- Email: [email protected]
Molecular basis for the temperature-dependent insolubility of cryoglobulins—IX
Molecular Immuno/ogy. Vol. 17. pp. 337-344. 0 Pergamon Press Ltd. 1980. Printed in Great Britain.
O161-5890/80/03OI-O337
t02.00/0
MOLECULAR BASIS FOR THE TEMPERATUREDEPENDENT INSOLUBILITY OF CRYOGLOBULINS-IX. PHYSICOCHEMICAL CHARACTERIZATION OF AN IgG,, K MONOCLONAL CRYOIMMUNOGLOBULIN EXHIBITING MARGINAL LOW TEMPERATURE-DEPENDENT INSOLUBILITY* G. W. LITMAN, B. GERBER-JENSON. R. LITMAN, C. R. MIDDAUGHP and C. SCHEFFEL
Sloan-Kettering
Institute for Cancer Research, Rye, NY 10580 and Department of Biochemistry, University of Wyoming. Laramie. WY 82070. U.S.A. (Received
I7 April 1979)
Abstract-A monoclonal IgG,, K immunoglobulin (Muk) exhibiting temperature-dependent insolubility is described. Limited N terminal amino acid seqp,encing indicates that the light chain belongs to the V,I subgroup and that the heavy chain is blocked. By comparison to other monoclonal cryoglobulins, protein Muk requires significant temperature reduction and/or elevated concentration to manifest cryoprecipitation. The solubility of the protein at O’C was calculated to be 2.05 mg/ml. In terms of subunit mass and sensitivity to proteolysis, Muk resembles noncryoglobulin IgG, reference proteins. Unlike earlier observations with certain other monoclonal cryoimmunoglobulins, CDS, fluorescence and analytical gel chromatography on agarose failed tp distinguish Muk from the IgG, references. The hydrophobic character of the Fab of Muk. as determined by chromatography on a phenyl substituted agarose, is within the range of noncryoglobulin reference Fabs. Amino acid composition of the protein indicates increased Asx and decreased Phe and Val. Hexosamine, fucose and, most significantly. sialic acid (4 20”, of normal content) are present in reduced amounts. Enzymatic removal of sialic acid from noncryoglobulin IgGs failed to induce detectable low temperature-dependent insolubility. Estimation ofcryoprecipitation in the presence of a variety of solutes suggest the involvement of electrostatic interactions in the cryoprecipitation of this protein. Cryoprecipitation of Muk could not be localized to the principal enzymatic fragments and does not appear to involve a typical antigen-antibody interaction. The studies with Muk further suggest the lack of a common structural basis for cryoglobulin behavior and implicate the general solubility properties of the Droteins rather than swcific molecular interactions as the critical factors in determining cold-dependent
INTRODUCTION
Immunoglobulins which become insoluble as the solution temperature is reduced from 37 to 0°C are commonly referred to as cryoimmunoglobulins (Lerner er al., 1947). While the occurrence and clinical significance of these proteins has been well documented (Grey & Kohler, 1973; Brouet er al., 1974), the molecular basis for their cold precipitation characteristics remains enigmatic. Our laboratory has l The studies were supported in part by NIH CA 08748 and NIH AI 13528 to G.W.L., to whom correspondence should be addressed. t Department of Biochemistry, University of Wyoming, Laramie, WY 82070, U.S.A. $ Abbreviations used: CD, circular dichroism; HPLC, high performance liquid chromatography; Mr, relative molecular phenylthiohydantoin; Tris-HCI, tris weight; PTH, (hydroxymethyl)-amino methane-HCI; V subgroup designates are from Kabat et al. (1976); i.,,, excitation wavelength.
approached the problem of defining a molecular basis for cryoglobulin behavior by:( 1) systematically investigating the physicochemical properties of the IgM monoclonal cryoimmunoglobulin, McE (Middaugh & Litman, 1977a,b; Middaugh et al., 1977; Middaugh & Litman, 19786);(2) comparing structural and functional properties of different monoclonal IgM and IgG cryoglobulins (Middaugh & Litman, 1978; Middaugh et al., 1978a). Both approaches also have utilized direct comparison of the cryoglobulins to a group of monoclonal immunoglobulin reference proteins lacking cryoglobulin properties. The results of these studies have suggested that: (.l) several different types of atypical conformation (presumably tertiary structure) distinguish certain cryoimmunoglobulins from noncryoglobulin reference proteins; (2) different functional mechanisms appear to distinguish groups of cryoglobulins; (3) the cryoglobulin phenomena may be related 337
338
G. W. LITMAN
to general aspects of solubility of the individual proteins. Recently we have encountered an IgG,, K monoclonal cryoimmunoglobulin exhibiting a marked reduction in the temperature and concentration-dependence of cryoprecipitation. It is the purpose of this communication to describe the results of a comprehensive physicochemical characterization of this variant molecule.
MATERIALS
AND METHODS
Protein Muk was isolated from the serum of a male patient aged 42 in the end stage of multiple myeloma unresponsive to a wide range of chemotherapeutic agents. Serum electrophoresis indicated the presence of a monoclonal yglobulin spike exceeding 10 g%. The following procedures were carried out essentially as described in earlier portions of this series: CD and fluorescence (Middaugh et al., 1977); isolation of noncryoglobulin IgG reference proteins, production of Fab’,, Fab and Fc fragments and N-terminal amino acid sequencing (Middaugh et al., 1978~); isolation of the cryoglobulin, immunochemical characterization, analytical gel filtration, complete reduction and alkylation, determination of amino acid and carbohydrate composition, and removal of sialic acid by treatment with
C 0 .-
et al.
neuraminidase (Middaugh et al., 1978b). In the N-terminal amino acid sequencing studies. PTH amino acids were identified by both HPLC (Zimmerman et al., 1977) and by back hydrolysis with HI to the free amino acid or degradation product and subsequent identification by amino acid composition analysis. Direct hemagglutination analyses were carried out in microtiter trays (Cooke Laboratory Products, Alexandria, VA) utilizing human type 0, RhC cells coated by the CrCl, method with immunoglobulin or immunoglobulin fragments (Wang et al., 1978). Analytical hydrophobic chromatography was carried out employing Phenyl Sepharose CL4BE (Pharmacia). In a typical experiment the cross-linked, phenyl-substituted agarose was first equilibrated extensively in 0.05 A4Tris-HCl, 4 M NaCl, pH 8.0, and then poured into a 0.9 x 15cm chromatography column to a bed height of 12 cm. An Fab fragment previously dialyzed against the 4 M NaCl containing buffer was applied to the column and the eluate monitoied for A,,,. In no instance was Azso absorbing material recovered under the initial elution conditions. and a 100 x 100 ml linear gradient diminishing from 0.05 M Tris-HCI, 4 M NaCl, pH 8.0, to 0.05 MTris-HCl, pH 8.0, was initated. After the gradient was exhausted, the column was developed in the limit buffer. The A,,, and conductivity of each 2.3 ml fraction were monitored separately.
80
‘5 .0.
cl aJ
z 0 ? ”
2 01
u
G
a
2 Protein Fig.
I,
4
6
concentration
8
10
(mg/ml
1
Effect of protein concentration on the cryoprecipitation of Muk (0) and the reference cryoglobulin McE (m) at 0 C. The assays were carried out in 0.15 IM Tris-HCI, pH 8.0.
Physjc~hemical
Characterization
Temperature
339
of an IgG Cryoglobulin
(“Cl
Fig. 2. Effect of temperature on the cryopr~jpitatjon ofprotein Muk @) and McE (m f. Assays werecarried out on the proteins at 3.7 m&ml, 0.15 M Tris-HCl, pH 8.0. RESULTS
Immunochemical analyses indicated that the monoclonal protein isolated from patient Muk was an IgG, K monoclonal cryoimmunoglobulin (we are indebted to Dr. A. C. Wang for carrying out the subclass determination). N-terminal amino acid sequencing of the light chain yielded a single residue at each of the first 12 positions determined and the sequence Asp-Ile-Gln-MetThr-Gln-Ser-Pro-Ser-Ser-Leu-Ser permitted tentative assignment of the light chain to the V,I subgroup (Kabat et al., 1976). The heavy chain
was found to be blocked. Analytical gel filtration of the intact protein as well as its Fab’, and Fab components under neutral aqueous conditions and its heavy and light chains in the presence of 6 M guanidine-HCl failed to distinguish intact Muk or its constituent chains and fragments ’ 251-labeled noncryoglobulin from IgG references. The concentration dependence of cryoprecipitation at 0°C is markedly reduced when compared to other cryoimmunoglobulins (Middaugh et al., 197%~)and is only 81% at a protein concentration of 11 mg/ml (Fig. 1). Above thisconcentration, the protein isolated by
Table 1. Solute variation assay” Addition No addition
Cryoprecipitation 49
Neutral salts 0.2 M NaCl 1.0 M NaCl 2.0 M NaCI 0.2 M NaSCN 1.0 M NaSCN 0.2 M M&I, 1.0 M MgCl,
16 17 16 17 1.5 13 10
Formamide 0.5 M formamide 1.0 M formamide
15 14
(%)
Addition
Cryoprecipitation
Ureas 0.2 M urea 1.0 M urea 1 .O M ethyl urea 1.0 M propyl urea
10 11 6 11
Alcohols 0.2 M 1.O M 0.2 M 1.0 M
methanol methanol propanol propanol
58 84 60 89
Sugars 0.5 M @lactose 1.O A4 galactose 1.O N ribose
6 IO 6
(%)
“Assays were carried out as described (Middaugh & Litman, 1978; Middaugh et al., 19780) in 0.15 M Tris-HCI. pH 8.0. Assay concentration was 4.3 mg/ml and the uncertainty in the values is 2-3:/,.
340
G. W. LITMAN
cryoprecipitation appeared to be only partially soluble at 37°C in the standard 0.15 M Tris-HCI, pH 8.0 buffer, complicating analysis of reversibility and thus confusing estimation of cryoprecipitation. The extrapolated pH optimum of the Muk protein at 7.2 mgjml was found to be 8.4 with cryoprecipitation being abolished below pH 4 and above pH 11. The temperature dependence of cryoprecipitation at 3.7 mg/ml was found to be markedly depressed in comparison to other cryoglobulins (Fig. 2, and Middaugh ef al., 1978~). Table 1 illustrates the behavior of the Muk protein in a solute variation assay which has proven useful in evaluating forces involved in cryoprecipitation. The cryprecipitation of Muk is effectively inhibited by the neutral salts. ureas, formamides and sugars. Cryoprecipitation is effectively enhanced by high concentrations (1 .O M) of 1 and 3 carbon primary alcohols. In terms of overall similarity or dissimilarity to other proteins. Muk aopears to most closely resemble two other IgG, K cryoglobulins Mow and Ger and the IgM cryoglobulins Mel and Pel. It differs most from the IgM cryoglobulins McE and Gre (Middaugh et al., 197%~). There were no apparent gross differences in acid composition (including the amino tryptophan) of heavy and light chains derived from protein Muk when compared to the heavy and light chains of the noncryoglobulin reference proteins McC and Penn analyzed in parallel or to the composition of protein Eu (Edelman et al., 1969). However. the value for heavy chain Asx was slightly increased (43.7 vs 35.7 residues/mol) and the values for Phe and Val were decreased (9.0 vs 12.6 and 33.5 vs 39.4 residues,/mol, respectively) relative to the reference proteins examined. If the data base is expanded to include other equivalent IgG, heavy chains, these differences become less significant (not illustrated). The carbohydrate composition of protein Muk expressed as weight percent was found to be: neutral hexose = 1.23, hexosamine = 0.62, fucose = 0.13 and sialic acid = 0.05. The value for hexose is within accepted limits for noncryoglobulin IgG, while the values obtained for fucose. hexosamine and sialic acid are reduced ( _ 50”” reduction for hexosamine and fucose: _ SO”,, for sialic acid). Values for sialic acid of noncryoglobulin references determined in parallel consistently fell within normal limits. Prolonged digestion with neuraminidase was utilized to remove sialic acid from a group of four
rr ul
I
01
1
I Elui~on
volume
1,.
I
(ml)
Fig. 3. Analytical hydrophobic chromatography of the Fab (papain) fragments of Muk and the reference cryoglobulin Ger (H) and noncryoglobulin IgGs Bau (A), McC (0) and Penn (0). Elution conditions were as described in the text. The peak in Azao for the references is indicated by the symbols and the continuous line represents the elution profile of Muk.
noncryoglobulin IgG,, ti reference proteins. During the course of digestion at 37’C. a slight white precipitate accounting for -5”, of the total protein was noted to form. When the proteins at c 10 mg/ml were equilibrated in 0.15 M Tris-HCl, pH 8.0, and incubated at 0°C for 48 hr, no cryoprecipitation was evident, and it was concluded that removal of sialic acid failed to induce cryoglobulin behavior. Analytical hydrophobic chromatography of the Fab fragment of Muk IgG on a phenylsubstituted agarose matrix revealed the presence of a single homogeneous chromatographic boundary eluting at a conductance between _ 140 and 75 mS’ with the peak elution centered at 105 mS’ (Fig. 3). Included for reference in the figure are the conductance values for the A,,, elution maxima of Fab fragments isolated from the noncryoglobulin reference proteins Bau. McC and Penn. as well as the value for the Ger. In monoclonal IgG,, K cryoglobulin terms of hydrophobic character, as assessed by the nature of binding to and elution from the phenyl-substituted matrix, it may be concluded that protein Muk is within the range observed for non-cryoglobulin references, while a second cryoglobulin appears to be unique. CD studies of protein Muk at 37 C indicated a prominent negative ellipticity band centered at degrees cm’ 217 nm, 0 = -2920 -. which is within d mol the range for noncryoglobulin monoclonal IgGs. The spectrum was distinguished by a slightly
Physicochemical
Characrerization
of an IgG Crgoglobulin
341
protein concentration, it was noted that the amount of protein present in the supernatant plateaued at 2.05 mglml. thus defining the solubility of Muk in 0.15 MTris-HCI. pH 8.0, at reduced experience, the o-c. In our cryoprecipitability of this protein is unique and appears to be distinct from other proteins described in the literature, although such comparisons are admittedly difficult. In comparative solute variation assays, Muk closely resembles protein Mow, another IgG, cryoglobulin. The effect of low concentrations of NaCl on the cryoprecipitation of Muk suggests that electrostatic interactions are involved in the cryoprecipitation of this protein, although the possibility exists that significant contributions from van der Waals and additional unspecified weak noncovalent interactions are involved. As has been observed with the majority of other cryoimmunoglobulins examined to date, localization of cryoprecipitation to the major enzymatically derived fragments of Muk has been unsuccessful. This finding, coupled with both the failure of high molar excesses of these fragments and noncryoglobulin IgG to inhibit cryoprecipitation of the parent protein, and the inability to detect cold-dependent intermolecular associations using a sensitive direct hemagglutination technique makes it unlikely that antigen-antibody complex formation is involved in cryoprecipitation. Structural characterization of the Muk protein failed to distinguish Muk from a group of noncryoglobulin reference IgG, proteins with respect to (I) subunit mass and arrangement (sensitivity to proteolysis) and (2) conformation (analytical gel filtration of the intact protein or Fab, intrinsic fluorescence and far ultraviolet CD). In this sense, Muk differs from the two DlSCUSSION other IgG, cryoglobulins (Ger and Mow) which Cryoglobulins thus far included in our series both exhibited a marked reduction in the have been rather similar in terms of fluorescence emission maxima when excited at concentration and temperature dependence of 275 nm (or 295 nm) and protein Mow which cryoprecipitation (Middaugh et al., 1978a). exhibited a decreased negative ellipticity at 217 Detectable insolubility began to occur below 2.5 nm (Middaugh er al., 1978a). Other mg/ml under established assay procedures and in distinguishing features of the protein are the every case at least 50% relative cryoprecipitation somewhat atypical amino acid composition and. was encountered above 1.0 mg/ml. The Muk more important, the greatly reduced content of protein, however, did not undergo precipitation sialic acid. The latter point previously has been above 6°C at a concentration of 3.7 mgjml .111d suggested as a basis for the cold dependent monoclonal 50”” cryoprecipitation did not occur until a insolubility other of IgG protein concentration of 4 mg/ml was reached :II cryoglobulins (Hansson & Lindstrom, 1973; O-C. By quantitating the amount of protein (AS; a Zinneman & Caperton. 1977). Whether the function of total protein) in the supcrnatant and reduced content of sialic acid, fucose and hexosamine in Muk affects the intrinsic precipitate at O’C as a function of increasing
more pronounced secondary ellipticity minimum centered near 225-230 nm than typically observed with IgGs. When the CD spectrum of Muk was determined at OC at a sufficiently low concentration of protein (0.1 mg/ml) to prevent cryoprecipitation, no change was noted. The intrinsic fluorescence emission spectrum (&, = 275: 295 nm) of Muk at 23C (protein concentration = 0.1 mgiml) was indistinguishable from that of the reference noncryoglobulin IgGs. Typical digestion kinetics were noted when the intact protein was incubated at 37°C with papain and the progress of digestion was monitored by immunoelectrophoresis employing antiserum to Kand ;‘chains. When the isolated Fab’,. Fab and Fc fragments of Muk at concentrations of 5-50 mg/ml in 0.15 M Tris-HCl, pH 8.0, were placed at 0°C for 48 hr. cryoprecipitation was not observed. Incubation of varying proportions (molar excesses of I-30 times that of the intact protein) of these or noncryoglobulin Fab’z, Fab and Fc fragments with the intact Muk protein similarly failed to quantitatively affect cryoprecipitation. In a further attempt to detect intermolecular low temperature-mediated associations with the Muk protein, a direct hemagglutination assay was employed. Hemagglutination at O’C was not detected when erythrccytescoated with Muk IgG and Fab were incubated with varying dilutions of homologous I&. Fab and Fc or heterologous IgG, although the cells were apparently coated with the intact protein and Fab fragments, as evidenced by agglutination at a high dilution (214) of antiserum directed at the K chain determinant of the Fab.
_
_
;‘,; V,,B
Muk
_
_
+
gel filtration
Agarose
Elevated Asx, reduced Phe and Val? see text
-
_
Amino acid composition
Sialic acid-reduced
_
-
+
+
Inhibition in 0.2 M NaCl
+
+
-
0.5nM formamide
Inhibition
t
+
-
Inhibition in 0.5 M galactose
Effect of solute on cryoprecipitation”
cryoimmunoglobulins”
Carbohydrate composition
K monoclonal
” Data in Table 2 were obtained from expcrimentsdescribed in this paper or in Middaugh et nl. (19780). The data presented has been chosen to reflect differences rather than simifaritrcs bctwccn the tndividual proteins. h ( + ) Indicates a positive tinding (signficantly different from normal) and (--) indicates a negative finding (indistinguishable from normal). ’ V,B indicates the presence of a blocked amino terminus. “( +) indicates that near complete inhibition takes place: (- ) Indicates that inhibition is only partial or does not take place
+
+
+
_
i’,; v,B’
Reduced intrinsic fluorescence (i._ = 275 nm)
;‘,; V,,lH
9’ (217 nm)
Reduced
Mow
L chain class & subgroup
of three IgG,,
properties”
properties
Atypical
of physicochemical
Cer
Protein
H chain class & subgroup
Table 2. Comparison
R
w
Physicochemical Characterization
solubility of the protein and influences cryoprecipitation in this way is unclear. The removal of sialic acid from noncryoglobulin reference proteins fails to induce cryoglobulin behavior. These studies have introduced results with an additional probe of macromolecular conformation, hydrophobic chromatography. The principle of this technique is based on the increased hydrophobic interaction which occurs between a protein and an immobilized ligand at high salt concentration (Hofstee, 1976). High ionic strength tends to simultaneously stabilize the hydrophobic interaction and minimize electrostatic interactions. In these studies, a phenyl substituted agarose, with an estimated hydrophobicity between 4 and 5 straight chain carbons, was employed (Hjerten, 1976). The elution characteristics of Muk suggest it to be within the range of the three reference proteins examined. For the sake of comparison, the Fab component of the cryoglobulin Ger was also included. Its unique elution characteristics suggest it to be of a less hydrophobic character than the other four proteins examined. The Fab component of Ger exhibits an anomalously high Mr in analytical gel filtration on agarose, and we have suggested that this effect may be due to a lack of interaction of certain cryoglobulin proteins with agarose, resulting in their elution ahead of the noncryoglobulin references (Middaugh et al., 1978~). If this interaction with agarose depends on the hydrophobic nature of the proteins, then it could be predicted that the Ger protein with a reduced hydrophobic character should emerge first, while the Muk protein which possesses a more typical net hydrophobicity should emerge at a normal Mr position. This was observed; however, a number of other possibilities exist for explaining the gel filtration abnormality. One of the strategies we have employed in an effort to discern a molecular basis forcryoglobulin behavior has been to compare the structure and functional behavior of as large a group of cryoglobulins as reasonable. When the physicochemical properties of the Muk protein and two additional cryoglobulins of the same heavy and light chain isotype are compared, no common pattern emerges which relates the individual proteins (Table 2). Findings such as these make it increasingly more likely that a variety of subtle alterations in macromolecular structure account for anomalous may temperature insolubility characteristic of
of an IgG Cryoglobulin
343
monoclonal cryoimmunoglobulins. We have suggested that cryoglobulin behavior may reflect a heterogeneity in solubility of immunoglobulins; cryoglobulins the may represent intrinsically less soluble members of the population (Middaugh et al., 1978~). If such were the case, then a comprehensive survey of cryoglobulins should detect proteins with only marginal cryoglobulin behavior. Protein Muk may represent one such variant and in this way lend support to this hypothesis.
REFERENCES
Brouet J., Clauvel J., Danon F., Klein M St Seligman M. (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am. J. Med. S7,77>788. Edelman G. M., Cunningham B. A., Gall W. E., Gottlieb P. D., Rutishauser U & Waxdal M. J. (1969) The covalent structure of an entire $i immunoglobulin molecule. Proc. natn. Acad. Sri. U.S.A. 63, 78-85. Grey H. M. & Kohler P. F. (1973) Cryoimmunoglobulins. Semin. hematol. 10, 87-112. Hansson U. & Lindstrom F. D. (1973) Some factors affecting precipitation and complex formation of an IgG cryoglobulin. C/in. exn. Immun. 14. 427-435. Hjer& S. (1976) Zone klectrophoresis, isoelectric focusing and displacement electrophoresis (isotachophoresis) in carrier-free solution. In Methods of Protein Separation (Edited by Catsimpoolas N.), Vol. 2, pp. 219-232. Pknum Press, New York. Hofstee B. H. J. (1976) Hydrophobic adsorption chromatography of proteins. In Methoa!s of Protein Separation (Edited by Catsimpoolas N.), Vol. 2, pp. 245-278. Plenum Press, New York. Kabat E. A., Wu T. T. & Bilofsky H. (1976) Chemical/Biological Information-Handling Program. Division of Research Resources, National Institutes of Health, Bethesda, Maryland. Lemer A. B., Barnum C. P. &Watson C. J. (1947) Studies of cryoglobulins; spontaneous precipitation of protein from serum at 5°C in various disease states. Am. J. med. Sci. 214, 416-421. Middaugh C. R. & Litman G. W. (19770) Effect of D,O on the temperature-dependent solubility of cryoimmunoglobulin and noncryoimmunoglobulin IgM. F’BS Lptt. 79, 200-202. Middaugh C. R. & Litman G. W. (19776) Effect of solutes on the cold-induced insolubility of monoclonal cryoimmunoglobulins. J. biol. Chem. 252, 8002-8006. Middaugh C. R. & Litman G. W.( 1978) Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobulins. VI. Quenching by acrylamide of the intrinsic tryptophan fluorescence of cryoglobulin and noncryoglobulin IgM proteins. Biochim. biophys. Acta 535, 33-43. Middaugh C. R., Thomas G. J., Prescott B., Aberlin M. E. & Litman G. W. (1977) Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobuhns. II. Spectroscopic studies of the IgM moncclonal cryoglobulin McE Biochemistry 16, 2986-2994. Middaugh C. R., Gerber-Jenson B., Hurvitz A., Palusxek A., Scheffel C. & Litman G. W. (1978a) Physicochemical characterization of six monoclonal cryoimmunoglobulins. Possible basis for cold-dependent insolubility. Proc. nkn. Acad. Sci. U.S.A. 75, 3440-3444. Middaugh C. R., Kehoe J. M., Prystowsky M. B., GerberJenson B., Jenson J. C. & Litman G. W. (39786) Molecular
344
G. W. LITMAN et al.
basis for the temperature dependent insolubility of cryoglobulins. IV. Structural studies of the IgM monoclonal cryoglobulin McE. immunochemistry 15, 171-187. Wang A. C., Mathur S., Pandey J., Siegal F. P., Middaugh C. R.. and Litman G. W. (1978) Hv(l), a variable-region genetic marker of human immunoglobulin heavy chains. Science 200, 327-329.
Zimmerman C. L., Appella E. & Pisano J. (1977) Rapid analysis of amino acid phenythiohydantoins by highperformance liquid chromatography. Analyf. Eiochem. 77, 569-573. Zinneman H. H. & Caperton E. (1977) Cryoglobulinemia in a patient with Sjogrens syndrome and factors of cryoprecipitation. J. lab. clin. Med. 89, 483-487.
O161-5890/80/03OI-O337
t02.00/0
MOLECULAR BASIS FOR THE TEMPERATUREDEPENDENT INSOLUBILITY OF CRYOGLOBULINS-IX. PHYSICOCHEMICAL CHARACTERIZATION OF AN IgG,, K MONOCLONAL CRYOIMMUNOGLOBULIN EXHIBITING MARGINAL LOW TEMPERATURE-DEPENDENT INSOLUBILITY* G. W. LITMAN, B. GERBER-JENSON. R. LITMAN, C. R. MIDDAUGHP and C. SCHEFFEL
Sloan-Kettering
Institute for Cancer Research, Rye, NY 10580 and Department of Biochemistry, University of Wyoming. Laramie. WY 82070. U.S.A. (Received
I7 April 1979)
Abstract-A monoclonal IgG,, K immunoglobulin (Muk) exhibiting temperature-dependent insolubility is described. Limited N terminal amino acid seqp,encing indicates that the light chain belongs to the V,I subgroup and that the heavy chain is blocked. By comparison to other monoclonal cryoglobulins, protein Muk requires significant temperature reduction and/or elevated concentration to manifest cryoprecipitation. The solubility of the protein at O’C was calculated to be 2.05 mg/ml. In terms of subunit mass and sensitivity to proteolysis, Muk resembles noncryoglobulin IgG, reference proteins. Unlike earlier observations with certain other monoclonal cryoimmunoglobulins, CDS, fluorescence and analytical gel chromatography on agarose failed tp distinguish Muk from the IgG, references. The hydrophobic character of the Fab of Muk. as determined by chromatography on a phenyl substituted agarose, is within the range of noncryoglobulin reference Fabs. Amino acid composition of the protein indicates increased Asx and decreased Phe and Val. Hexosamine, fucose and, most significantly. sialic acid (4 20”, of normal content) are present in reduced amounts. Enzymatic removal of sialic acid from noncryoglobulin IgGs failed to induce detectable low temperature-dependent insolubility. Estimation ofcryoprecipitation in the presence of a variety of solutes suggest the involvement of electrostatic interactions in the cryoprecipitation of this protein. Cryoprecipitation of Muk could not be localized to the principal enzymatic fragments and does not appear to involve a typical antigen-antibody interaction. The studies with Muk further suggest the lack of a common structural basis for cryoglobulin behavior and implicate the general solubility properties of the Droteins rather than swcific molecular interactions as the critical factors in determining cold-dependent
INTRODUCTION
Immunoglobulins which become insoluble as the solution temperature is reduced from 37 to 0°C are commonly referred to as cryoimmunoglobulins (Lerner er al., 1947). While the occurrence and clinical significance of these proteins has been well documented (Grey & Kohler, 1973; Brouet er al., 1974), the molecular basis for their cold precipitation characteristics remains enigmatic. Our laboratory has l The studies were supported in part by NIH CA 08748 and NIH AI 13528 to G.W.L., to whom correspondence should be addressed. t Department of Biochemistry, University of Wyoming, Laramie, WY 82070, U.S.A. $ Abbreviations used: CD, circular dichroism; HPLC, high performance liquid chromatography; Mr, relative molecular phenylthiohydantoin; Tris-HCI, tris weight; PTH, (hydroxymethyl)-amino methane-HCI; V subgroup designates are from Kabat et al. (1976); i.,,, excitation wavelength.
approached the problem of defining a molecular basis for cryoglobulin behavior by:( 1) systematically investigating the physicochemical properties of the IgM monoclonal cryoimmunoglobulin, McE (Middaugh & Litman, 1977a,b; Middaugh et al., 1977; Middaugh & Litman, 19786);(2) comparing structural and functional properties of different monoclonal IgM and IgG cryoglobulins (Middaugh & Litman, 1978; Middaugh et al., 1978a). Both approaches also have utilized direct comparison of the cryoglobulins to a group of monoclonal immunoglobulin reference proteins lacking cryoglobulin properties. The results of these studies have suggested that: (.l) several different types of atypical conformation (presumably tertiary structure) distinguish certain cryoimmunoglobulins from noncryoglobulin reference proteins; (2) different functional mechanisms appear to distinguish groups of cryoglobulins; (3) the cryoglobulin phenomena may be related 337
338
G. W. LITMAN
to general aspects of solubility of the individual proteins. Recently we have encountered an IgG,, K monoclonal cryoimmunoglobulin exhibiting a marked reduction in the temperature and concentration-dependence of cryoprecipitation. It is the purpose of this communication to describe the results of a comprehensive physicochemical characterization of this variant molecule.
MATERIALS
AND METHODS
Protein Muk was isolated from the serum of a male patient aged 42 in the end stage of multiple myeloma unresponsive to a wide range of chemotherapeutic agents. Serum electrophoresis indicated the presence of a monoclonal yglobulin spike exceeding 10 g%. The following procedures were carried out essentially as described in earlier portions of this series: CD and fluorescence (Middaugh et al., 1977); isolation of noncryoglobulin IgG reference proteins, production of Fab’,, Fab and Fc fragments and N-terminal amino acid sequencing (Middaugh et al., 1978~); isolation of the cryoglobulin, immunochemical characterization, analytical gel filtration, complete reduction and alkylation, determination of amino acid and carbohydrate composition, and removal of sialic acid by treatment with
C 0 .-
et al.
neuraminidase (Middaugh et al., 1978b). In the N-terminal amino acid sequencing studies. PTH amino acids were identified by both HPLC (Zimmerman et al., 1977) and by back hydrolysis with HI to the free amino acid or degradation product and subsequent identification by amino acid composition analysis. Direct hemagglutination analyses were carried out in microtiter trays (Cooke Laboratory Products, Alexandria, VA) utilizing human type 0, RhC cells coated by the CrCl, method with immunoglobulin or immunoglobulin fragments (Wang et al., 1978). Analytical hydrophobic chromatography was carried out employing Phenyl Sepharose CL4BE (Pharmacia). In a typical experiment the cross-linked, phenyl-substituted agarose was first equilibrated extensively in 0.05 A4Tris-HCl, 4 M NaCl, pH 8.0, and then poured into a 0.9 x 15cm chromatography column to a bed height of 12 cm. An Fab fragment previously dialyzed against the 4 M NaCl containing buffer was applied to the column and the eluate monitoied for A,,,. In no instance was Azso absorbing material recovered under the initial elution conditions. and a 100 x 100 ml linear gradient diminishing from 0.05 M Tris-HCI, 4 M NaCl, pH 8.0, to 0.05 MTris-HCl, pH 8.0, was initated. After the gradient was exhausted, the column was developed in the limit buffer. The A,,, and conductivity of each 2.3 ml fraction were monitored separately.
80
‘5 .0.
cl aJ
z 0 ? ”
2 01
u
G
a
2 Protein Fig.
I,
4
6
concentration
8
10
(mg/ml
1
Effect of protein concentration on the cryoprecipitation of Muk (0) and the reference cryoglobulin McE (m) at 0 C. The assays were carried out in 0.15 IM Tris-HCI, pH 8.0.
Physjc~hemical
Characterization
Temperature
339
of an IgG Cryoglobulin
(“Cl
Fig. 2. Effect of temperature on the cryopr~jpitatjon ofprotein Muk @) and McE (m f. Assays werecarried out on the proteins at 3.7 m&ml, 0.15 M Tris-HCl, pH 8.0. RESULTS
Immunochemical analyses indicated that the monoclonal protein isolated from patient Muk was an IgG, K monoclonal cryoimmunoglobulin (we are indebted to Dr. A. C. Wang for carrying out the subclass determination). N-terminal amino acid sequencing of the light chain yielded a single residue at each of the first 12 positions determined and the sequence Asp-Ile-Gln-MetThr-Gln-Ser-Pro-Ser-Ser-Leu-Ser permitted tentative assignment of the light chain to the V,I subgroup (Kabat et al., 1976). The heavy chain
was found to be blocked. Analytical gel filtration of the intact protein as well as its Fab’, and Fab components under neutral aqueous conditions and its heavy and light chains in the presence of 6 M guanidine-HCl failed to distinguish intact Muk or its constituent chains and fragments ’ 251-labeled noncryoglobulin from IgG references. The concentration dependence of cryoprecipitation at 0°C is markedly reduced when compared to other cryoimmunoglobulins (Middaugh et al., 197%~)and is only 81% at a protein concentration of 11 mg/ml (Fig. 1). Above thisconcentration, the protein isolated by
Table 1. Solute variation assay” Addition No addition
Cryoprecipitation 49
Neutral salts 0.2 M NaCl 1.0 M NaCl 2.0 M NaCI 0.2 M NaSCN 1.0 M NaSCN 0.2 M M&I, 1.0 M MgCl,
16 17 16 17 1.5 13 10
Formamide 0.5 M formamide 1.0 M formamide
15 14
(%)
Addition
Cryoprecipitation
Ureas 0.2 M urea 1.0 M urea 1 .O M ethyl urea 1.0 M propyl urea
10 11 6 11
Alcohols 0.2 M 1.O M 0.2 M 1.0 M
methanol methanol propanol propanol
58 84 60 89
Sugars 0.5 M @lactose 1.O A4 galactose 1.O N ribose
6 IO 6
(%)
“Assays were carried out as described (Middaugh & Litman, 1978; Middaugh et al., 19780) in 0.15 M Tris-HCI. pH 8.0. Assay concentration was 4.3 mg/ml and the uncertainty in the values is 2-3:/,.
340
G. W. LITMAN
cryoprecipitation appeared to be only partially soluble at 37°C in the standard 0.15 M Tris-HCI, pH 8.0 buffer, complicating analysis of reversibility and thus confusing estimation of cryoprecipitation. The extrapolated pH optimum of the Muk protein at 7.2 mgjml was found to be 8.4 with cryoprecipitation being abolished below pH 4 and above pH 11. The temperature dependence of cryoprecipitation at 3.7 mg/ml was found to be markedly depressed in comparison to other cryoglobulins (Fig. 2, and Middaugh ef al., 1978~). Table 1 illustrates the behavior of the Muk protein in a solute variation assay which has proven useful in evaluating forces involved in cryoprecipitation. The cryprecipitation of Muk is effectively inhibited by the neutral salts. ureas, formamides and sugars. Cryoprecipitation is effectively enhanced by high concentrations (1 .O M) of 1 and 3 carbon primary alcohols. In terms of overall similarity or dissimilarity to other proteins. Muk aopears to most closely resemble two other IgG, K cryoglobulins Mow and Ger and the IgM cryoglobulins Mel and Pel. It differs most from the IgM cryoglobulins McE and Gre (Middaugh et al., 197%~). There were no apparent gross differences in acid composition (including the amino tryptophan) of heavy and light chains derived from protein Muk when compared to the heavy and light chains of the noncryoglobulin reference proteins McC and Penn analyzed in parallel or to the composition of protein Eu (Edelman et al., 1969). However. the value for heavy chain Asx was slightly increased (43.7 vs 35.7 residues/mol) and the values for Phe and Val were decreased (9.0 vs 12.6 and 33.5 vs 39.4 residues,/mol, respectively) relative to the reference proteins examined. If the data base is expanded to include other equivalent IgG, heavy chains, these differences become less significant (not illustrated). The carbohydrate composition of protein Muk expressed as weight percent was found to be: neutral hexose = 1.23, hexosamine = 0.62, fucose = 0.13 and sialic acid = 0.05. The value for hexose is within accepted limits for noncryoglobulin IgG, while the values obtained for fucose. hexosamine and sialic acid are reduced ( _ 50”” reduction for hexosamine and fucose: _ SO”,, for sialic acid). Values for sialic acid of noncryoglobulin references determined in parallel consistently fell within normal limits. Prolonged digestion with neuraminidase was utilized to remove sialic acid from a group of four
rr ul
I
01
1
I Elui~on
volume
1,.
I
(ml)
Fig. 3. Analytical hydrophobic chromatography of the Fab (papain) fragments of Muk and the reference cryoglobulin Ger (H) and noncryoglobulin IgGs Bau (A), McC (0) and Penn (0). Elution conditions were as described in the text. The peak in Azao for the references is indicated by the symbols and the continuous line represents the elution profile of Muk.
noncryoglobulin IgG,, ti reference proteins. During the course of digestion at 37’C. a slight white precipitate accounting for -5”, of the total protein was noted to form. When the proteins at c 10 mg/ml were equilibrated in 0.15 M Tris-HCl, pH 8.0, and incubated at 0°C for 48 hr, no cryoprecipitation was evident, and it was concluded that removal of sialic acid failed to induce cryoglobulin behavior. Analytical hydrophobic chromatography of the Fab fragment of Muk IgG on a phenylsubstituted agarose matrix revealed the presence of a single homogeneous chromatographic boundary eluting at a conductance between _ 140 and 75 mS’ with the peak elution centered at 105 mS’ (Fig. 3). Included for reference in the figure are the conductance values for the A,,, elution maxima of Fab fragments isolated from the noncryoglobulin reference proteins Bau. McC and Penn. as well as the value for the Ger. In monoclonal IgG,, K cryoglobulin terms of hydrophobic character, as assessed by the nature of binding to and elution from the phenyl-substituted matrix, it may be concluded that protein Muk is within the range observed for non-cryoglobulin references, while a second cryoglobulin appears to be unique. CD studies of protein Muk at 37 C indicated a prominent negative ellipticity band centered at degrees cm’ 217 nm, 0 = -2920 -. which is within d mol the range for noncryoglobulin monoclonal IgGs. The spectrum was distinguished by a slightly
Physicochemical
Characrerization
of an IgG Crgoglobulin
341
protein concentration, it was noted that the amount of protein present in the supernatant plateaued at 2.05 mglml. thus defining the solubility of Muk in 0.15 MTris-HCI. pH 8.0, at reduced experience, the o-c. In our cryoprecipitability of this protein is unique and appears to be distinct from other proteins described in the literature, although such comparisons are admittedly difficult. In comparative solute variation assays, Muk closely resembles protein Mow, another IgG, cryoglobulin. The effect of low concentrations of NaCl on the cryoprecipitation of Muk suggests that electrostatic interactions are involved in the cryoprecipitation of this protein, although the possibility exists that significant contributions from van der Waals and additional unspecified weak noncovalent interactions are involved. As has been observed with the majority of other cryoimmunoglobulins examined to date, localization of cryoprecipitation to the major enzymatically derived fragments of Muk has been unsuccessful. This finding, coupled with both the failure of high molar excesses of these fragments and noncryoglobulin IgG to inhibit cryoprecipitation of the parent protein, and the inability to detect cold-dependent intermolecular associations using a sensitive direct hemagglutination technique makes it unlikely that antigen-antibody complex formation is involved in cryoprecipitation. Structural characterization of the Muk protein failed to distinguish Muk from a group of noncryoglobulin reference IgG, proteins with respect to (I) subunit mass and arrangement (sensitivity to proteolysis) and (2) conformation (analytical gel filtration of the intact protein or Fab, intrinsic fluorescence and far ultraviolet CD). In this sense, Muk differs from the two DlSCUSSION other IgG, cryoglobulins (Ger and Mow) which Cryoglobulins thus far included in our series both exhibited a marked reduction in the have been rather similar in terms of fluorescence emission maxima when excited at concentration and temperature dependence of 275 nm (or 295 nm) and protein Mow which cryoprecipitation (Middaugh et al., 1978a). exhibited a decreased negative ellipticity at 217 Detectable insolubility began to occur below 2.5 nm (Middaugh er al., 1978a). Other mg/ml under established assay procedures and in distinguishing features of the protein are the every case at least 50% relative cryoprecipitation somewhat atypical amino acid composition and. was encountered above 1.0 mg/ml. The Muk more important, the greatly reduced content of protein, however, did not undergo precipitation sialic acid. The latter point previously has been above 6°C at a concentration of 3.7 mgjml .111d suggested as a basis for the cold dependent monoclonal 50”” cryoprecipitation did not occur until a insolubility other of IgG protein concentration of 4 mg/ml was reached :II cryoglobulins (Hansson & Lindstrom, 1973; O-C. By quantitating the amount of protein (AS; a Zinneman & Caperton. 1977). Whether the function of total protein) in the supcrnatant and reduced content of sialic acid, fucose and hexosamine in Muk affects the intrinsic precipitate at O’C as a function of increasing
more pronounced secondary ellipticity minimum centered near 225-230 nm than typically observed with IgGs. When the CD spectrum of Muk was determined at OC at a sufficiently low concentration of protein (0.1 mg/ml) to prevent cryoprecipitation, no change was noted. The intrinsic fluorescence emission spectrum (&, = 275: 295 nm) of Muk at 23C (protein concentration = 0.1 mgiml) was indistinguishable from that of the reference noncryoglobulin IgGs. Typical digestion kinetics were noted when the intact protein was incubated at 37°C with papain and the progress of digestion was monitored by immunoelectrophoresis employing antiserum to Kand ;‘chains. When the isolated Fab’,. Fab and Fc fragments of Muk at concentrations of 5-50 mg/ml in 0.15 M Tris-HCl, pH 8.0, were placed at 0°C for 48 hr. cryoprecipitation was not observed. Incubation of varying proportions (molar excesses of I-30 times that of the intact protein) of these or noncryoglobulin Fab’z, Fab and Fc fragments with the intact Muk protein similarly failed to quantitatively affect cryoprecipitation. In a further attempt to detect intermolecular low temperature-mediated associations with the Muk protein, a direct hemagglutination assay was employed. Hemagglutination at O’C was not detected when erythrccytescoated with Muk IgG and Fab were incubated with varying dilutions of homologous I&. Fab and Fc or heterologous IgG, although the cells were apparently coated with the intact protein and Fab fragments, as evidenced by agglutination at a high dilution (214) of antiserum directed at the K chain determinant of the Fab.
_
_
;‘,; V,,B
Muk
_
_
+
gel filtration
Agarose
Elevated Asx, reduced Phe and Val? see text
-
_
Amino acid composition
Sialic acid-reduced
_
-
+
+
Inhibition in 0.2 M NaCl
+
+
-
0.5nM formamide
Inhibition
t
+
-
Inhibition in 0.5 M galactose
Effect of solute on cryoprecipitation”
cryoimmunoglobulins”
Carbohydrate composition
K monoclonal
” Data in Table 2 were obtained from expcrimentsdescribed in this paper or in Middaugh et nl. (19780). The data presented has been chosen to reflect differences rather than simifaritrcs bctwccn the tndividual proteins. h ( + ) Indicates a positive tinding (signficantly different from normal) and (--) indicates a negative finding (indistinguishable from normal). ’ V,B indicates the presence of a blocked amino terminus. “( +) indicates that near complete inhibition takes place: (- ) Indicates that inhibition is only partial or does not take place
+
+
+
_
i’,; v,B’
Reduced intrinsic fluorescence (i._ = 275 nm)
;‘,; V,,lH
9’ (217 nm)
Reduced
Mow
L chain class & subgroup
of three IgG,,
properties”
properties
Atypical
of physicochemical
Cer
Protein
H chain class & subgroup
Table 2. Comparison
R
w
Physicochemical Characterization
solubility of the protein and influences cryoprecipitation in this way is unclear. The removal of sialic acid from noncryoglobulin reference proteins fails to induce cryoglobulin behavior. These studies have introduced results with an additional probe of macromolecular conformation, hydrophobic chromatography. The principle of this technique is based on the increased hydrophobic interaction which occurs between a protein and an immobilized ligand at high salt concentration (Hofstee, 1976). High ionic strength tends to simultaneously stabilize the hydrophobic interaction and minimize electrostatic interactions. In these studies, a phenyl substituted agarose, with an estimated hydrophobicity between 4 and 5 straight chain carbons, was employed (Hjerten, 1976). The elution characteristics of Muk suggest it to be within the range of the three reference proteins examined. For the sake of comparison, the Fab component of the cryoglobulin Ger was also included. Its unique elution characteristics suggest it to be of a less hydrophobic character than the other four proteins examined. The Fab component of Ger exhibits an anomalously high Mr in analytical gel filtration on agarose, and we have suggested that this effect may be due to a lack of interaction of certain cryoglobulin proteins with agarose, resulting in their elution ahead of the noncryoglobulin references (Middaugh et al., 1978~). If this interaction with agarose depends on the hydrophobic nature of the proteins, then it could be predicted that the Ger protein with a reduced hydrophobic character should emerge first, while the Muk protein which possesses a more typical net hydrophobicity should emerge at a normal Mr position. This was observed; however, a number of other possibilities exist for explaining the gel filtration abnormality. One of the strategies we have employed in an effort to discern a molecular basis forcryoglobulin behavior has been to compare the structure and functional behavior of as large a group of cryoglobulins as reasonable. When the physicochemical properties of the Muk protein and two additional cryoglobulins of the same heavy and light chain isotype are compared, no common pattern emerges which relates the individual proteins (Table 2). Findings such as these make it increasingly more likely that a variety of subtle alterations in macromolecular structure account for anomalous may temperature insolubility characteristic of
of an IgG Cryoglobulin
343
monoclonal cryoimmunoglobulins. We have suggested that cryoglobulin behavior may reflect a heterogeneity in solubility of immunoglobulins; cryoglobulins the may represent intrinsically less soluble members of the population (Middaugh et al., 1978~). If such were the case, then a comprehensive survey of cryoglobulins should detect proteins with only marginal cryoglobulin behavior. Protein Muk may represent one such variant and in this way lend support to this hypothesis.
REFERENCES
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344
G. W. LITMAN et al.
basis for the temperature dependent insolubility of cryoglobulins. IV. Structural studies of the IgM monoclonal cryoglobulin McE. immunochemistry 15, 171-187. Wang A. C., Mathur S., Pandey J., Siegal F. P., Middaugh C. R.. and Litman G. W. (1978) Hv(l), a variable-region genetic marker of human immunoglobulin heavy chains. Science 200, 327-329.
Zimmerman C. L., Appella E. & Pisano J. (1977) Rapid analysis of amino acid phenythiohydantoins by highperformance liquid chromatography. Analyf. Eiochem. 77, 569-573. Zinneman H. H. & Caperton E. (1977) Cryoglobulinemia in a patient with Sjogrens syndrome and factors of cryoprecipitation. J. lab. clin. Med. 89, 483-487.