. Immunoprecipitations Indirect precipitations were performed with 10 ~1 of rabbit anti-rat L, p or 6 chain serum or normal rabbit serum (Bazin et al., 1974 and 1978) and 200 ~1 of goat anti-rabbit immunoglobulin (Ig) serum. Antibody precipitates were washed twice with ice cold 1% NP 40 in PBS, once in PBS and then in 50 mA4 sodium phosphate, pH 7. Analysis

of the precipitates

Direct analysis was performed in 4.5% (w/v) polyacrylamide (Merck) gel containing 0.1% (w/v) sodium dodecyl sulfate (SDS, Sigma Chem. Co.) (Della Corte & Parkhouse, 1973). Chemical reduction was carried out on fractions slices by an original eluted from gel electrophoretic method (to be published) allowing 70-90% extraction in 100 ~1 of buffer.

Bourgois, Centre Correspondence to: Alain d’Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, F-13288 Marseille Cedex 9, France. 249

2.50

GISCLE

ALCARAZ

Samples were reduced in 2 mM dithiothre~tol-50 mM sodium phosphate buffer pH 7-2”/,, (w/v) sodium dodecyl sulfate at IOO’C for 10 min., alkylated by addition of 100 mM final concentration iodoacetamide, then resolved on 7.57: (w/v) polyacrylamide gels containing 0. I “h (w/v) SDS. Various internal 13‘I-labelled (Hunter Br Greenwood, 1962) markers were added: A = unreduced mouse Ig (150,000 mol. wt.); B = reduced transfer-tin (80,000 mol. wt.) C = reduced creatine phosphokinase (40,000 mol. wt.); D = reduced murine myeloma x chain (23,000 mol. wt.); E = reduced cytochrome C (12,000 mol. wt). Reduction of standard proteins was performed as mentioned earlier. After electrophoresis, the gels were sliced into 2 mm segments and the radioactivity was determined. Values were corrected for cross-channel spill and plotted with the top of the gel on the left hand side of the figure. Isolation

of’the nz)wloma ig0

er al KESL! LTS

Immunoprecipitation receptors

of’ rut

IgD

and

IgM

Surface immunoglobuiins from a Iysate of radiolabelled rat spleen cells were analysed by SDS polyacrylamide gel electrophoresis (SDSPAGE) of appropriate immunoprecipitates (Fig. 1). Two major fractions (mol. wts = 200~00~) and 165,000) were obtained in an anti-rat k’ chain precipitate, but not in a control precipitate (with normal serum). In addition. both precipitates reproducibly contained a small fraction exhibiting a molecular weight (115,000 daltons) similar to that of the mouse and rabbit Fc receptors (Bourgois et al., 1977b: Sire t’l ui., 1980).

IR 731

The IgD protein was isolated by gel filtration on acrylamide agarose ACA 3.4 (Ultragel LKB) column and QAE A50 Sephadex column from pooled sera collected from LOUjCjWSL rats bearing the plasmacytoma IR 73 I, as previously described (Bazin et al.. 1978; Alcaraz et al., 1980). Tryptir cleuvuge ~?~‘~~l~,~~lonl~i IgD IR 731 The digestion was performed in an ice bath with TPCK treated trypsin (Worthington) with an ~nzyme~protei~ ratio of 17; (w/w). Incubation time was I min and proteolysis was stopped by addition of soybean trypsin inhibitor (final concentration I mgiml). The substrate (IR 731 molecules) was iodinated in presence of chloramine T (Hunter & Greenwood, 1962) and dialysed against PBS (final concentration of protein 0.2 mg/ml).

Plasmin digestion

of m~elorw

IgD IR 731

The digestion was performed at 37’“C with mouse plasmin (enzyme~protein ratio equivalent to that used for trypsin) for the time indicated in the different assays. Plasminogen was purified as previously described (Maillard & Favreau, 1977). Plasmin was produced by activation of I pg of plasminogen by 1 unit of urokinase (1 hr at 37°C in PBS). The iodinated substrate (IR 731 molecules) was prepared as indicated in the previous section.

Frac:ion

N”

Ftg. I Analysis of surface 1~ on rat ~plenocytes. Spleen cell suspensions were labeled wtth tz51 by the lactoperoxidase procedure, washed and then lysed m ice-cold l”,,(a:v) NP40 in PBS in the presence of I mM phcnyl methyl sulfonyl fluoride and 100 mM iodoacctamide. All further operations were done at 0’ C. This lysate was centrifuged (10,000g 30 mini and then passed over Sephadex G-25 equilibrated with ‘*‘I-labeled surface Ig were the same solution. Then, precipitated with various antisera. Precipitates w’ere washed twice with I”,, (MI/~) NP 40 in PBS and once with PBS. Precipitates were dissolved and analvsed in 4.5”,, fu:vi poiyacryiamide~elsw~thout reductton. Various internal ’ “Ilabeled markers were added: A = unreduced mouse IgG (mol. wt.. 150,000): B = reduced transferrtn (80.000): C = reduced cr’eatine phosphokinase (40.000); D = reduced mouse myeloma h- chain (23.000). The position of the “‘I marker proteins is indicated hy arrows. After electrophorrsis. the gels were sliced into 2 mm qments and r-adioacti\ity determined, Values were corrected for cross-channel spill of ’ “I and plotted with the top ofthe gel on the left-hand side of ~1 Molecules precipated with antiserum the @n-e. (-specific for rat z cham. (---.I Control precipitate with normal strum.

Tryptic

4

and Plasmic

Cleavage

of a Rat Myeloma

(a)

251

IgD

(b)

‘: 0 x E

2

e

* .? .> t

2

.-x B IY

0

A

B

C

A

D Fraction

B

C

D

E

No

Fig. 2. Analysis of rat IgM receptor. The cell lysate was prepared as described in the legend to Fig. 1. The molecules were precipitated with anti-serum specific for rat p chain. The precipitate was washed, dissolved and analyzed in 4.5% (w/v) polyacrylamide gel (a) as described in the legend to Fig. 1. Various internal r3iIlabeled markers were added: A, B, C, D. The Ig fraction (mol. wt. 200,000) was extracted, reduced, alkylated and re-electrophoresed in 7.5% (w/v) polyacrylamide gel; (b). Various internal r3’I-labeled markers were added: A, B, C, D, E (E: reduced cytochrome c: 12,000 dalton).

Further analysis revealed that the 200,000 dalton and 165,000 dalton molecules were precipitated by anti-rat p and anti-rat 6 antisera respectively (Fig. 2a and 3a) and would thus represent membrane IgM and IgD. After reduction (Fig. 2b and 3b), these molecules were separated into heavy chains (80,000 daltons and 60,000 daltons respectively for P and S) and light chains (23,000 daltons).

Tryptic and plasmic cleavages of a rat myeloma IgD The rat myeloma IgD IR 731 was markedly susceptible to tryptic digestion. Indeed, an almost complete conversion into two types of fragments was obtained after one minute at 0°C (Fig. 4). The heaviest fragment (mol. wt. 50,000) was identified as a Fab fragment as it was

(a)

(b)

Fraction

No

Fig. 3. Analysis of rat IgD receptor. The cell lysate was prepared as described in the legend to Fig. 1, The molecules were precipitated with anti-serum specific for rat 6 chain. The precipitate was washed, dissolved, and analysed in 4.5’j/, (w/v) polyacrylamide gel: (a) as described in the legend to Fig. 1. Various internal r3iI. labeled markers were added: A, B, C, D. The Ig fractions (mol. wt. 165,000) was extracted, reduced, alkylated and re-electrophoresed in 7.5% (w/v) polyacrylamide gel; (b). Various internal r3’I-labeled markers were added: A, B, C, D, E.

252

GISELE

A

C

B Fraction

D

ALCARAZ

E

N”

Fig 4. Tryptic digestion of rat myeloma IgD (IR 731). The IR 731 rat myeloma IgD was labeled by the chloramin T procedure then dialysed against PBS. Trypsin was added with an enzyme/substrate ratio of I”,, WIW. Digestion was performed in an ice bath during one minute and stopped by addition of soybean trypsin inhibitor. The tryptic products were analysed in 4.5”‘; (w/v) polyacrylamide gels as described in legend to Fig. I Various internal i3’I-labeled markers were added: A. B, C, D. E. (---) Direct analysis. (---) Analysis of an anti-K chain precipitate. (. .) Analysis of an anti-6 chain precipitate.

specifically precipitated by an anti-L chain antiserum (Fig. 4) and could be reduced (Fig. 5) into two polypeptides (mol. wt. 30,000 and 23,000). The other fragment (mol. wt. 36,000) precipitated (Fig. 4) by the anti-6 antiserum (and

ef al.

not by the anti-L chain) should represent the Fc region and was reduced in a unique 19,000 dalton molecule (Fig. 5). The unexpectedly low molecular weight of the complete molecule (Fig. 6) and the Fc fragment (respectively 150,000 and 36,000 daltons) suggest that the isolated myeloma IgD protein has lost a part of its Fc fragment. As plasminogen can react with an activator located on B cell membranes (Maillard & Favreau, 1977), the possible physiological cleavage of IgD (Vitetta & Uhr, 1975; Bourgois rt al., 1977~ and c) could be realised by plasmin on the B cell surface. The kinetics of plasmin digestion of IR 731 was then studied (Fig. 6 and Table 1). The results indicate that: (1) Several hours are necessary to obtain an almost complete conversion in Fab and Fc fragments. (2) An intermediary 100,000 dalton fragment is produced containing an heavy chain, a light chain plus a 19,000 dalton chain (Fig. 7). This intermediary molecule could be derived from IgD by loss of an Fab fragment, thus resulting in a Fabc like product (Nelson, 1964; Goodman 1965; Michaelsen & Natvig, 1972). This result indicates that the plasmic cleavage of IgD differs from that of rabbit IgG molecules degraded into an Facb fragment by loss of the Fc’ region (Connell & Porter, 1971). (3) The rate of digestion (1.4 x IO x moles 1. ’ min ‘) is similar for the two sides of the symmetrical hinge region in the conditions used (Table 1). In addition. Fabc fragments were observed

(a)

4

(b)

io,ooo I9.OOC

3,000 2

0

b

A

B

C

D

E Froctvm

No

Fig. 5. Reduction of the tryptic fragments of rat myeloma IgD (IR 731). The I * 5I-labeled tryptic products described in Fig. 4 and precipitated by an anti-k chain (a) or an anti-s chain (b) were reduced, alkylated and analysed in 7.5”);,(w/v) polyacrylamidegels. Various internal iJII-labeled markers were added: A. B, C, D, E.

Tryptic

(c)

and Plasmic

Cleavage

of a Rat Myeloma

253

IgD

(d)

8

4

li50 ~

AB

C

DE

50

d

Fraction

A B

L&cl

Fraction

Fig. 7. Reduction of the l~,OOO daltons intermediary fragment produced by digestion of IR 731 rat IgD with plasmin. The 100,000 daltons intermediary fragment described in Fig. 6c was extracted, reduced, alkylated and reelectrophoresed in 7.5% (w/v) polyacrylamide gel. Various internal 1311-labeled markers were added: A, B, C, D, E.

N’

Fig. 6. Digestion of rat myeloma IgD (IR 731) by plasmin. The ‘2SI-labeled IR 731 rat IgD was digested by plasmin at 37°C in the conditions described in Materials and Methods for different periods of time: 0 minute (a), 40 minutes fb), 140 minutes(c) and 480 minutes (d). The digestion products were analysed in 4.5% (w/v) polyacrylamide gels as described in legend to Fig. 1. Various internal i3iI-labeled markers were added: A, B, C, D, E.

dalton molecule specifically precipitated by an antiserum directed against a rat 6 chain from a myeloma (IR 731) producing IgD molecules (Bazin et at., 1978). This result confirms then the previous observations suggesting that, as for other species (Van Boxel et al., 1972; Rowe et al., 1973; Abney & Parkhouse, 1974; Melcher et al., 1974; Martin et al., 1976; Fiebig & Ambrosius, 1976; Sire et al., 1979) the main rat membrane immunoglobulins are IgM and IgD (Ruddick & Leslie, 1977; Yurin et al., 1977; Bazin et al., 1978). The reduction products consisted of 80,000 dalton and 23,000 dalton chains for the IgM molecule and 40,000 dalton and 23,000 dalton chains for the IgD molecule. The molecular weight observed for the chain is close to that of

(result not shown) in IR 731 preparations obtained after rough purification from the serum. This result is in agreement with the trypsin-like of spontaneous observation cleavages in the hinge region of human IgD by one or more plasma enzymes (Goyert et af., 1977) and suggests that plasmin could be the enzyme involved. DISCUSSION

Two main celi surface immunoglobulins were observed on the surface of rat lymphocytes:-a 200,000 dalton molecule specifically precipitated by an anti-rat p chain antiserum, and a 165,000 Table

I. Kinetic

Incubation time in mn ____-~~--~.--.-Percentage of IgD converted in Fabc fragments (a) Percentage of IgD converted in Fab and Fc fragments (a)

of conversion

N’

DE

of IgD IR 731 in Fabc,

Fab and Fe fragments

by digestion

with plasmin

20

40

60

80

100 -_____

120

14.2

19.1

21’.7

23.9

24.8

25.5

25.8

14.2

7.4

1.3

7.6

12.6

19.3

25.0

31.0

38.3

40.8

70.0

79.8

98.7

140 240 ..-___-..-

480

900

(a) The relative amount of IgD, Fabc and Fab + Fc was deduced from the radioactivity of the corresponding peaks (see Fig. 6) and the figures obtained verify the following formulas: C, o = eKzO*, Cralx: = 2(e-“‘+ zai), Crc = 1 +e-““‘-2e-“’ in which c of IgD, Fabc and?% at a given time(t) and in which a is the rate constant estimated ) CW!c’ C designate the concentration a#& calculat%r (a = 1.4 x 10-s moles l_ ’ min- i). The concentration of Fc was considered to be the third of the concentration of Fe + Fab.

254

GISfiLE

ALCARAZ

the rat 6 chain described by Yurin rt al. (1977) which was found between the 7 chain (50,000 daltons) and the p chain (80,000 daltons) markers. It is also similar to the molecular weight (65,000 daltons) of the minor form of fi chain described by Ruddick and Leslie (1977) but smaller than the molecular weight of the major form (73,000) found by those authors. The rat IgD molecule produced by the myeloma IR 731 was found to have a molecular weight of 150,000 daltons, smaller than that of the surface IgD and this seems to be due to a smaller Fc fragment. This result has been confirmed by sequence study (Alcaraz et al., 1980). The tryptic digestion of this molecule leads to the production of Fc (mol. wt. 36,000) and Fab (mol. wt. 50,000) fragments (reducing into 19,000 dalton and 30,000 dalton plus 23,000 dalton molecules, respectively). This splitting occurs in one minute in an ice bath, giving a new example of the extreme susceptibility to proteolysis of the hinge region of IgD (Spiegelberg et al., 1970; Jefferis & Matthews, 1977; Bourgois et al., 19770; Sire rt al., 1979). As plasmin is likely to emerge as an active enzyme on the surface of B lymphocytes (Maillard & Favreau, 1977) and could split surface IgD under physiological conditions, the plasmic cleavage of the rat myeloma IgD IR 731 was studied. Although trypsin and plasmin possess the same specificity of cleavage (Cterminal side of arginyl and lysyl residues), their splitting activity on the hinge region of IgD was markedly different. Indeed the conversion of IgD in Fab and Fc fragments by plasmin took several hours at 37°C (instead of 1 min at 0°C when trypsin was used with a similar enzyme/substrate ratio). In addition, it was possible during the conversion to observe an intermediary fragment (Fabc) already observed in rare cases of low rate splitting of immunoglobulin molecules (Nelson, 1964; Goodman, 1965; Michaelsen & Natvig, 1972). This low rate of splitting of the IgD molecule by plasmin allows the coexistence of the two molecules on the B lymphocyte membrane. Nevertheless, if, as previously proposed (Vitetta & IJhr. 1975; Bourgois or ul.. 1977u), the IgD receptor is physiologically split after binding of the antigen, a possible explanation would be that the rate of fragmentation of the IgD molecule by plasmin is considerably enhanced when the antigen is bound to the immunoglobulin. It would be thus of great interest to extend such studies to an IgD molecule possessing a known

et al.

specificity in order to verify if the binding of the antigen modifies the kinetics of cleavage by plasmin. AcknoM?ledgements~We are grateful to Dr. J. Maillard for helpful discussion and for providing the plasminogen and urokinase preparations. We thank our colleagues from the Centre d’Immunologie for critical reading of the manuscript.

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Tryptic

and Plasmic

Cleavage

Martin L. N., Leslie G. A. & Hinder R. (1976) Lymphocyte surface IgD in non human primates. ht. Arch. Allergy Appl. Immunol., 51, 320-329. Melcher V., Vitetta E. S., MacWilliams M., Lamm M. F., Phillips Quagliata J. M. & Uhr J. W. (1974) Cell surface immunoglobulins. X-Identification of an IgD like molecule on the surface of murine splenocytes. J. Exp. Med., 140, 1427-1431. Michaelsen T. E. & Natvig J. B. (1972) Three new fragments: F(ab),, F(c), and Fab/c obtained by papain proteolysis of normal human IgG. Stand. J. Immunol.. 1. 255-268. Nelson C. A. (1964) Isolation of a new intermediate in the papain cleavage of rabbit y-globulin. J. biol. Chem., 239, 3727-3732. Pauwels R., Bazin H., Platteau B. & Van der Straeten M. (1979) The influence of different adjuvants on the production of IgD and IgE antibodies. Ann. Zmmunol. (Inst. Pasteur), 13OC, 49-58. Rowe D. S., Fahey J. L. (1965a) A new class ofhuman Ig. I. A unique myeloma protein. J. exp. Med., 121, 171-184. Rowe D. S. & Fahey J. L. (1965b) A new class of human Ig. II. Norma1 serum IgD. J. exp. Med., 121, 185-199. Rowe D. S., Hug K., Forni L., & Pernis B. (1973) Immunoglobulin D as a lymphocyte receptor. J. exp. Med.

of a Rat Myeloma

IgD

255

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