Bence jones proteins and light chains of immunoglobulins—XXVII

Inmuno~hrmurr~ Vol. 15. pp 453-458 tr Pergamon Press Ltd 1978. Printed m Great Bntm

BENCE

UNIQUE

JONES PROTEINS AND LIGHT IMMUNOGLOBULINS-XVII.

SUSCEPTIBILITY OF CERTAIN GRANULOCYTE-DERIVED ALAN

’ The University

of Tennessee 379210, U.S.A.

SOLOMON1

CHAINS

K-CHAINS TO PROTEOLYSIS NEUTRAL PROTEASES* and

KLAUS

OF

BY HUMAN

HAVEMANN

Center for the Health Sciences, Knoxville Units, Memorial Research Center, and LThe Medical Clinic, University of Marburg, Marburg, West Germany

Knoxville,

TN

(Received 22 December 1977) Abstract-Light chains of human immunoglobulins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (V,) portion. and to the carboxyl-terminal, constant (CL) portion. We have investigated the effect of two human polymorphonuclear leucocyte-derived neutral proteases, the elastase-like protease (ELP) and the chymotrypsin-like protease (CLP), on Bence Jones proteins representative of the four structurally and immunochemically-defined groups of human kappa light chains. When ~1, ~11, ~111 and KIV proteins were incubated with ELP or CLP, the result was an extensive proteolysis of the CL portion of these molecules and the generation of VL-related fragments. However, the VL portions of certain K-chains classified immunologically as a subgroup of XIchains. h-1-1, were also extraordinarily susceptible to proteolysis by these granulocyte-derived neutral proteases. These findings have provided new evidence relating specific structural and antigenic features of human kappa light polypeptide chains.

INTRODUCTION

1969). Our recent studies on the effect on Bence Jones proteins of the elastase-like (ELP) and chymotrypsinThe relationship between structure and antigenicity of like (CLP) neutral proteases derived from human the light polypeptide chain of immunoglobulins has polymorphonuclear leucocytes demonstrated the been elucidated through physico-chemical and extraordinary susceptibility of the CL portion of these immunochemical studies on Bence Jones proteins. molecules to proteolysis. Differences in the Thus, four basic groups of human K light chains, susceptibility of the V, portion to digestion were also designated ICI,K-II, ~111 and KIV, have been defined on evident (Solomon et al., 1976). the basis of characteristic sequences of the first 23 In order to determine whether the sensitivity of amino acid residues (Kabat et al., 1976). The certain light chains to extensive proteolysis by ELP or structural definition for each of the four groups has CLP could be related to specific structural features of been confirmed immunochemically (Solomon & the VL portion of these molecules, we have McLaughlin, 1969~; McLaughlin & Solomon, 1974; investigated the effect of these enzymes on 3 1 human K Sletten et al., 1974). The findings of distinct intragroup representative of the four and intergroup antigenic heterogeneity among IC- Bence Jones proteins structurally and immunochemically-defined groups of chains (Solomon & McLaughlin, 1971; McLaughlin & K chains: ICI, KII, ~111 and KIV. Our results indicate Solomon, 1971) led to the localization and association that whereas VL-related fragments were formed from of distinct regions in the amino-terminal or variant each of the proteins studied, KI light chains of an half (V,) of the K-chain with specific antigenic immunologically-defined subgroup, ~-1-l (Solomon & sites (McLaughlin & Solomon, 1972). The immunoMcLaughlin, 1971), are especially susceptible to chemically-classified groups or subgroups of K-chains digestion by the granulocyte-derived neutral have not as yet been linked with specific tertiary proteases. structural features. Differences in the three-dimensional folding of MATERIALSAND METHODS immunoglobulin molecules have been inferred from studies demonstrating differences in susceptibility of Bence Jones proteins these proteins to cleavage by proteolytic enzymes The procedures followed for the isolation and purification (Putnam er al... 1962). Light chains of immunoof Bence Jones proteins, the cleavage of Bence Jones proteins globulins can be cleaved by pepsin into two fragments by pepsin (Worthington Biochemical Corp., Freehold, NJ), corresponding to the amino-terminal or variant half and the isolation. purification and characterization of V, and (V L) and to the carboxyl-terminal or constant half (C,) CL pepsin-derived fragments have been previously described (Solomon & McLaughlin, 1969b; Karlsson et al., (Solomon & McLaughlin, 19696). Immunologic techniques *This work was supported in part by USPHS Research Grant CA 10056-I 2 from the National Cancer Institute and the Stein Cancer Research Fund.

The preparation in albino New Zealand rabbits of antisera to Bence Jones proteins and the characterization of the specificity of these antisera for V, and CL antigenic __

45.1

t

t

CL

BJP

-

anti-C

-

anti-BJP

-

anti-V

VL

Fig. 1. Immunoclectrophoretic analysis ofa pepsin-treated Rcnce Jones protein. The antigen wells contain a K Bence Jones protein cleaved partially by pepsin into constant IialflC, ) andvariant haIf fragments; the upper, lower and middle antisera troughs contained anti-Rence Jones protein antisera with specificity either for C, (anti-C) V, (anti-V). or both C, and V,_ (anti-BJP) antigenic determinants, respectively.

0A

PEPSIN

-

ELP

CONTROL

-

t

t

CL

BJP

VL

4

+

+

t

0f3 PEPSIN

ELP

CONTROL

-

-

-

Fig. 2. Comparison of the cffcct of pepsinand tLP on a Hence Jonca protein (A) Im~~~unoelcctrophoresis. The lower antigen well contained the untreated Bcncc Jones protein (control), the upper hell contained the sample obtained after partial dlgcstion of the Bencc Jones protein for 3 min by pepsin (pepsin) and the middle well contained the sample obtained after J-hr digestion of the protein by ELP. The antisera troughs contained an antiserum prepared against the homologous native Bcncc Jones protein. (B) Cellulose acetate electrophoresis. The control and treated samples al-t‘ as indlcatcd above. The position of the intact Bence Jones protein (BJP). constant half (C, ) and variant half IV, j l’ragment~ are as indicated.

K-Chain

Cleavage

by Granulocyte

determinants have both been described (Solomon & were 1969rr). Immunoelectrophoreses McLaughlin, performed on microscope slides coated with a 1”” agar medium prepared in a barbital buffer, pH 8.6. ionic strength 0.10. Cellulose acetate electrophoreses were accomplished with a Microzone apparatus (Beckman Instruments, Inc.. Palo Alto, CA). Optimum resolution was obtained by electrophoresis for 40 min at 250 V in 0.075 ionic strength barbital buffer, pH 8.6. After Ponceau S staining, visualization of the protein bands was enhanced by keeping the membrane opaque. i.e. destaining with 5”,, acetic acid and drying without further treatment.

The methods for isolating and purtfying ELP neutral N-tactivjity against esterase exhibiting proteases butyloxycarbonylalanme p-nitrophenyl ester and CLP proteases with esterase activity il’-acetylagainst phenylalanine 2-napthyl eater were as described (Schmidt & Havemann. 1974). The purified enzymes were dialyzed against 0.05 M Tris-HCI-0.05 M glycine buffer. pH 7.5, and then adjusted to a concentration of 20 pg of protein per ml as determined by a modification of the Lowry method (Geiger & Bessman, 1972) using bovine serum albumin as a protein standard. 0.5 ml of enzyme sample was lyophilized in individual vials and reconstituted with 0.15 A4 NaCl prior to use. Lyophilization resulted in an _ 5?,, loss of esterase activity. RESL’LW AND DISCUSSION

Ejyect Q‘pepsirl tmd ELP on light chains qf immunoglohidirzs A sample of a K Bence Jones protein was incubated at 37 C in a 0.05 M Tris-HCI-0.05 M glycine buffer,

455

Proteases

pH 3.4, with pepsin (enzyme:substrate ratio, 1:50). Aliquots were obtained at designated intervals and the digestion process was terminated by inactivating the pepsin with the addition of 0.1 M Tris base of sufficient quantity to increase the pH of the reaction mixture to 8.0. After the extent of light chain proteolysis had been determined by cellulose acetate electrophoresis. the proteolytic fragments were identified immunochemically with antisera capable of recognizing constant (C) region and variant (V) region light chain antigenic determinants. An immunoelectrophoretic analysis of a 3-min pepsin digest of a K Bence Jones protein is shown in Fig. 1. When the digest was tested with an antiserum prepared against the native protein, three distinct precipitin arcs were evident. The middle arc corresponded to that of the native Bence Jones protein; the antigenically deficient anodal and cathodal arcs were identified as reactions of Ct.- and Vr-related components, respectively. Specific identification of the C, component was made with an antiserum prepared against a heterologous ti Bence Jones protein (anti-C); the homologous antiserum, absorbed with a heterologous K Bence Jones protein (anti-v), was used to identify the V, component. Strikingly different results were obtained when the same Bence Jones protein was incubated with ELP at 37 ‘C in a 0.15 M NaCI. 0.05 M Tris-HCI-0.05 M glycine buffer, pH 7.5 (enzyme:substrate ratio, 1:X)). The amount of intact Bence Jones protein decreased progressively throughout the incubation period. After 8 hr, no intact Bence Jones protein could be detected electrophoreticallyorimmunochemically. Concomitant

“PEP L

ELP h

BJP

anti-BJP

anti-V

anti-C

Fig. 3. Comparison of the immunochemical reactivity of a Bencc Jones protein (BJP) and Its V,-related fragments derived by pepsin (V FE”) and by ELP (VkLp) digestion. The proteins contained in the outer antigen wells in the lower two Ouchterlony immunodiffusion analyses were as designated in the upper portion of the tigurc. As indicated. the central wells contatncd anti-Bence Jones protein antisera having specificity either for C region (anti-C). V region (antt-V), or both c‘ and V region (anti-BJP) anttgenic determinants.

456

ALAN

SOLOMON

and KLAUS

with this decrease was the appearance of a new component antigenically deficient to the Bence Jones protein. The new component was identified immunochemically as having antigenic determinants associated with the V region of the native molecule; no C region antigenic determinants were detectable. Although the electrophoretic mobility of the new V,related component differed from that of the intact Bence Jones protein, it was comparable to the V, fragment derived by pepsin cleavage of the native protein (Fig. 2). The site of cleavage by ELP on the light chain which results in the generation of the V,-related fragment is not presently known. The V and C regions of human light chains exist as two compact domains linked by an extended - loresidue polypeptide termed the ‘switch’ region; the V-C junction is located between residues at positions 107 and 108 (Schiffer et al., 1973). Seon and coworkers (1973) have shown that the specific site of cleavage by pepsin of K light chains is between residues at positions 116 and 117 in the CL; thus, the carboxylterminus of the pepsin-derived V,_-related fragment (V,PEP, extends into the C region 8 residues beyond the V-C ‘switch’ region. Whether the carboxyl-terminus of the ELP-derived V,-related fragments (VfL4

0A

0 hr

-

4

hr

-

8

hr

-

HAVEMANN

corresponds to that of the VeEP fragment or is closer to the V-C junction remains to be determined. No antigenic differences were apparent between VtEP and VELp (Fig. 3). No proteolysis was observed when i. Bence Jones proteins representative of 4 of the 5 structurallydefined A: chain groups (Kabat et al., 1976) were incubated with pepsin or ELP under identical conditions employed for the K Bence Jones proteins. As previously reported (Solomon et al.. 1976), only after reduction and alkylation of the 1 Bence Jones proteins were they rendered susceptible to proteolysis by ELP. Reduction and alkylation of l-chains also rendered these proteins susceptible to cleavage by pepsin (Solomon & McLaughlin, 1969b; Karlsson et al., 1969). Efjtict of ELP on

~1,

~11, KIII and

KIV

light chains

Differences in the extent of proteolysis of certain K light chains by ELP were evident in our initial studies with this enzyme (Solomon et al., 1976). For example, as shown in Fig. 4, when two K Bence Jones proteins were treated with ELP under identical conditions, a V,-related fragment was formed from one protein but not from the second. Within a 4-hr period of incubation with ELP, the K Bence Jones protein EPP

16 hr -

t

t

BJP

VL

1

4

0B 16 hr

-

8

hr

-

4

hr

-

0

hr

-

KEPP

KMCC

Fig. 4. Comparison of the effect of ELP on two K Bence Jones proteins. (A) Immunoelectrophoresis. The antigen wells contained samples of Bence Jones proteins EPP and McC incubated with ELP for 0,4,8 and 16 hr. The antisera prepared against Bence Jones proteins EPP and McC were contained in the antisera troughs on the left and right. respectively. (B) Cellulose acetate electrophoresis. The samples were as described above. The positions of intact Bence Jones protein (BJP) and V,,-related fragment (V,) are as indicated.

K-chain

Cleavage

by Granulocyte

had undergone extensive proteolysis and no V,related component was evident by immunochemical or electrophoretic analyses. In contrast, the Vr-related component formed by ELP digestion of protein McC was cieariy evident in the 4-, S- and 16hr samples. Proteins EPP and McC were classified immunochemically as members of the tiI and ~111 groups, respectively. In order to determine if the differences in susceptibility to proteolysis by ELP ofcertain K-chains were group-related, we studied the effect of this enzyme on the Bence Jones proteins which were representative of the four structurally and immunochemically-defined K-chain groups. Incubation of these proteins with ELP generated Vi,related fragments from ti1, ~11, till1and k-IV proteins; however, certain ~1 proteins were particularly susceptible to proteoiysis and no V,-related fragment could be demonstrated immunochemically or efectrophoretically (Table I). Table

I, Effect of ELP on x Bence Jones proteins 4 hr

x-chainh h.1

CR0 BRUS JOH* HUS H3J4h EDW GALh HAU EPP REI” BIh DIE”

CLA A@ KIN ROYh WMS SCWh tilI

KfiI

FIN PAT CUM* LAYM TIh COL McC McK OAKh xoc WASH’

KIV LENh KAL

UJP 0 0 0 0 0 +

0 0 0

If0 + f i

0 + 0

0 0 0 -t

0 0 0 -t”

0 Ik F0

0 0

Incubation period” 8 hr I6 hr VLd BJP V,_ BJP Vt 0 0 0 0 0

0 0 0 0 + + + + + + + f + + + + + + + + + + + + + +

0 0 0 0 0 0

0 0

0 0 0 0

0 0 0 0 0

0 0

0

0

0

0

0

0 0 0 0 0 0 0 0 0

0

0

+ + + + + + + + +

+ 0 0 0 0 0

+ t + +

0 0 + 0

0 0 0

+ + + + + + +

0 0 0 0 0 0 0

I& It + + +

0

+

0

+

0

?I

0

0

0 + 0

Ik 0 + 0 + 0 0 0

0 + 0 0 0

k

0

0 0 0

+ 0 +

2 0 + + -t + +

r!z + -!-

0 +

“Period of incubation at 37°C of ELP with h--chain (enzymesubstrate ratio, I:50). *h--chains for which partial or complete amino acid sequence data are available (J. D. Capra, unpubfished data. 1974; Kabat et al., 1976). The assignment of h--chains as ICI, ~11, k-111 and rcIV was determined structurally and/or immunochemically (Solomon, 1976). ’ BJP = Bence Jones protein. ‘V, = Variant half, i.e. amino-terminal portion, of the light chain.

Proteases

457

Our previous demonstration of antigenic heterogeneity among proteins of each of the k-chain groups led to the immunochemical definition of two distinct subgroups within each major group; for KI proteins these were designated ~1-1 and ~1-2 (Solomon & McLaughlin, 1971). Among the k-I-chains listed in Table 1, proteins HAU, GAL and HBJ4 have been classified immunochemically as KI- 1, and proteins REI, AU and ROY as k-1-2. Immunochemical analyses of the ~1 proteins most susceptible to ELP digestion, i.e. extensive proteolysis of the Vi_-portion of these molecules, revealed that they were all of the ~1-1 subgroup. Alternatively, the ~1 proteins cleaved by ELP into Vi+-related fragments were classified as x1-2 chains, Seon et d. (1972) have shown that the yield and stability of V, or CL fragments derived from the cleavage of Bence Jones protein by pepsin can be influenced markedly by the temperature at which the enzymatic digestion is performed. At 37‘C, the V, fragment is relatively stable to further pepsin digestion, whereas the C, is degraded extensively to smaller peptides. At 55”C, the opposite phenomenon occurs; the C, fragment is relatively stable and the V, is extremely susceptible to proteolysis. Because of the marked susceptibility to proteolysis of a ‘pepsinstable’ C, fragment (derived from a Bence Jones protein digest performed at 55°C) when incubated at 37’C with ELP (Solomon et al., 1976), we compared the effect of a 55°C incubation temperature on digestion by pepsin and ELP of KI- I and x1-2 proteins. As shown in Fig. 5, pepsin treatment of the k-I-1 Bence Jones protein CR0 resulted in the formation of the expected Cr-related component; however, at 55°C no C, component could be demonstrated immunochemically or electrophoretically in ELP digests of this protein. Incubation at 55°C of ~-1-2, ~11, h-111and KIV proteins with ELP resulted in extensive proteolysis and neither V, nor C, components could be detected. Effect of CLP on fight chains The proteolytic effects of CLP on Bence Jones proteins were similar to those achieved with ELP; however, the rate of proteolysis with CLP was approximately one-third less than that obtained with ELP (Solomon ef af., 1976). The increased susceptibility of ~1-1 chains (as compared to other types of K--chains) to proteolysis by ELP was also evident when these proteins were incubated with CLP. The marked susceptibility of the V, portion of x1-1 chains to proteolysis by ELP suggests differences in the tertiary structure of the V region of XI- 1 proteins as compared with the V regions of tiI-2, ~11, k-111and KIV proteins. The three-dimensional structure of the V, portion of only three K light chains have been determined by X-ray crystallographic analyses (Epp ef al., 1974; Fehlhammer et al., 1975; Colman et (I/., 1977). All three of these fragments have the aminoterminal sequences characteristic for ~1 proteins. and we have classified them immunologically as representative of the h-I-2 subgroup. It is not now known what structural features of tiI-1 light chains contribute to the enhanced susceptibility of the V, portion of these molecules to proteolysis by the granulocyte-derived neutral proteases. Such information will provide a mofecular basis for the

458

ALAN

SOLOMON

and KLAUS

HAVEMANN

PEPSIN

0+ 5

min

-

3

min

-

1 min

-

CONTROL

-

Fig. 5. Comparison of the effect of incubation of a xl-1 Bcnce Jones protein at 55 C with pepsin and with ELP. Cellulose acetate electropherogram of samples obtained I. 3 and 5 min after incubation of ;I Bence Jones protein (control) with the two enzymes. The positions of the intact protein (BJP) and CL-related fragment (C,) arc as indicated

in~munoch~mical heterogeneity observed among Kchains and add to our knowledge of the relationship between antibody structure and specificity. REFERENCES

Colman P. M., Schramm H. J. 6i Guss J. M. (1077) J. m&c,. Biol, 116, 73. Epp O., Colman I’., Fehlhamnler H ., Bode W.. Schiffer M, & Huber R. f 1974) Eur. J. Biochetir. 45, 5 13. Fehlhammer H., Schiffer M., Epp 0.. Cohuan P. M.. Lattman E. E.. Schwaper P. & Steigemann W. (1975) Biq&j~. Srrucr. MeehuZsn* 1, 139. Geiger P. J. & Bessman S. P. (1972) Analvr. Biocf7cm. 49,467 Kabat E. A.. Wu T. T. & Bit&sky H. (1976) Vurinhlr R&m.s of In7rnu~#%l~~i~li~ Chains: T~hulat~u~.~ cwd Anu/y.ses of An7ino Acid Sequences. Medical Computer Systems. Cambridge, MA. Karlsson F. A., Peterson P. A. & BerggIrd 1. (1969) Proc. mtn. Acad. Sci. U.S.A. 64, 1257. McLaughlin C. L. &Solomon A. (1971) J. Irntnun. 107, 169’). McLaughlin C. L. & Solomon A. (1972) /. hio1. Chm7. 247, 5017.

McLaughlin c‘. L. & Solomon A. (1974) .I. hnmu~7. 113, 1369. Putnam F. W.. Easlcy c‘. W. & Lynn L. 7‘. (1962) Biochim. hio~ph~x 4(./u 58, 27Y. Schiffer M., Girling R. L., Ely K. R. & Edmundson A. B. ( 1973) Bioc~heniistt:~*12, 4620. Schmidt W. & Haremann K. (1974) ~j~p~f,-Se~l~~‘.s 2. ~‘h,rxicil. C;iiWi. 355, i 07:. Seon 8. K.. Grossberg A. L.. Roholt 0. A. & Pressman D. (1973) .I. Ir17niuri. lil, 269. Seon B. K.. Roholt 0. A. 6i Pressman D. (1972) J. hiul. C/7a77. 247, 21.51. Sletten K.. Hannestad K. & Harboe M. (1974) Smnd. J. rt17t777ct10/. 3 2 19. Solorimt A. (‘IY76) Xeic Ggi. J. :Mrif. 294, 17, 9 I. Solomon A. gi McLaughlin C. L. (1969~) ‘1. c”p. Med. 130, 1295. Solomon A. Rr McLaughlin C. L. ( 1969h) J. hiol. Chem. 244, 3393. Solomon A. & McLaughlin C. L. (1971) J. Zmmun. 106, 120. Solomon A., Schmidt W. & Havemann K. (1976) J. Immun. 117, LOlO.