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Immunochemical studies of an immunoglobulin M-immunoglobulin G mixed cryoglobulin
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
9,67-74
(1978)
lmmunochemical Studies of an lmmunoglobulin lmmunoglobulin G Mixed Cryoglobulinl FRANCOIS CHENAIS, Department
H. HUGH FUDENBERG,
of Basic and Clinical of South Carolina,
Immunology Charleston,
AND AN-CHUAN
and Microbiology, South Carolina
Medical 29401
M-
WANG~ University
Received April 4, 1977 A mixed IgG-IgM cryoglobulin was isolated from a patient with arthritis and glomerulonephritis. Electron microscopic examination of both cryoprecipitate and glomerular deposits revealed unusual structures designated as “cylindrical and annular bodies.” The IgM component was monoclonal and had antibody activity against IgG molecules. Ultracentrifugation showed that immune complex formation was not temperature dependent. From amino acid analysis, the IgM component of the cryoglobulin was classified as V,I-VJI. In terms of chemical characteristics, this cryoglobulin was not different from most of the others previously described. These findings raise the possibility that the ultrastructural organization of the antigen-antibody complex could be related to its cryoprecipitation properties and could be a general property of cryoglobulins.
INTRODUCTION
Cryoglobulins are serum proteins, usually immunoglobulins, which reversibly precipitate in the cold. Purification and immunochemical analysis have led to a classification of this group of proteins (1, 2) into three categories: type I, consisting of a single monoclonal immunoglobulin; type II, mixed cryoglobulins in which one of the components is a monoclonal immunoglobulin; and type III, mixed polyclonal cryoglobulins. Cryoglobulins may be associated with several diseases, such as immunoproliferative disorders or autoimmune diseases, though an appreciable number of them are idiopathic. Type II and III cryoglobulins are believed to represent immune complexes, and indeed many of the clinical manifestations of cryoglobulinemia resemble those of immune complex diseases; i.e., chronic vascular purpura, arthritis, and glomerulonephritis are frequent symptoms. Type II cryoglobulins represent remarkable immune complexes where the antibody is a monoclonal immunoglobulin and is directed against a well-defined antigen; most often the antibody is a monoclonal IgM with anti-IgG activity. Such an immunoglobulin is especially suitable for chemical studies of its antigen-binding site. The four-polypeptide-chain structure of immunoglobulin molecules is now well established. The carboxy-terminal portion of each chain is prone to restricted variations, specific for a given class or subclass (with the exception of allotypic variations). The amino-terminal portion is much more variable, though several 1 This is publication No. 121 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by ACS Grant IM161, USPHS Grants AM-19201 and AI-13388, and National Science Foundation Grant BMS-75-09513. A.C.W. is the recipient of an American Cancer Society Faculty Research Award (125). 2 Kindly address correspondence to Dr. Wang. 67 0090-1229/78/0091-0067$01.00/O Copyright All rights
@ 1978 by Academic F’ress,inc. of reproduction in any form reserved
68
CHENAIS,
FUDENBERG,
AND
WANG
subgroups have been defined, based on the degree of amino acid sequence homology (3). It is established that the variable region of an immunoglobulin is responsible for its antibody specificity (4). The present paper reports chemical studies of an IgM-IgG cryoglobulin isolated from a patient with glomerulonephritis. The clinical and histological studies of this patient have been reported elsewhere (5). In brief, the patient presented with arthritis and evidence of renal disease. A kidney biopsy evidenced a proliferative endocapillary glomerulonephritis. This clinical pattern fits with the syndrome first described by Meltzer ef al. (6) in patients with mixed cryoglobulins. The most remarkable feature was the ultrastructural appearance of the cryoglobulin: Structures designated as “cylindrical and annular bodies” were found both in glomerular deposits and in the cryoprecipitate isolated from serum. Amino acid analyses and sequence studies of this peculiar cryoglobulin were undertaken to compare it with others previously studied and to determine whether the ultrastructural aspect was associated with unusual chemical characteristics. MATERIALS
AND METHODS
Isolation ofcryoglobulin. Blood from the patient studied (Cha) was drawn in a warm syringe and allowed to clot at 37°C for 2 hr. Serum was kept at 4°C for 48 hr and centrifuged, and the supernatant was removed. The precipitate was dissolved in phosphate-buffered saline, pH 8.0, at 37°C. Insoluble material was removed by centrifugation. The cryoglobulin was reprecipitated at 4°C. then washed three times with cold phosphate-buffered saline. The precipitate was finally suspended in 0.2 M sodium acetate buffer, pH 4.0. The IgM and IgG components of the cryoglobulin were separated by gel filtration on a Sephadex G-200 column equilibrated with 0.2 M sodium acetate buffer, pH 4.0, and lyophilized. The purity of the fractions was controlled by immunoelectrophoresis. Ultracentrifugation. A Beckman Model E analytical ultracentrifuge, equipped with Schlieren optics and an RITC temperature control unit, was used. Sedimentation runs were performed at 60,000 rpm at various temperatures. The rotor and cells were preincubated at the desired temperature. Photographs were taken at 8-min intervals after two-thirds of the speed was reached. Enzymatic digestion ofZgG. Fab and Fc fragments were obtained from the IgG by papain digestion, using the method of Gergely et al. (7). The IgG was dissolved in 0.075 M phosphate buffer, pH 7.0, containing 0.075 M NaCl and 0.002 M EDTA. The solution was incubated at 37°C for 2 hr with papain at an enzyme-toprotein ratio of 1: 100, in the presence of cysteine. The digestion was stopped by the addition of a 50% excess of iodoacetamide. The fragments were separated by chromatography on DEAE-cellulose. Fab fragments were eluted in 0.005 M phosphate buffer, pH 8.0. Antibody activity of the IgM against the IgG and its Fc fragments was assayed by double immunodiffusion. As the formation of precipitin lines was not significantly affected by temperature, the migration was carried out at room temperature. Separation of heavy and light chains. Reduction of the IgM component was carried out with 0.1 M dithiothreitol in 7 M guanidine hydrochloride dissolved in 0.5 M Tris-HCl buffer, pH 8.5, for 2 hr at room temperature. Alkylation was done with 0.25 M iodoacetamide for 1 hr at room temperature. The heavy and light
IcM-ICC
MIXED
CRYOGtOBULIN
69
chains were separated by gel filtration through a Sephadex G-100 column equilibrated with 6 M urea in 1.0 M acetic acid. Amino acid analyses. Amino acid compositions were determined by analysis of hydrolyzed proteins on a Beckman amino acid analyzer. Hydrolysis was performed in 6 N HCl under reduced pressure at 110°C for 22 hr. Amino acid sequences were determined by Edman degradation (8) with a Beckman Model 890 protein sequencer as previously described (9). The N-terminal residue of the heavy chain of the IgM was determined by the dansyl chloride method (10). Dansyl amino acids were identified by thin-layer chromatography on silica gel ( 11). The pyrrolidone carboxylic acid (PCA)-blocked N-terminal peptide was obtained by Nagase digestion and isolated as previously described (12). RESULTS
Gel filtration allowed purification of the two components of the cryoglobulin. The IgM component was a monoclonal protein with a K-type light chain. The IgG component, as expected, was polyclonal and reacted with antisera directed (Fig. l), the IgM against human K and A chains. On double immunodiffusion
FIG. 1. Precipitation 3. Fc fragment of IgG. gel.
of the IgM component with IgG component and its Fc fragment. 1, IgM; 2, IgG; The line above and to the right of well 1 is an artifact resulting from a fold in the
70
CHENAIS,
FUDENBERG,
AND
WANG
component formed precipitin lines with the IgG component and with its Fc fragment, confirming the anti-IgG antibody activity of the IgM, which was shown previously by agglutination studies (5). The IgM had specificity for the Fc fragment of the IgG. Ultracentrifugation of the whole serum of the patient at 10°C and at 37°C showed the presence of a component sedimenting more rapidly than the IgM and representing immune complexes. This fast component was also seen when a mixture of the IgM and the IgG was ultracentrifuged (Fig. 21. When the same mixture was centrifuged at 37°C and at lO”C, no appreciable difference was seen in the amount of complexes formed.
FIG. 2. Analytical ultracentrifugation of the isolated cryoglobulin. Top, control containing normal IgG and IgM: bottom, cryoglobulin. The photograph was taken 16 min after two-thirds speed was reached. The run was done at 37°C. A small fraction sedimented faster than the IgM. S.C. indicates the soluble complex.
IcM-ICC
MIXED
71
CRYOGLOBULIN
Amino acid analysis was performed for the IgM molecule only. The amino acid compositions of the heavy and light chains are shown in Table 1. The amino acid contents of the heavy and the light chains of the IgM are consistent with those of other cryoglobulins in that their light chains appear to have fewer serine residues (12). The V-region subgroups of the heavy chain (V,) were determined according to the composition of the N-terminal peptide (3), which is PCA-blocked. The amino acid sequence of this peptide is PCA (Val-GlxLeu), which is characteristic of the VnI subgroup. The N-terminal amino acid sequence of the light chain is shown in Table 2. Comparison with the prototpye amino acid sequences of various human V, subgroups (13) indicates that this light chain belongs to the VJI subgroup. TABLE AMINO
ACID
COMPOSITIONS IcM COMPONENTS
1
OF HEAVY OF THREE
AND LIGHT CHAINS CRYOGLOBULINS
OF THE
Cryoglobulin Cha
PO1
Nev
Amino acid0
u
K
w
A
P
K
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
6.4 2.0 4.9 7.9 9.0 10.5 9.1 7.7 6.8 5.8 3.0 8.8 1.8 2.4 6.8 3.6 3.3
5.5 1.3 4.5 8.2 6.1 6.9 12.0 8.4 8.4 6.9 3.0 8.7 0.5 2.7 8.2 4.3 3.8
3.6 1.4 3.3 9.3 9.3 7.4 10.9 7.4 7.1 6.9 2.5 10.1 1.3 3.7 8.2 3.8 3.8
5.2 1.5 3.8 8.7 7.8 8.1 10.5 7.5 8.6 8.7 2.7 8.1 3.2 7.9 5.4 2.3
3.4 1.4 3.3 10.1 8.7 8.8 10.2 8.5 7.2 7.5 2.5 9.6 1.1 3.2 7.6 2.9 4.1
4.8 1.1 4.4 8.4 7.9 8.2 12.8 6.6 7.2 7.2 2.7 7.9 2.1 9.7 4.6 4.3
o The percentage of each amino acid is expressed in terms of molar fraction, the total molar content of each chain taken as unity. Tryptophan was decomposed during the hydrolysis and thus is not included in the calculation of amino acid composition. Cha is the cryoglobulin of the present study. The results for Nev and Pol are taken from Wang et al. (12).
DISCUSSION
Despite its peculiar ultrastructural appearance, the IgM-IgG cryoglobulin reported here does not differ chemically from others previously reported. From amino acid analysis, the IgM component of the cryoglobulin is classified as V,IVJI. The amino acid sequences of other immunoglobulins with anti-IgG activity have also been studied and they do not seem to be associated with a unique light-chain subgroup. Capra and Kunkel (14) studied anti-y-globulin antibodies isolated from four patients with hyperglobulinemic purpura. One of them had a
Ser
VAV
Glu
Ile Val
Leu
Val
ri Prototype sequences sequences to that listed
Nev
Asp
chain 1 Glu Glx
VJI Cha Pol Ber
Light
Gln
5 Thr
Asp Asx
Gln Glx Glu Glx
of light chain subgroup? on the top line
Thr
Leu
N-TERMINAL
Pro \‘,I1
Ser
Val
Ala
Gly
ACID
and V,V
Ala
Pro
AMINO
TABLE
Val
Leu
2
Ala
Ser
Gly
Ser
CHAINS
by WHO
Leu
Leu
OF LIGHT
are those accepted
Ser
10 Thr
SEQUENCES
Thr
Gly
nomenclature
Gin Glx
15 Pro
Arg
Arg
( 13). Solid
Val
Glx
Glu
OF CRYOGLOBULINS”
Thr
20 Thr
lines indicate
Ile
Ala
identity
Cys
Leu
Glx
Cys
of amino
Gin
Ser
acid
25
IcM--ICC
MIXED
CRYOGLOBULIN
73
A-type light chain, one had a K-type light chain of VJI subgroup, and the two others belonged to the V,I subgroup and had identical amino acid sequences up to residue 40. In contrast, the IgM anti-IgG isolated from mixed cryoglobulin seems to have more restricted light-chain subgroups. Previous reports from our own and other laboratories (12, 15, 16) show that subgroup VJI is found in more than 90% of the IgM components of cryoglobulins (Table 2). Thus the VJI subgroup does not appear to be associated necessarily with anti-IgG activity but is closely related with cryoprecipitation properties of the antibody. On this point the cryoglobulin studied in this paper does not differ from the rule. Heavy-chain subgroups are not as well defined. Though all the cryoglobulins we have studied so far belong to the VJ subgroup, others have reported IgM-IgG cryoglobulins as being VnII or VJII (15, 17). Thus, the ultrastructural appearance of the cryoglobulin reported here is not associated with peculiar chemical characteristics. This finding does not exclude the possibility of a relationship between variable-region subgroups and the cylindrical ultrastructure, as this is the first report of both amino acid analysis and ultrastructure of a cryoglobulin. It is not unreasonable to believe that the characteristics of the variable region of an immunoglobulin could give to the antigenbinding site such a quaternary structure that the antigen-antibody complex gets a defined ultrastructure. Whether or not this could be a general property of cryoglobulins is not known. The number of reports of ultrastructure of cryoprecipitates is very limited. Two reports of single cases described monoclonal IgG cryoglobulins with a tubular structure (18, 19), and one concerned a mixed IgM-IgG cryoglobulin similar to that reported here (20). In a more extensive study (21) it was found that, when a defined structure was visible on electron microscopic examination of cryoprecipitates, tubules corresponded to monoclonal IgGs and “cylindrical and annular bodies” corresponded to mixed IgM-IgG cryoglobulins with monoclonal IgM. Comparison of the diameters of these structures suggested that the IgG composed the dense wall of the tubules or cylindrical bodies, surrounded by a ring of IgM (21). Variable-region subgroups of those cryoglobulins were not determined. It would be of interest, then, to determine whether this ultrastructure is restricted to V,I-VJI molecules or is a more general property of cryoglobulins. The clinical course of patients with mixed essential cryoglobulinemia and glomerulonephritis is variable; some have acute renal failure, which is a major cause of death, while others are found to have chronic glomerulonephritis, not threatening their life (22). Thus one might suspect that the course of the disease could be related to the V-region subgroups of the cryoglobulin. Our results argue against such an assumption. The patient reported here had a reversible renal disease, while other patients that we described previously (12), who had the same V-region subgroups, died from renal failure. Ultracentrifugation studies of the cryoglobulin showed that soluble complexes were present in the patient’s serum at 37°C. Hence it appears that binding of the two components is not synonymous with cryoprecipitation. Cold induces a conformational change of preexisting soluble immune complexes, which become insoluble. A different explanation has been given by Saluk and Clem (23) for cryo-
74
CHENAIS,
FUDENBERG,
AND
WANG
precipitation of an IgGB type I cryoglobulin. In their hands, binding did not occur above 25°C and precipitation in the cold was thought to be a direct consequence of complex formation. The mechanism of cryoprecipitation may differ for type I and type II cryoglobulins. Indeed, our results are in agreement with those of others concerning type II cryoglobulins (6). Circulating immune complexes have been detected in the serum of some patients with cryoglobulinemia by ultracentrifugation at 37°C. Physicochemical studies of a type II cryoglobulin (24) suggested that primary binding was not temperature dependent and that cryoprecipitation was a property of preexisting complexes. This applies to cryoglobulins found in patients with progressive renal failure as well as less severe disease. These findings support the conclusion that thermal instability is not the primary factor in the etiology and progress of at least some of the lesions present in patients with type II cryoglobulins. No relationship exists between exposure to the cold and the onset of symptoms; vascular deposits of immunoglobulins are found in visceral vessels protected from thermal variations. Thus the pathogenicity of mixed cryoglobulins is probably related more to the formation of immune complexes than to the cryoprecipitation. REFERENCES 1. Brouet, J. C., Clauvel, J. P., Danon, F., Klein, M.. and Seligmann. M., Amer. J. Med. 57. 775. 1974. 2. Grey. H. M.. and Kohler, P. F.. Semin. Hemoiol. 10, 87. 1973. 3. Pink, R.. Wang, A. C., and Fudenberg, H. H., Annu. Rev. Med. 22, 145. 1971. 4. Pressman, D., and Grossberg, A. L., “The Structural Basis of Antibody Specificity,” W. A. Benjamin, New York, 1968. 5. Cordonnier, D., Martin, H., Groslambert, P.. Micouin. C.. Chenais, F.. and Stoebner, P.. Amer. J. Med. 59, 867. 1975. 6. Meltzer, M.. Franklin, E. C.. Elias. K., McCluskey. R. T.. and Cooper. N.. Amer. J. Med. 40, 837, 1966. 7. Gergely. J.. Medgyesi, G. A., and Stanworth, D. R., Immunachemisfry 4, 369. 1967. 8. Edman, P., and Begg, G., Eur. J. Biochem. 1, 80, 1967. 9. Wang, A. C., Gergely. J., and Fudenberg, H. H.. Biochemistry 12, 528, 1973. 10. Gros, C., and Labouesse, B., Eur. J. Biochem. 7, 463, 1969. 11. Seiler, N., and Wiechmann, H., Experientia 20, 559. 1964. 12. Wang, A. C., Wells, J. V., Fudenberg. H. H., and Gergely, J.. Immunochemistry 11, 341. 1974. 13. World Health Organization, Bull. WHO 41, 975. 1969. 14. Capra. J. D., and Kunkel, H. G.. Proc. Nat. Acad. Sci. USA 67, 87, 1970. 15. Kunkel, H. G., Winchester, R. J., Joslin, F. G., and Capra, J. D.. J. Exp. Med. 139, 128, 1974. 16. Johnston, S. L., Abraham, G. N., and Welch. E. H., Biochem. Biophys. Res. Commun. 66,842, 1975. 17. Capra, J. D., and Kehoe, J. M., Pror. Nat. Acad. Sri. USA 71. 4032, 1974. 18. Bogaars, H. A., Kalderon, A. E.. Cummings, F. J., Kaplan. S., Melnicoff. I., Park. C.. Diamond, I.. and Calabresi, P., Nature (London) 245, 117, 1973. 19. Bengtsson, U., Larsson. O., Lindstedt. G., and Svalander. C., Quart. J. Med. 44, 491, 1975. 20. Cruchaud. A., Widgren. S.. Nicod, I.. and Vassali, P., In “Protides of the Biological Fluids, 20th Colloquium.” Bruges, Belgium, 1972. (H. Peeters. Ed.), Vol. 20. pp. 39-41. Pergamon Press, New York, 1973. 21. Cordonnier, O., Viahel, P., Martin. H., Renversez, J. C.. Chenais, F.. Micouin, C.. and Stoebner. P.. Advan. Nephrol. 7, in press, 1977. 22. Morel-Maroger, L., Mery, J.Ph.. In “Proceedings, Fifth lnternationa] Congress on Nephrology.. Mexico, 1972,” (H. Villarreol. Ed.), Vol. 1. pp. 173- 178, S. Karger, Bascl, 1974. 23. Saluk. P. H.. and Clem, W.. Immunochemistry 12, 29. 1975. 24. Stone, M. J., and Metzger, H., J. Biol. Chcm. 243, 5977, 1968.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
9,67-74
(1978)
lmmunochemical Studies of an lmmunoglobulin lmmunoglobulin G Mixed Cryoglobulinl FRANCOIS CHENAIS, Department
H. HUGH FUDENBERG,
of Basic and Clinical of South Carolina,
Immunology Charleston,
AND AN-CHUAN
and Microbiology, South Carolina
Medical 29401
M-
WANG~ University
Received April 4, 1977 A mixed IgG-IgM cryoglobulin was isolated from a patient with arthritis and glomerulonephritis. Electron microscopic examination of both cryoprecipitate and glomerular deposits revealed unusual structures designated as “cylindrical and annular bodies.” The IgM component was monoclonal and had antibody activity against IgG molecules. Ultracentrifugation showed that immune complex formation was not temperature dependent. From amino acid analysis, the IgM component of the cryoglobulin was classified as V,I-VJI. In terms of chemical characteristics, this cryoglobulin was not different from most of the others previously described. These findings raise the possibility that the ultrastructural organization of the antigen-antibody complex could be related to its cryoprecipitation properties and could be a general property of cryoglobulins.
INTRODUCTION
Cryoglobulins are serum proteins, usually immunoglobulins, which reversibly precipitate in the cold. Purification and immunochemical analysis have led to a classification of this group of proteins (1, 2) into three categories: type I, consisting of a single monoclonal immunoglobulin; type II, mixed cryoglobulins in which one of the components is a monoclonal immunoglobulin; and type III, mixed polyclonal cryoglobulins. Cryoglobulins may be associated with several diseases, such as immunoproliferative disorders or autoimmune diseases, though an appreciable number of them are idiopathic. Type II and III cryoglobulins are believed to represent immune complexes, and indeed many of the clinical manifestations of cryoglobulinemia resemble those of immune complex diseases; i.e., chronic vascular purpura, arthritis, and glomerulonephritis are frequent symptoms. Type II cryoglobulins represent remarkable immune complexes where the antibody is a monoclonal immunoglobulin and is directed against a well-defined antigen; most often the antibody is a monoclonal IgM with anti-IgG activity. Such an immunoglobulin is especially suitable for chemical studies of its antigen-binding site. The four-polypeptide-chain structure of immunoglobulin molecules is now well established. The carboxy-terminal portion of each chain is prone to restricted variations, specific for a given class or subclass (with the exception of allotypic variations). The amino-terminal portion is much more variable, though several 1 This is publication No. 121 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by ACS Grant IM161, USPHS Grants AM-19201 and AI-13388, and National Science Foundation Grant BMS-75-09513. A.C.W. is the recipient of an American Cancer Society Faculty Research Award (125). 2 Kindly address correspondence to Dr. Wang. 67 0090-1229/78/0091-0067$01.00/O Copyright All rights
@ 1978 by Academic F’ress,inc. of reproduction in any form reserved
68
CHENAIS,
FUDENBERG,
AND
WANG
subgroups have been defined, based on the degree of amino acid sequence homology (3). It is established that the variable region of an immunoglobulin is responsible for its antibody specificity (4). The present paper reports chemical studies of an IgM-IgG cryoglobulin isolated from a patient with glomerulonephritis. The clinical and histological studies of this patient have been reported elsewhere (5). In brief, the patient presented with arthritis and evidence of renal disease. A kidney biopsy evidenced a proliferative endocapillary glomerulonephritis. This clinical pattern fits with the syndrome first described by Meltzer ef al. (6) in patients with mixed cryoglobulins. The most remarkable feature was the ultrastructural appearance of the cryoglobulin: Structures designated as “cylindrical and annular bodies” were found both in glomerular deposits and in the cryoprecipitate isolated from serum. Amino acid analyses and sequence studies of this peculiar cryoglobulin were undertaken to compare it with others previously studied and to determine whether the ultrastructural aspect was associated with unusual chemical characteristics. MATERIALS
AND METHODS
Isolation ofcryoglobulin. Blood from the patient studied (Cha) was drawn in a warm syringe and allowed to clot at 37°C for 2 hr. Serum was kept at 4°C for 48 hr and centrifuged, and the supernatant was removed. The precipitate was dissolved in phosphate-buffered saline, pH 8.0, at 37°C. Insoluble material was removed by centrifugation. The cryoglobulin was reprecipitated at 4°C. then washed three times with cold phosphate-buffered saline. The precipitate was finally suspended in 0.2 M sodium acetate buffer, pH 4.0. The IgM and IgG components of the cryoglobulin were separated by gel filtration on a Sephadex G-200 column equilibrated with 0.2 M sodium acetate buffer, pH 4.0, and lyophilized. The purity of the fractions was controlled by immunoelectrophoresis. Ultracentrifugation. A Beckman Model E analytical ultracentrifuge, equipped with Schlieren optics and an RITC temperature control unit, was used. Sedimentation runs were performed at 60,000 rpm at various temperatures. The rotor and cells were preincubated at the desired temperature. Photographs were taken at 8-min intervals after two-thirds of the speed was reached. Enzymatic digestion ofZgG. Fab and Fc fragments were obtained from the IgG by papain digestion, using the method of Gergely et al. (7). The IgG was dissolved in 0.075 M phosphate buffer, pH 7.0, containing 0.075 M NaCl and 0.002 M EDTA. The solution was incubated at 37°C for 2 hr with papain at an enzyme-toprotein ratio of 1: 100, in the presence of cysteine. The digestion was stopped by the addition of a 50% excess of iodoacetamide. The fragments were separated by chromatography on DEAE-cellulose. Fab fragments were eluted in 0.005 M phosphate buffer, pH 8.0. Antibody activity of the IgM against the IgG and its Fc fragments was assayed by double immunodiffusion. As the formation of precipitin lines was not significantly affected by temperature, the migration was carried out at room temperature. Separation of heavy and light chains. Reduction of the IgM component was carried out with 0.1 M dithiothreitol in 7 M guanidine hydrochloride dissolved in 0.5 M Tris-HCl buffer, pH 8.5, for 2 hr at room temperature. Alkylation was done with 0.25 M iodoacetamide for 1 hr at room temperature. The heavy and light
IcM-ICC
MIXED
CRYOGtOBULIN
69
chains were separated by gel filtration through a Sephadex G-100 column equilibrated with 6 M urea in 1.0 M acetic acid. Amino acid analyses. Amino acid compositions were determined by analysis of hydrolyzed proteins on a Beckman amino acid analyzer. Hydrolysis was performed in 6 N HCl under reduced pressure at 110°C for 22 hr. Amino acid sequences were determined by Edman degradation (8) with a Beckman Model 890 protein sequencer as previously described (9). The N-terminal residue of the heavy chain of the IgM was determined by the dansyl chloride method (10). Dansyl amino acids were identified by thin-layer chromatography on silica gel ( 11). The pyrrolidone carboxylic acid (PCA)-blocked N-terminal peptide was obtained by Nagase digestion and isolated as previously described (12). RESULTS
Gel filtration allowed purification of the two components of the cryoglobulin. The IgM component was a monoclonal protein with a K-type light chain. The IgG component, as expected, was polyclonal and reacted with antisera directed (Fig. l), the IgM against human K and A chains. On double immunodiffusion
FIG. 1. Precipitation 3. Fc fragment of IgG. gel.
of the IgM component with IgG component and its Fc fragment. 1, IgM; 2, IgG; The line above and to the right of well 1 is an artifact resulting from a fold in the
70
CHENAIS,
FUDENBERG,
AND
WANG
component formed precipitin lines with the IgG component and with its Fc fragment, confirming the anti-IgG antibody activity of the IgM, which was shown previously by agglutination studies (5). The IgM had specificity for the Fc fragment of the IgG. Ultracentrifugation of the whole serum of the patient at 10°C and at 37°C showed the presence of a component sedimenting more rapidly than the IgM and representing immune complexes. This fast component was also seen when a mixture of the IgM and the IgG was ultracentrifuged (Fig. 21. When the same mixture was centrifuged at 37°C and at lO”C, no appreciable difference was seen in the amount of complexes formed.
FIG. 2. Analytical ultracentrifugation of the isolated cryoglobulin. Top, control containing normal IgG and IgM: bottom, cryoglobulin. The photograph was taken 16 min after two-thirds speed was reached. The run was done at 37°C. A small fraction sedimented faster than the IgM. S.C. indicates the soluble complex.
IcM-ICC
MIXED
71
CRYOGLOBULIN
Amino acid analysis was performed for the IgM molecule only. The amino acid compositions of the heavy and light chains are shown in Table 1. The amino acid contents of the heavy and the light chains of the IgM are consistent with those of other cryoglobulins in that their light chains appear to have fewer serine residues (12). The V-region subgroups of the heavy chain (V,) were determined according to the composition of the N-terminal peptide (3), which is PCA-blocked. The amino acid sequence of this peptide is PCA (Val-GlxLeu), which is characteristic of the VnI subgroup. The N-terminal amino acid sequence of the light chain is shown in Table 2. Comparison with the prototpye amino acid sequences of various human V, subgroups (13) indicates that this light chain belongs to the VJI subgroup. TABLE AMINO
ACID
COMPOSITIONS IcM COMPONENTS
1
OF HEAVY OF THREE
AND LIGHT CHAINS CRYOGLOBULINS
OF THE
Cryoglobulin Cha
PO1
Nev
Amino acid0
u
K
w
A
P
K
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
6.4 2.0 4.9 7.9 9.0 10.5 9.1 7.7 6.8 5.8 3.0 8.8 1.8 2.4 6.8 3.6 3.3
5.5 1.3 4.5 8.2 6.1 6.9 12.0 8.4 8.4 6.9 3.0 8.7 0.5 2.7 8.2 4.3 3.8
3.6 1.4 3.3 9.3 9.3 7.4 10.9 7.4 7.1 6.9 2.5 10.1 1.3 3.7 8.2 3.8 3.8
5.2 1.5 3.8 8.7 7.8 8.1 10.5 7.5 8.6 8.7 2.7 8.1 3.2 7.9 5.4 2.3
3.4 1.4 3.3 10.1 8.7 8.8 10.2 8.5 7.2 7.5 2.5 9.6 1.1 3.2 7.6 2.9 4.1
4.8 1.1 4.4 8.4 7.9 8.2 12.8 6.6 7.2 7.2 2.7 7.9 2.1 9.7 4.6 4.3
o The percentage of each amino acid is expressed in terms of molar fraction, the total molar content of each chain taken as unity. Tryptophan was decomposed during the hydrolysis and thus is not included in the calculation of amino acid composition. Cha is the cryoglobulin of the present study. The results for Nev and Pol are taken from Wang et al. (12).
DISCUSSION
Despite its peculiar ultrastructural appearance, the IgM-IgG cryoglobulin reported here does not differ chemically from others previously reported. From amino acid analysis, the IgM component of the cryoglobulin is classified as V,IVJI. The amino acid sequences of other immunoglobulins with anti-IgG activity have also been studied and they do not seem to be associated with a unique light-chain subgroup. Capra and Kunkel (14) studied anti-y-globulin antibodies isolated from four patients with hyperglobulinemic purpura. One of them had a
Ser
VAV
Glu
Ile Val
Leu
Val
ri Prototype sequences sequences to that listed
Nev
Asp
chain 1 Glu Glx
VJI Cha Pol Ber
Light
Gln
5 Thr
Asp Asx
Gln Glx Glu Glx
of light chain subgroup? on the top line
Thr
Leu
N-TERMINAL
Pro \‘,I1
Ser
Val
Ala
Gly
ACID
and V,V
Ala
Pro
AMINO
TABLE
Val
Leu
2
Ala
Ser
Gly
Ser
CHAINS
by WHO
Leu
Leu
OF LIGHT
are those accepted
Ser
10 Thr
SEQUENCES
Thr
Gly
nomenclature
Gin Glx
15 Pro
Arg
Arg
( 13). Solid
Val
Glx
Glu
OF CRYOGLOBULINS”
Thr
20 Thr
lines indicate
Ile
Ala
identity
Cys
Leu
Glx
Cys
of amino
Gin
Ser
acid
25
IcM--ICC
MIXED
CRYOGLOBULIN
73
A-type light chain, one had a K-type light chain of VJI subgroup, and the two others belonged to the V,I subgroup and had identical amino acid sequences up to residue 40. In contrast, the IgM anti-IgG isolated from mixed cryoglobulin seems to have more restricted light-chain subgroups. Previous reports from our own and other laboratories (12, 15, 16) show that subgroup VJI is found in more than 90% of the IgM components of cryoglobulins (Table 2). Thus the VJI subgroup does not appear to be associated necessarily with anti-IgG activity but is closely related with cryoprecipitation properties of the antibody. On this point the cryoglobulin studied in this paper does not differ from the rule. Heavy-chain subgroups are not as well defined. Though all the cryoglobulins we have studied so far belong to the VJ subgroup, others have reported IgM-IgG cryoglobulins as being VnII or VJII (15, 17). Thus, the ultrastructural appearance of the cryoglobulin reported here is not associated with peculiar chemical characteristics. This finding does not exclude the possibility of a relationship between variable-region subgroups and the cylindrical ultrastructure, as this is the first report of both amino acid analysis and ultrastructure of a cryoglobulin. It is not unreasonable to believe that the characteristics of the variable region of an immunoglobulin could give to the antigenbinding site such a quaternary structure that the antigen-antibody complex gets a defined ultrastructure. Whether or not this could be a general property of cryoglobulins is not known. The number of reports of ultrastructure of cryoprecipitates is very limited. Two reports of single cases described monoclonal IgG cryoglobulins with a tubular structure (18, 19), and one concerned a mixed IgM-IgG cryoglobulin similar to that reported here (20). In a more extensive study (21) it was found that, when a defined structure was visible on electron microscopic examination of cryoprecipitates, tubules corresponded to monoclonal IgGs and “cylindrical and annular bodies” corresponded to mixed IgM-IgG cryoglobulins with monoclonal IgM. Comparison of the diameters of these structures suggested that the IgG composed the dense wall of the tubules or cylindrical bodies, surrounded by a ring of IgM (21). Variable-region subgroups of those cryoglobulins were not determined. It would be of interest, then, to determine whether this ultrastructure is restricted to V,I-VJI molecules or is a more general property of cryoglobulins. The clinical course of patients with mixed essential cryoglobulinemia and glomerulonephritis is variable; some have acute renal failure, which is a major cause of death, while others are found to have chronic glomerulonephritis, not threatening their life (22). Thus one might suspect that the course of the disease could be related to the V-region subgroups of the cryoglobulin. Our results argue against such an assumption. The patient reported here had a reversible renal disease, while other patients that we described previously (12), who had the same V-region subgroups, died from renal failure. Ultracentrifugation studies of the cryoglobulin showed that soluble complexes were present in the patient’s serum at 37°C. Hence it appears that binding of the two components is not synonymous with cryoprecipitation. Cold induces a conformational change of preexisting soluble immune complexes, which become insoluble. A different explanation has been given by Saluk and Clem (23) for cryo-
74
CHENAIS,
FUDENBERG,
AND
WANG
precipitation of an IgGB type I cryoglobulin. In their hands, binding did not occur above 25°C and precipitation in the cold was thought to be a direct consequence of complex formation. The mechanism of cryoprecipitation may differ for type I and type II cryoglobulins. Indeed, our results are in agreement with those of others concerning type II cryoglobulins (6). Circulating immune complexes have been detected in the serum of some patients with cryoglobulinemia by ultracentrifugation at 37°C. Physicochemical studies of a type II cryoglobulin (24) suggested that primary binding was not temperature dependent and that cryoprecipitation was a property of preexisting complexes. This applies to cryoglobulins found in patients with progressive renal failure as well as less severe disease. These findings support the conclusion that thermal instability is not the primary factor in the etiology and progress of at least some of the lesions present in patients with type II cryoglobulins. No relationship exists between exposure to the cold and the onset of symptoms; vascular deposits of immunoglobulins are found in visceral vessels protected from thermal variations. Thus the pathogenicity of mixed cryoglobulins is probably related more to the formation of immune complexes than to the cryoprecipitation. REFERENCES 1. Brouet, J. C., Clauvel, J. P., Danon, F., Klein, M.. and Seligmann. M., Amer. J. Med. 57. 775. 1974. 2. Grey. H. M.. and Kohler, P. F.. Semin. Hemoiol. 10, 87. 1973. 3. Pink, R.. Wang, A. C., and Fudenberg, H. H., Annu. Rev. Med. 22, 145. 1971. 4. Pressman, D., and Grossberg, A. L., “The Structural Basis of Antibody Specificity,” W. A. Benjamin, New York, 1968. 5. Cordonnier, D., Martin, H., Groslambert, P.. Micouin. C.. Chenais, F.. and Stoebner, P.. Amer. J. Med. 59, 867. 1975. 6. Meltzer, M.. Franklin, E. C.. Elias. K., McCluskey. R. T.. and Cooper. N.. Amer. J. Med. 40, 837, 1966. 7. Gergely. J.. Medgyesi, G. A., and Stanworth, D. R., Immunachemisfry 4, 369. 1967. 8. Edman, P., and Begg, G., Eur. J. Biochem. 1, 80, 1967. 9. Wang, A. C., Gergely. J., and Fudenberg, H. H.. Biochemistry 12, 528, 1973. 10. Gros, C., and Labouesse, B., Eur. J. Biochem. 7, 463, 1969. 11. Seiler, N., and Wiechmann, H., Experientia 20, 559. 1964. 12. Wang, A. C., Wells, J. V., Fudenberg. H. H., and Gergely, J.. Immunochemistry 11, 341. 1974. 13. World Health Organization, Bull. WHO 41, 975. 1969. 14. Capra. J. D., and Kunkel, H. G.. Proc. Nat. Acad. Sci. USA 67, 87, 1970. 15. Kunkel, H. G., Winchester, R. J., Joslin, F. G., and Capra, J. D.. J. Exp. Med. 139, 128, 1974. 16. Johnston, S. L., Abraham, G. N., and Welch. E. H., Biochem. Biophys. Res. Commun. 66,842, 1975. 17. Capra, J. D., and Kehoe, J. M., Pror. Nat. Acad. Sri. USA 71. 4032, 1974. 18. Bogaars, H. A., Kalderon, A. E.. Cummings, F. J., Kaplan. S., Melnicoff. I., Park. C.. Diamond, I.. and Calabresi, P., Nature (London) 245, 117, 1973. 19. Bengtsson, U., Larsson. O., Lindstedt. G., and Svalander. C., Quart. J. Med. 44, 491, 1975. 20. Cruchaud. A., Widgren. S.. Nicod, I.. and Vassali, P., In “Protides of the Biological Fluids, 20th Colloquium.” Bruges, Belgium, 1972. (H. Peeters. Ed.), Vol. 20. pp. 39-41. Pergamon Press, New York, 1973. 21. Cordonnier, O., Viahel, P., Martin. H., Renversez, J. C.. Chenais, F.. Micouin, C.. and Stoebner. P.. Advan. Nephrol. 7, in press, 1977. 22. Morel-Maroger, L., Mery, J.Ph.. In “Proceedings, Fifth lnternationa] Congress on Nephrology.. Mexico, 1972,” (H. Villarreol. Ed.), Vol. 1. pp. 173- 178, S. Karger, Bascl, 1974. 23. Saluk. P. H.. and Clem, W.. Immunochemistry 12, 29. 1975. 24. Stone, M. J., and Metzger, H., J. Biol. Chcm. 243, 5977, 1968.