Br. J. exp. Path. (1976) 57, 582.
CONFORMATIONAL FLEXIBILITY OF THE SERUM AMYLOID PRECURSOR SAA J. D. SIPE, K. P. W. J. McADAM*, B. F. TORAIN AND G. G. GLENNER From the Laboratory of Experimental Pathology, National In8titute of Arthriti8, Metaboli8m and Dige8tive Disease8, *Immunology Branch, National Cancer In8titute, National In8titutes of Health, Bethe8da, Maryland, 20014 Received for publication June 4, 1976
Summary.-SAA is a normal acute-phase serum protein and is thought to be the precursor of amyloid protein AA which is deposited as insoluble P-pleated sheet fibrils in secondary amyloidosis. Native SAA has a molecular weight of 160,000 and has not been isolated; it has been most frequently purified as a species (designated SAAL) of 12,500 mol. wt. by gel filtration in dissociating solutions. The conformational properties of SAA proteins in patients with and without amyloidosis have been compared in an effort to determine the factors involved in the induction of the P,pleated sheet conformation in the amyloid SAA protein prior to fibril deposition. Amyloid and nonamyloid SAA proteins are similar in that they readily undergo conformational changes which result in the formation of heterogeneous mol. wt. SAA species and in an increased exposure of antigenic determinants which cross-react with AA fibril proteins. Amyloid and nonamyloid SAA are different, however, in that amyloid SAA is more resistant to dissociation to SAAL. Amyloid SAAL, while similar to nonamyloid SAAL in immunoreactivity, shows a greater tendency toward aggregation. The relative resistance of both amyloid SAA and SAAL to complete dissociation may play an important role in amyloid fibril formation from these precursors.
AMYLOIDOSIS is the generic term given to a group of diseases characterized by the extracellular deposition of protein fibrils having a /-pleated sheet conformation (Glenner and Page, 1976). As a result of their conformation, the fibrils are insoluble and resistant to proteolysis. It is also the ,3-pleated sheet conformation which is responsible for the characteristic Congo red birefringence of amyloid deposits. Amyloid fibril proteins are thought to be derived from normally innocuous polypeptide precursors by proteolysis or other physicochemical mechanisms. The first amyloid precursor to be identified was the immunoglobulin light chain (Glenner, Terry and Isersky, 1973). Variable region fragments and intact light chains have been found to be the major fibril protein in amyloid of immunoglobulin origin (AIO) which is found in association with myeloma and primary amyloid syndromes. It is
now recognized that other normal proteins can be the precursor of fibrils having the /3-pleated sheet conformation characteristic of amyloid. A second class of amyloid precursors originates from cells of neural crest origin, which have been designated APUD cells because of the common cytochemical characteristics they share. An amyloid fibril protein larger than calcitonin, but sharing a common amino acid sequence with it, has been isolated from amyloid deposits of a medullary carcinoma of the thyroid (Sletten, Westermark and Natvig, 1976). The amyloid deposits associated with the neuroendocrine system have been termed apudamyloid (Pearse, Ewen and Polak, 1972). A third type of amyloid precursor is a normal serum protein SAA, which has the properties of an acute-phase reactant. SAA appears to be the precursor of amyloid AA, which is the major fibril protein
THE SERUM AMYLOID PRECURSOR SAA
associated with amyloid secondary to recurrent acute inflammatory conditions, such as rheumatoid arthritis, familial Mediterranean fever, and chronic infections (Benditt et al., 1971). As amyloid AA protein has an amino acid sequence which is different from that of any known protein, it was designated amyloid of unknown origin (AUO). The deposition in tissues of the insoluble ,8-pleated sheet amyloid fibrils may involve molecular defects in one or more aspects of precursor metabolism, including (1) the amount of precursor synthesized, (2) the amino acid sequence of the precursor, and (3) the catabolic processing of the precursor by its functional target cells. The physiological processes involved in the induction of the /3-pleated sheet conformation and/or fibril formation from amyloid precursors are poorly understood. X-ray crystallographic studies of immunoglobulin polypeptide chains have shown that both variable and constant region domains consist of layers of antiparallel /-pleated sheet segments (Davies, Padlon and Segal, 1974), and amyloid fibrils have been formed from Bence Jones proteins and immunoglobulin light chains in vitro (Glenner et al., 1971; Tan and Epstein, 1972) by proteolytic digestion. Synthetic /3-pleated sheet fibrils have been formed also from insulin and glucagon, in acid solution at moderately elevated temperatures (Glenner et al., 1974). Glucagon is a molecule with considerable conformational flexibility; in dilute neutral solution it exists in a random coil conformation (Panijpan and Gratzer, 1974), in alkaline solution or in the presence of detergents it adopts an ac-helical conformation (Bornet and Edelhoch, 1971) and in acid solution it forms gels and eventually antiparallel /3-pleated sheet fibrils (Beaven, Gratzer and Davis, 1969). The kinetics of gel and fibril formation from glucagon are sensitive to protein and salt concentration, and temperature, as well as to pH. This communication is concerned with the conformational flexibility of SAA, the putative precursor of amyloid AA fibrils
583
of unknown origin. SAA has been studied intensively since it was identified by Levin and coworkers (1973) in the serum of patients with chronic diseases. SAA has been detected as well in mouse (Isersky et al., 1971), guinea pig (Skinner et al., 1974) and in mink (Anders et al., 1976) with antibodies raised to denatured AA fibrils from the corresponding species. SAA levels are often found to be elevated in patients with chronic and neoplastic diseases, although very few of these patients develop secondary amyloidosis (Rosenthal and Franklin, 1975). SAA levels have been shown to rise within a few hours of an acute phase stimulus (McAdam and Sipe, 1976). The cell of origin and function of SAA are unknown; however it has been reported that murine SAA has immunosuppressive activity (Benson et al., 1975). The structure of SAA is also unknown; the native species has a mol. wt. of 160,000 to 180,000 (Sipe et al., 1976) which has not been isolated to date. SAA has been most frequently purified as a species (designated SAAL) of approximately 12,500 mol. wt., by gel filtration of serum or serum fractions in guanidine hydrochloride or formic acid (Linke et al., 1975; Anders et al., 1975). The first 11 NH2terminal amino acids are identical in SAAL and AA (Rosenthal et al., 1976), supporting the idea that the 12,500 mol. wt. SAAL is the precursor of the 5300-8500 AA fragment found in tissues. It is possible that SAA consists of SAAL complexed through noncovalent bonds to a high mol. wt. polypeptide which does not cross-react with anti-AA antibodies, or that SAA is a polymer of SAAL moieties arranged in an unknown fashion. We now report evidence that SAA exhibits conformational flexibility. During storage of serum at 40, AA crossreacting species of heterogeneous mol. wt. are formed and AA cross-reacting determinants are exposed during this process. Because this conformational flexibility may play an important role in the induction of the /-pleated sheet confor-
584
J. D. SIPE, K. P. W. J. McADAM, B. F. TORAIN AND G. G. GLENNER
mation prior to fibril deposition, we have compared SAA from patients with and without secondary amyloidosis, and have found that both types of precursor exhibit conformational flexibility. However, there are differences in susceptibility of the two types of SAA to SAAL formation. SAA from patients with secondary amyloidosis is less readily dissociated to SAAL by gel filtration in guanidine hydrochloride or formic acid than is SAA from patients without amyloidosis, under the same conditions. Furthermore, isolated SAAL preparations from patients with amyloidosis, although similar in immunoreactivity to that from patients without amyloidosis, have a markedly greater tendency toward reaggregation even in the presence of formic acid.
except that column dimensions were 3 x 90 cm. Those fractions from the low mol. wt. region on the chromatographic profile which were antigenically reactive in radioimmunoassay were pooled and concentrated by dialysis vs polyethylene glycol or by vacuum rotary evaporation at room temperature. Protein determinations were made according to the method of Lowry, Rosebrough and Farr (1951) using crystalline bovine serum albumin as standard. Sodium dodecyl sulphate polyacrylamide gel electrophoresis.-SAAL isolated by gel filtration in 10% formic acid and concentrated in the presence of formic acid to 0-1-0-5 mg/ml was further concentrated under a stream of nitrogen immediately prior to electrophoresis in 10% polyacrylamide gels in 0-1% sodium dodecyl sulphate, at pH 7-1 according to the method of Weber and Osborn (1969). Dithiothreitol (0-1%) was added to the sample solution to dissociate possible disulphide polymers in the sample. RESULTS
MATERIALS AND METHODS
SAA-containing serum.-Serum from patient PEK with Reiter's syndrome but without clinical evidence of amyloidosis was stored at 40 in the presence of thymol as preservative. Serum from patient AMB with amyloidosis secondary to leprosy was initially frozen for transit, and was then stored at 40 in the presence of thymol as preservative. Serum was not frozen during storage because it has been observed that SAA precipitates upon thawing after freezing and not all of the precipitate can be dissolved. Radioimmunoassay.-Both SAA and SAAL were determined by their cross-reaction in the solid phase radioimmunoassay for the tissue fibril protein AA as previously described (Sipe et al., 1976). Duplicate determinations were carried out on all samples. When samples were denatured in formic acid prior to assay, or when gel filtration samples eluted in formic acid were to be assayed, the acid was diluted to less than 4% (v/v) with water, frozen in dry ice and lyophilized. Preparation of SAA.-SAA was isolated from whole serum by gel filtration on Sephadex G-200 in 0-1 M phosphate-buffered saline pH 7-2 (Sipe et al., 1976). Aliquots of each fraction were tested by radioimmunoassay for crossreactivity with the tissue amyloid protein AA. The AA cross-reacting fractions were pooled and concentrated by negative pressure dialysis to a final volume of 2-4 ml. Preparation of SAAL.-SAAL was obtained by gel filtration of SAA isolated from whole serum on Sephadex G-100 in 10% (v/v) formic acid as previously described (Sipe et al., 1976),
Change in molecular weight distribution of SAA upon storage When the mol. wt. of SAA from the serum of patient PEK (without amyloidosis) was determined by gel filtration of fresh serum on Sephadex G-200 in 0-1 M phosphate-buffered saline, pH 7-2, it was approximately 160,000 as determined by its coelution with the IgG fraction of serum (Fig. la). When the fractionation was repeated under identical conditions after 5 months' storage of serum PEK at 40 in the presence of thymol as preservative, AA cross-reacting species of heterogeneous mol. wt. were observed (Fig. lb). Almost one-half of the observed immunoreactivity was in the void volume fractions after gel filtration of the stored serum, and a small fraction of AA cross-reacting material was also detected in the low mol. wt. region between albumin and the salt boundary, whereas no immunoreactivity was detected in these regions upon separation of fresh serum. Low mol. wt. AA-cross-reactivity under physiological conditions (Fig. 2) has also been observed in the serum from patient AMB with amyloidosis secondary to leprosy, which had been stored for several months at 40. Fresh serum from this patient was not
585
THE SERUM AMYLOID PRECURSOR SAA
300
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FIG. la.-Top: Descending Sephadex G-200 gel filtration of 2 ml of serum PEK carried out within 24 h after collection from the patient and removal of the fibrin clot by centrifugation. The serum was applied to the chromatographic column (110 x 2-5 cm) equilibrated with PBS and eluted at a flow rate of 15 ml/h with 2*5 ml fractions collected. The AA cross-reactivity was determined by diluting 100 ,ul of each fraction with 400 jul of 2% casein in 0 1 M barbital buffer, pH 8-6, and testing *, absorbance at 280 nm; 0 duplicate 200-,ul aliquots in RIA (II). * QO, AA crossreactivity in ng/fraction. FIG. lb.-Bottom: Descending Sephadex G-200 gel filtration of 2 ml of serum PEK after storage for approximately 5 months at 40 in the presence of thymol as preservative. Conditions for separation and assay are identical to those described above.
available; however, separation of serum within a few days of collection from a patient with amyloidosis secondary to rheumatoid arthritis showed only the 160,000 mol. wt. SAA species, and none of the void volume or post-albumin crossreacting material.
Increased AA cross-reactivity of serum upon storage When radioimmunoassay (RIA) inhibition curves were constructed from serial dilutions in casein barbital dilution buffer of serum PEK, they yielded an SAA concentration of 0.3 jig/ml when the serum was fresh and 8 ,ug/ml when the serum was 5 months old. The SAA concentration was determined as AA cross-reactivity by comparing the 50 % inhibition points of the RIA inhibition curves of AA and serum
PEK. Similarly, untreated serum AMB was found to increase in immunoreactivity from 0.4 to 20 ,tg/ml during 6 months of storage in the laboratory. Increased AA cross-reactivity upon denaturation of serum.-Because of the increases in immunoreactivity of sera upon storage, it seemed advantageous to minimize this effect by denaturation of serum samples before determination of their SAA levels by RIA. During the course of isolating SAAL by formic acid dissociation (Sipe et al., 1976), it was observed that there was a 150-fold increase in immunoreactivity relative to whole serum upon purification of the low mol. wt. species. Therefore, the effects of length of time and temperature of formic acid incubation on the AA cross-reactivity of serum AMB were investigated (Fig. 3).
586
J. D. SIPE, K. P. W. J. MCADAM, B. F. TORAIN AND G. G. GLENNER
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FIG. 2.- Descending Sephadex G-200 gel filtration of 3 ml of serumn AMB after approximately 5 months of storage at 4° in the presence of thymol as preservative. Conditions of separation and analysis are as described in Fig. 1, except that elution of fractions was interrupted for approximately 8 h in the region of fraction 60. Uninterrupted elution yielded a similar, but less well defined peak in this region.
Dilution of 1 ,tl of serum with 500 /il of 10% formic acid followed by immediate lyophilization increased the observed immunoreactivity from 20 ,ug/ml to 40 ,tg/ml, and there was no further increase over 72 h at 0°. Further increases in immunoreactivity were observed when incubation was carried out at 25° and 37°, however, and the highest value measured was about 120 ,tg/ml after 72 h of incubation at 37°. It was found that reproducible inhibition curves could be constructed from serum AMB and serum PEK by making serial dilutions of the sera in 10% formic acid, incubating them at 37° for 24 h, removing the formic acid by lyophilization
and assaying the lyophilized material by RIA (examples of these curves are shown in Fig. 4). By coincidence, these two sera have similar SAA levels after formic acid denaturation, which, as calculated from the 5000 inhibition points of the serum and AA inhibition curves, is 180 ,ag/ml of AA cross-reactivity. The SAA levels of the denatured sera are significantly greater than the 20 and 8 jig/ml observed for the untreated sera. Dissociation of SAA to SAAL in amyloid and nonamyloid sera When the SAA fraction isolated from serum PEK by neutral Sephadex G-200
587
THE SERUM AMYLOID PRECURSOR SAA
gel filtration was applied without pH adjustment to a Sephadex G-100 column equilibrated with 10% formic acid, all of the AA cross-reactivity was dissociated from the nonimmunoreactive serum pro-
teins which were eluted in the void volume fractions (Fig. 5a). Analysis of this SAAL preparation by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulphate showed it to be of similar
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INCUBATION TIME, HOURS FIG. 3.-Effect of length of time and temperature of formic acid denaturation on AA cross-reactivity of serum AMB. A dilution of serum AMB was made by mixing 40 PI of serum with 20 ml of 10% formic acid. Aliquots of 500 ,1 each were incubated in tightly stoppered tubes for varying times, after which 2 ml of water was added, the tubes were frozen, and the formic acid was removed by lyophilization. RIA was carried out on each lyophilized sample by adding 100 PI 0-1 M barbital buffer pH 8 6 and 400 ,ul 2% casein in the same barbital buffer (II). Immunoreactivi.ty is expressed in terms of AA cross-reactivity/ml of undiluted serum. #PWR. Vwi* .i
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FIG. 4.-Radioimmunoassay inhibition curves showing cross-reactivity of SAA and SAAL with AA. DuwMicate determinations were carried out using the indicated ng of SAAL: * -*, PEK; O, AMB; a, PEK and *--*, AMB; A- -A, AA; ,ul of undenatured serum: O Q, AMB. The inhibition curve and ,ul of formic acid denatured serum: C] E, PEK;and O of serum PEK shown was determined in a separate experiment from the others. Hence the mean of two duplicate inhibition curves for AA and SAAL-AMB is plotted and the horizontal lines represent the values for the individual experiments.
J. D. SIPE, K. P. W. J. MCADAM, B. F. TORAIN AND G. G. GLENNER
588
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FIG. 5b.-Descending gel filtration on Sephadex G-100 in 10% (v/v) formic acid of the SAA containing fractions from Sephadex G-200 PBS gel filtration of serum AMB. Conditions for separation and assay are the same as those described above.
THE SERUM AMYLOID PRECURSOR SAA
mobility (Fig. 6) to SAAL previously determined to be 12,500 in mol. wt. (Linke et al., 1975). The same treatment of the SAA fraction from serum AMB, however, results in only partial dissocia-
589
tion of the AA cross-reacting species from the other serum proteins which are eluted in the void volume. Analysis of the isolated SAAL by polyacrylamide gel electrophoresis showed in addition to the
FIG. 6. Polyacrylamide (10%) electrophoresis in 0-1% sodium dodecyl sulphate of SAAL preparations from serum AMB and PEK. Left: 40 ,ug SAAL-AMB (Fractions 59-73, Fig. 5b), Middle: 40 ,ug SAAL-PEK (Fraction 62-77, Fig. 5a), Right: mol. wt. markers (a) bovine serum albumin (67,000), (b) chymotrypsinogen (24,000), (c) cytochrome c (13,000), and (d) insulin (6000).
590
J. D. SIPE, K. P. W. J. McADAM, B. F. TORAIN AND G. G. GLENNER
expected low mol. wt. band, two bands which correspond to mol. wt. of approximately 25,000 and 75,000 (Fig. 6). In view of the degree of resolution evident in the gel filtration profile, these high mol. wt. bands would appear to have derived from material that was of lower mol. wt. at the time of formic acid gel filtration. High mol. wt. material of this type has also consistently been observed in SAAL preparations from patients with secondary amyloidosis which have been prepared by gel filtration on Sephadex G-100 in guanidine hydrochloride. In spite of their differences in susceptibility to dissociation, the SAAL preparations from the two types of sera show very similar RIA inhibition curves (Fig. 4), thus supporting the use of RIA to compare SAA levels in various types of disease sera. DISCUSSION
While the structure of SAA has not yet been elucidated, certain aspects of its nature have become apparent. SAA appears to consist of 12,500 mol. wt. SAAL species arranged in a labile, easily dissociable structure of approximately 160,000 mol. wt. It would appear that all of the AA cross-reacting determinants contained in SAA are due to the low mol. wt. material, since SAA from the serum of a patient without amyloidosis was completely dissociated to SAAL by gel filtration on Sephadex G-100 in formic acid. The existence of an AA crossreacting polypeptide chain intermediate in mol. wt. to SAAL and SAA cannot be excluded in patients with secondary amyloidosis. However, since the proportion of SAA dissociating to SAAL has varied between individual separations of the same serum sample, from patients with or without amyloidosis, it seems likely that the immunoreactivity observed in the void volume fractions after gel filtration on Sephadex G-100 in guanidine hydrochloride or formic acid represents undissociated SAA or aggregated SAAL. The arrangement of SAAL polypep-
tides in SAA is unknown. It may be that one or two SAAL proteins are complexed to a high mol. wt. protein which does not cross-react with anti-AA antibodies, or alternatively, SAA may be a polymer of SAAL chains which contains in addition, low mol. wt. nonantigenic material such as peptides or lipid. It appears that SAAL is contained in SAA in a conformation such that it is soluble and can be dissociated. Dissociation of SAAL from SAA occurs upon storage or various experimental manipulations of serum, and during this process determinants which cross-react with anti-AA antibodies are exposed. It seems highly significant that SAAL aggregates easily and is then difficult to dissociate when it has a conformation such that antigenic determinants which cross-react with antibodies raised to the insoluble fibrils deposited in tissues are maximally exposed. We have observed that SAAL preparations from both serum PEK and serum AMB migrate poorly into sodium dodecyl sulphate polyacrylamide gels after lyophilization, and we have found it necessary to concentrate and store these proteins in the presence of 10% formic acid, removing water and acid under a stream of nitrogen immediately before electrophoresis is carried out. While denaturation of SAA-containing serum and tissue seems advantageous from the standpoint of increased sensitivity and more reproducible RIA determinations, the problem of determining with precision the SAAL content of a serum sample remains. Complete exposure of AA cross-reacting determinants would appear both to be difficult to achieve, and to ascertain when it had been achieved. When denaturation was carried out for 24 h at 370 at a ratio of serum-toformic-acid of 0.002, the observed AA cross-reactivity was approximately 90 ,tg/ml (Fig. 3). When it was carried out at a serum-to-acid ratio of 0*00025 (Fig. 4), the observed immunoreactivity was approximately 180 ,lig/ml of AA. The observation that reproducible inhibition
THE SERUM AMYLOID PRECURSOR SAA
curves can be constructed after treatment of serum dilutions with formic acid for 24 h at 370 supports the use of formic acid denaturation for comparing SAA levels in different serum samples. Thus it appears that SAA proteins in amyloid and nonamyloid sera share certain characteristics, one of which is a tendency to undergo conformational changes which result in the formation of heterogeneous mol. wt. SAA species and in an increased oxposure of antigenic determinants which are common with the tissue fibril proteins. Amyloid and nonamyloid SAA are also similar in that each can be dissociated to a low mol. wt. fraction, SAAL, and the two types of SAAL are identical in immunoreactivity (Fig. 4). However, there are differences between amyloid and nonamyloid SAA proteins, in that amyloid SAA has consistently been more resistant to dissociation to SAAL. Furthermore, amyloid and nonamyloid SAAL preparations show different mol. wt. distributions when examined by SDS polyacrylamide gel electrophoresis; the amyloid SAAL contains material which aggregates under strong dissociating conditions. There is a need, therefore, to examine SAAL preparations from the two types of sera thoroughly. Complete amino acid sequence determinations on both amyloid and nonamyloid SAAL may reveal sequence heterogeneity near the carboxyl portions of the proteins as being important in amyloidogenesis, despite the similarities in the amino terminal sequence. The degree of homogeneity of SAAL needs to be established also, since the presence of only small amounts of nonimmunoreactive proteins of similar mol. wt. to the SAAL protein would be difficult to detect, but such proteins might play an important role in the aggregation of SAAL. The present studies have employed antibodies raised to a 5300 mol. wt., 45 amino acid residue polypeptide AA which presumably arises by proteolytic digestion of the 12,500 mol. wt. SAAL, consisting of approximately 114 residues. If there are some determinants in SAAL which are
591
completely exposed in the native SAA species, use of 125SAAL and anti-SAAL, when available, may facilitate detection of SAA without denaturation. However, the use of antibodies raised to the AA fragment has provided considerable information about the flexibility of the SAA species. This flexibility may play a role in the deposition of insoluble P-pleated sheet amyloid fibrils in some patients with recurrent acute or chronic inflammatory conditions. We thank Miss Janice Ryan for capable assistance with all aspects of this work. K. M. was the recipient of a Medical Research Council (U.K.) Travelling Fellowship. REFERENCES ANDERS, R. F., NATVIG, J. B., MICHAELSEN, T. E. & HUSBY, G. (1975) Isolation and Characterization of Amyloid-related Serum Protein SAA as a Low Molecular Weight Protein. Scand. J. Immunol., 4, 397. ANDERS, R. F., NORDSTOGA, K., NATVIG, J. B. & HUSBY, G. (1976) Amyloid-related Serum Protein SAA in Endotoxin Induced Amyloidosis of the Mink. J. exp. Med., 143, 678. BEAVEN, G. H., GRATZER, W. B. & DAVIEs, H.G. (1969) Formation and structura of Gals and Fibrils from Glucaoon. Eur. J. Biochem., 11, 37. BENDITT, E. P., ERIKSEN, N., HERMODSON, M. A. & ERICSSON, L. H. (1971) The Major Proteins of Human and Monkey Amyloid Substance: Common Properties Including Unusual N-terminal Amino Acid Sequences. FEBS Lett., 19, 169. BENSON, M. D., ALDO-BENsoN, M. A., SHIRAHAMA, T., BOREL, Y. & COHEN, A.S. (1975) Suppression of in vitro Antibody Response by a Serum Factor (SAA) in Experimentally Induced Amyloidosis. J. exp. Med., 142, 236. BORNET, M. & EDELHOCH, H. (1971) Polypeptide Hormone Interaction. J. Biol. Chem., 246, 1785. DAVIES, D. R., PADLAN, E. A. and SEGAL, D. M. (1975) Three-Dimensional Structure of Immunoglobulins. Ann. Rev. Biochem., 44, 639. GLENNER, G. G., EANES, E. D., BLADEN, H. A., LINKE, R. P. & TERMINE, J. D. (1974) f-Pleated Sheet Fibrils: A Comparison of Native Amyloid with Synthetic Protein Fibrils. J. Histochem. Cytochem., 22, 1141. GLENNER, G. G., EIN, D., EANES, E. D., BLADEN, H. A., TERRY, W. D. & PAGE, D. (1971) The Creation of " Amyloid ' Fibrils from Bence Jones Proteins in vitro. Science, N. Y., 174, 712. GLENNER, G. G. & PAGE, D. L. (1976) Amyloid, Amyloidosis and Amyloidogenesis. Int. Rev. exp. Pathol., 15, 1. GLENNER, G. G., TERRY, W. D. & ISERSKY, C. (1973) Amyloidosis: Its Nature and Pathogenesis. Semin. Hematol., 10, 65.
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ISERSKY, C., PAGE, D. L., CUATRECASAS, P., DE LELLIS, R. A., & GLENNER, G. G. (1971) Murine Amyloidosis: Immunologic Characterization of Amyloid Fibril Protein. J. Immunol., 107, 1690. LEVIN, M., PRAS, M., LINKE, R. & FRANKLIN, E. C. (1973) Immunologic Studies of ASF, the Major Nonimmunoglobulin Component of Certain Amyloid Fibrils. Arthriti8 Rheum., 16, 123. LINKE, R. P., SIPE, J. D., POLIOCK, P. S., IGNACZAK, T. F. & GLENNER, G. G. (1975) Isolation of a LowMolecular-Weight Serum Component Antigenically Related to an Amyloid Fibril Protein of Unknown Origin. Proc. natn. Acad. Sci., USA., 72, 1473. LowRY, 0. H., RoSEBROUGH, N. J. & FARR, A. L. (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265. McADAM, K. P. W. J. & SIPE, J. D. (1976) Serum Precursor of Murine Amyloid Protein: An Acute Phase Reactant in Response to Polyclonal B Cell Mitogens. Fed. Proc., 35, Abstract 1500. PANIJPAN, B. & GRATZER, W. B. (1974) Conformational Nature of Monomeric Glucagon. Eur. J. Biochem., 45, 547. PEARSE, A. G. E., EWEN, S. W. B. & POLAK, J. M. (1972) The Genesis of Apudamyloid in Endocrine Polypeptide Tumours: Histochemical Distinction from Immunamyloid. Virchow8 Arch. Abt. B Zellpath., 10, 93. ROSENTHAL, C. J. and FRANKLIN, E. C. (1975) Variation with Age and Disease of an Amyloid A
Protein-Related Serum Component. J. Clin. Invest., 55, 746. ROSENTHAL, C. J., FRANKLIN, E. C., FRANGIONE, B. & GREENSPAN, J. (1976) Isolation and Partial Characterization of SAA-an Amyloid Related Protein from Human Serum. J. Immunol., 116, 1415. SIPE, J. D., IGNACZAK, T. F., POLLOCK, P. S. & GLENNER, G. G. (1976) Amyloid Fibril Protein AA: Purification and Properties of the Antigenically Related Serum Component as Determined by Solid Phase Radioimmunoassay. J. Immunol., 116, 1151. SKINNER, M., CATHCART, E. S., COHEN, A. S. & BENSON, M. D. (1974) Isolation and Identification by Sequence Analysis of Experimentally Induced Guinea-Pig Amyloid Fibrils. J. exp. Med., 140, 871. SLETTEN, K., WESTERMARK, P. & NATVIG, J. B. (1976) Characterization of Amyloid Fibril Proteins from Medullary Carcinoma of the Thyroid. J. exp. Med., 143, 993. TAN, M. & EPSTEIN, W. (1972) Polymer Formation During Degradation of Human Light Chain and Bence Jones Proteins by an Extraet of the Lysosomal Fraction of Normal Human Kidney. Immunochemistry, 9, 9. WEBER, K. & OSBORN, M. (1969) The Reliability of Molecular Weight Determination by Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. J. Biol. Chem., 244, 4406.
CONFORMATIONAL FLEXIBILITY OF THE SERUM AMYLOID PRECURSOR SAA J. D. SIPE, K. P. W. J. McADAM*, B. F. TORAIN AND G. G. GLENNER From the Laboratory of Experimental Pathology, National In8titute of Arthriti8, Metaboli8m and Dige8tive Disease8, *Immunology Branch, National Cancer In8titute, National In8titutes of Health, Bethe8da, Maryland, 20014 Received for publication June 4, 1976
Summary.-SAA is a normal acute-phase serum protein and is thought to be the precursor of amyloid protein AA which is deposited as insoluble P-pleated sheet fibrils in secondary amyloidosis. Native SAA has a molecular weight of 160,000 and has not been isolated; it has been most frequently purified as a species (designated SAAL) of 12,500 mol. wt. by gel filtration in dissociating solutions. The conformational properties of SAA proteins in patients with and without amyloidosis have been compared in an effort to determine the factors involved in the induction of the P,pleated sheet conformation in the amyloid SAA protein prior to fibril deposition. Amyloid and nonamyloid SAA proteins are similar in that they readily undergo conformational changes which result in the formation of heterogeneous mol. wt. SAA species and in an increased exposure of antigenic determinants which cross-react with AA fibril proteins. Amyloid and nonamyloid SAA are different, however, in that amyloid SAA is more resistant to dissociation to SAAL. Amyloid SAAL, while similar to nonamyloid SAAL in immunoreactivity, shows a greater tendency toward aggregation. The relative resistance of both amyloid SAA and SAAL to complete dissociation may play an important role in amyloid fibril formation from these precursors.
AMYLOIDOSIS is the generic term given to a group of diseases characterized by the extracellular deposition of protein fibrils having a /-pleated sheet conformation (Glenner and Page, 1976). As a result of their conformation, the fibrils are insoluble and resistant to proteolysis. It is also the ,3-pleated sheet conformation which is responsible for the characteristic Congo red birefringence of amyloid deposits. Amyloid fibril proteins are thought to be derived from normally innocuous polypeptide precursors by proteolysis or other physicochemical mechanisms. The first amyloid precursor to be identified was the immunoglobulin light chain (Glenner, Terry and Isersky, 1973). Variable region fragments and intact light chains have been found to be the major fibril protein in amyloid of immunoglobulin origin (AIO) which is found in association with myeloma and primary amyloid syndromes. It is
now recognized that other normal proteins can be the precursor of fibrils having the /3-pleated sheet conformation characteristic of amyloid. A second class of amyloid precursors originates from cells of neural crest origin, which have been designated APUD cells because of the common cytochemical characteristics they share. An amyloid fibril protein larger than calcitonin, but sharing a common amino acid sequence with it, has been isolated from amyloid deposits of a medullary carcinoma of the thyroid (Sletten, Westermark and Natvig, 1976). The amyloid deposits associated with the neuroendocrine system have been termed apudamyloid (Pearse, Ewen and Polak, 1972). A third type of amyloid precursor is a normal serum protein SAA, which has the properties of an acute-phase reactant. SAA appears to be the precursor of amyloid AA, which is the major fibril protein
THE SERUM AMYLOID PRECURSOR SAA
associated with amyloid secondary to recurrent acute inflammatory conditions, such as rheumatoid arthritis, familial Mediterranean fever, and chronic infections (Benditt et al., 1971). As amyloid AA protein has an amino acid sequence which is different from that of any known protein, it was designated amyloid of unknown origin (AUO). The deposition in tissues of the insoluble ,8-pleated sheet amyloid fibrils may involve molecular defects in one or more aspects of precursor metabolism, including (1) the amount of precursor synthesized, (2) the amino acid sequence of the precursor, and (3) the catabolic processing of the precursor by its functional target cells. The physiological processes involved in the induction of the /3-pleated sheet conformation and/or fibril formation from amyloid precursors are poorly understood. X-ray crystallographic studies of immunoglobulin polypeptide chains have shown that both variable and constant region domains consist of layers of antiparallel /-pleated sheet segments (Davies, Padlon and Segal, 1974), and amyloid fibrils have been formed from Bence Jones proteins and immunoglobulin light chains in vitro (Glenner et al., 1971; Tan and Epstein, 1972) by proteolytic digestion. Synthetic /3-pleated sheet fibrils have been formed also from insulin and glucagon, in acid solution at moderately elevated temperatures (Glenner et al., 1974). Glucagon is a molecule with considerable conformational flexibility; in dilute neutral solution it exists in a random coil conformation (Panijpan and Gratzer, 1974), in alkaline solution or in the presence of detergents it adopts an ac-helical conformation (Bornet and Edelhoch, 1971) and in acid solution it forms gels and eventually antiparallel /3-pleated sheet fibrils (Beaven, Gratzer and Davis, 1969). The kinetics of gel and fibril formation from glucagon are sensitive to protein and salt concentration, and temperature, as well as to pH. This communication is concerned with the conformational flexibility of SAA, the putative precursor of amyloid AA fibrils
583
of unknown origin. SAA has been studied intensively since it was identified by Levin and coworkers (1973) in the serum of patients with chronic diseases. SAA has been detected as well in mouse (Isersky et al., 1971), guinea pig (Skinner et al., 1974) and in mink (Anders et al., 1976) with antibodies raised to denatured AA fibrils from the corresponding species. SAA levels are often found to be elevated in patients with chronic and neoplastic diseases, although very few of these patients develop secondary amyloidosis (Rosenthal and Franklin, 1975). SAA levels have been shown to rise within a few hours of an acute phase stimulus (McAdam and Sipe, 1976). The cell of origin and function of SAA are unknown; however it has been reported that murine SAA has immunosuppressive activity (Benson et al., 1975). The structure of SAA is also unknown; the native species has a mol. wt. of 160,000 to 180,000 (Sipe et al., 1976) which has not been isolated to date. SAA has been most frequently purified as a species (designated SAAL) of approximately 12,500 mol. wt., by gel filtration of serum or serum fractions in guanidine hydrochloride or formic acid (Linke et al., 1975; Anders et al., 1975). The first 11 NH2terminal amino acids are identical in SAAL and AA (Rosenthal et al., 1976), supporting the idea that the 12,500 mol. wt. SAAL is the precursor of the 5300-8500 AA fragment found in tissues. It is possible that SAA consists of SAAL complexed through noncovalent bonds to a high mol. wt. polypeptide which does not cross-react with anti-AA antibodies, or that SAA is a polymer of SAAL moieties arranged in an unknown fashion. We now report evidence that SAA exhibits conformational flexibility. During storage of serum at 40, AA crossreacting species of heterogeneous mol. wt. are formed and AA cross-reacting determinants are exposed during this process. Because this conformational flexibility may play an important role in the induction of the /-pleated sheet confor-
584
J. D. SIPE, K. P. W. J. McADAM, B. F. TORAIN AND G. G. GLENNER
mation prior to fibril deposition, we have compared SAA from patients with and without secondary amyloidosis, and have found that both types of precursor exhibit conformational flexibility. However, there are differences in susceptibility of the two types of SAA to SAAL formation. SAA from patients with secondary amyloidosis is less readily dissociated to SAAL by gel filtration in guanidine hydrochloride or formic acid than is SAA from patients without amyloidosis, under the same conditions. Furthermore, isolated SAAL preparations from patients with amyloidosis, although similar in immunoreactivity to that from patients without amyloidosis, have a markedly greater tendency toward reaggregation even in the presence of formic acid.
except that column dimensions were 3 x 90 cm. Those fractions from the low mol. wt. region on the chromatographic profile which were antigenically reactive in radioimmunoassay were pooled and concentrated by dialysis vs polyethylene glycol or by vacuum rotary evaporation at room temperature. Protein determinations were made according to the method of Lowry, Rosebrough and Farr (1951) using crystalline bovine serum albumin as standard. Sodium dodecyl sulphate polyacrylamide gel electrophoresis.-SAAL isolated by gel filtration in 10% formic acid and concentrated in the presence of formic acid to 0-1-0-5 mg/ml was further concentrated under a stream of nitrogen immediately prior to electrophoresis in 10% polyacrylamide gels in 0-1% sodium dodecyl sulphate, at pH 7-1 according to the method of Weber and Osborn (1969). Dithiothreitol (0-1%) was added to the sample solution to dissociate possible disulphide polymers in the sample. RESULTS
MATERIALS AND METHODS
SAA-containing serum.-Serum from patient PEK with Reiter's syndrome but without clinical evidence of amyloidosis was stored at 40 in the presence of thymol as preservative. Serum from patient AMB with amyloidosis secondary to leprosy was initially frozen for transit, and was then stored at 40 in the presence of thymol as preservative. Serum was not frozen during storage because it has been observed that SAA precipitates upon thawing after freezing and not all of the precipitate can be dissolved. Radioimmunoassay.-Both SAA and SAAL were determined by their cross-reaction in the solid phase radioimmunoassay for the tissue fibril protein AA as previously described (Sipe et al., 1976). Duplicate determinations were carried out on all samples. When samples were denatured in formic acid prior to assay, or when gel filtration samples eluted in formic acid were to be assayed, the acid was diluted to less than 4% (v/v) with water, frozen in dry ice and lyophilized. Preparation of SAA.-SAA was isolated from whole serum by gel filtration on Sephadex G-200 in 0-1 M phosphate-buffered saline pH 7-2 (Sipe et al., 1976). Aliquots of each fraction were tested by radioimmunoassay for crossreactivity with the tissue amyloid protein AA. The AA cross-reacting fractions were pooled and concentrated by negative pressure dialysis to a final volume of 2-4 ml. Preparation of SAAL.-SAAL was obtained by gel filtration of SAA isolated from whole serum on Sephadex G-100 in 10% (v/v) formic acid as previously described (Sipe et al., 1976),
Change in molecular weight distribution of SAA upon storage When the mol. wt. of SAA from the serum of patient PEK (without amyloidosis) was determined by gel filtration of fresh serum on Sephadex G-200 in 0-1 M phosphate-buffered saline, pH 7-2, it was approximately 160,000 as determined by its coelution with the IgG fraction of serum (Fig. la). When the fractionation was repeated under identical conditions after 5 months' storage of serum PEK at 40 in the presence of thymol as preservative, AA cross-reacting species of heterogeneous mol. wt. were observed (Fig. lb). Almost one-half of the observed immunoreactivity was in the void volume fractions after gel filtration of the stored serum, and a small fraction of AA cross-reacting material was also detected in the low mol. wt. region between albumin and the salt boundary, whereas no immunoreactivity was detected in these regions upon separation of fresh serum. Low mol. wt. AA-cross-reactivity under physiological conditions (Fig. 2) has also been observed in the serum from patient AMB with amyloidosis secondary to leprosy, which had been stored for several months at 40. Fresh serum from this patient was not
585
THE SERUM AMYLOID PRECURSOR SAA
300
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FIG. la.-Top: Descending Sephadex G-200 gel filtration of 2 ml of serum PEK carried out within 24 h after collection from the patient and removal of the fibrin clot by centrifugation. The serum was applied to the chromatographic column (110 x 2-5 cm) equilibrated with PBS and eluted at a flow rate of 15 ml/h with 2*5 ml fractions collected. The AA cross-reactivity was determined by diluting 100 ,ul of each fraction with 400 jul of 2% casein in 0 1 M barbital buffer, pH 8-6, and testing *, absorbance at 280 nm; 0 duplicate 200-,ul aliquots in RIA (II). * QO, AA crossreactivity in ng/fraction. FIG. lb.-Bottom: Descending Sephadex G-200 gel filtration of 2 ml of serum PEK after storage for approximately 5 months at 40 in the presence of thymol as preservative. Conditions for separation and assay are identical to those described above.
available; however, separation of serum within a few days of collection from a patient with amyloidosis secondary to rheumatoid arthritis showed only the 160,000 mol. wt. SAA species, and none of the void volume or post-albumin crossreacting material.
Increased AA cross-reactivity of serum upon storage When radioimmunoassay (RIA) inhibition curves were constructed from serial dilutions in casein barbital dilution buffer of serum PEK, they yielded an SAA concentration of 0.3 jig/ml when the serum was fresh and 8 ,ug/ml when the serum was 5 months old. The SAA concentration was determined as AA cross-reactivity by comparing the 50 % inhibition points of the RIA inhibition curves of AA and serum
PEK. Similarly, untreated serum AMB was found to increase in immunoreactivity from 0.4 to 20 ,tg/ml during 6 months of storage in the laboratory. Increased AA cross-reactivity upon denaturation of serum.-Because of the increases in immunoreactivity of sera upon storage, it seemed advantageous to minimize this effect by denaturation of serum samples before determination of their SAA levels by RIA. During the course of isolating SAAL by formic acid dissociation (Sipe et al., 1976), it was observed that there was a 150-fold increase in immunoreactivity relative to whole serum upon purification of the low mol. wt. species. Therefore, the effects of length of time and temperature of formic acid incubation on the AA cross-reactivity of serum AMB were investigated (Fig. 3).
586
J. D. SIPE, K. P. W. J. MCADAM, B. F. TORAIN AND G. G. GLENNER
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FIG. 2.- Descending Sephadex G-200 gel filtration of 3 ml of serumn AMB after approximately 5 months of storage at 4° in the presence of thymol as preservative. Conditions of separation and analysis are as described in Fig. 1, except that elution of fractions was interrupted for approximately 8 h in the region of fraction 60. Uninterrupted elution yielded a similar, but less well defined peak in this region.
Dilution of 1 ,tl of serum with 500 /il of 10% formic acid followed by immediate lyophilization increased the observed immunoreactivity from 20 ,ug/ml to 40 ,tg/ml, and there was no further increase over 72 h at 0°. Further increases in immunoreactivity were observed when incubation was carried out at 25° and 37°, however, and the highest value measured was about 120 ,tg/ml after 72 h of incubation at 37°. It was found that reproducible inhibition curves could be constructed from serum AMB and serum PEK by making serial dilutions of the sera in 10% formic acid, incubating them at 37° for 24 h, removing the formic acid by lyophilization
and assaying the lyophilized material by RIA (examples of these curves are shown in Fig. 4). By coincidence, these two sera have similar SAA levels after formic acid denaturation, which, as calculated from the 5000 inhibition points of the serum and AA inhibition curves, is 180 ,ag/ml of AA cross-reactivity. The SAA levels of the denatured sera are significantly greater than the 20 and 8 jig/ml observed for the untreated sera. Dissociation of SAA to SAAL in amyloid and nonamyloid sera When the SAA fraction isolated from serum PEK by neutral Sephadex G-200
587
THE SERUM AMYLOID PRECURSOR SAA
gel filtration was applied without pH adjustment to a Sephadex G-100 column equilibrated with 10% formic acid, all of the AA cross-reactivity was dissociated from the nonimmunoreactive serum pro-
teins which were eluted in the void volume fractions (Fig. 5a). Analysis of this SAAL preparation by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulphate showed it to be of similar
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INCUBATION TIME, HOURS FIG. 3.-Effect of length of time and temperature of formic acid denaturation on AA cross-reactivity of serum AMB. A dilution of serum AMB was made by mixing 40 PI of serum with 20 ml of 10% formic acid. Aliquots of 500 ,1 each were incubated in tightly stoppered tubes for varying times, after which 2 ml of water was added, the tubes were frozen, and the formic acid was removed by lyophilization. RIA was carried out on each lyophilized sample by adding 100 PI 0-1 M barbital buffer pH 8 6 and 400 ,ul 2% casein in the same barbital buffer (II). Immunoreactivi.ty is expressed in terms of AA cross-reactivity/ml of undiluted serum. #PWR. Vwi* .i
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FIG. 4.-Radioimmunoassay inhibition curves showing cross-reactivity of SAA and SAAL with AA. DuwMicate determinations were carried out using the indicated ng of SAAL: * -*, PEK; O, AMB; a, PEK and *--*, AMB; A- -A, AA; ,ul of undenatured serum: O Q, AMB. The inhibition curve and ,ul of formic acid denatured serum: C] E, PEK;and O of serum PEK shown was determined in a separate experiment from the others. Hence the mean of two duplicate inhibition curves for AA and SAAL-AMB is plotted and the horizontal lines represent the values for the individual experiments.
J. D. SIPE, K. P. W. J. MCADAM, B. F. TORAIN AND G. G. GLENNER
588
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FiG. 5a.-Descendirng gel filtration on Sephadex G-100 in 10% (v/v) formic acid of the SAA containing absorbance at 280 nm; fractions from Sephadex G-200 PBS gel filtration of serum PEK: * 0, AA cross-reactivity ,sg/fraction. The SAA containing fractions were concentrated to 0 approximately 3 ml by negative pressure dialysis. The concentrated fraction was applied to a 3 x 90-cm column of Sephadex G-100 equilibrated in 10% formic acid and fractions of 4-1 ml were collected at a flow rate of 10 ml/h. The AA cross-reactivity of each fraction was determined from a 50-,ul aliquot which was diluted with water, lyophilized and tested by RIA. 3.0
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FIG. 5b.-Descending gel filtration on Sephadex G-100 in 10% (v/v) formic acid of the SAA containing fractions from Sephadex G-200 PBS gel filtration of serum AMB. Conditions for separation and assay are the same as those described above.
THE SERUM AMYLOID PRECURSOR SAA
mobility (Fig. 6) to SAAL previously determined to be 12,500 in mol. wt. (Linke et al., 1975). The same treatment of the SAA fraction from serum AMB, however, results in only partial dissocia-
589
tion of the AA cross-reacting species from the other serum proteins which are eluted in the void volume. Analysis of the isolated SAAL by polyacrylamide gel electrophoresis showed in addition to the
FIG. 6. Polyacrylamide (10%) electrophoresis in 0-1% sodium dodecyl sulphate of SAAL preparations from serum AMB and PEK. Left: 40 ,ug SAAL-AMB (Fractions 59-73, Fig. 5b), Middle: 40 ,ug SAAL-PEK (Fraction 62-77, Fig. 5a), Right: mol. wt. markers (a) bovine serum albumin (67,000), (b) chymotrypsinogen (24,000), (c) cytochrome c (13,000), and (d) insulin (6000).
590
J. D. SIPE, K. P. W. J. McADAM, B. F. TORAIN AND G. G. GLENNER
expected low mol. wt. band, two bands which correspond to mol. wt. of approximately 25,000 and 75,000 (Fig. 6). In view of the degree of resolution evident in the gel filtration profile, these high mol. wt. bands would appear to have derived from material that was of lower mol. wt. at the time of formic acid gel filtration. High mol. wt. material of this type has also consistently been observed in SAAL preparations from patients with secondary amyloidosis which have been prepared by gel filtration on Sephadex G-100 in guanidine hydrochloride. In spite of their differences in susceptibility to dissociation, the SAAL preparations from the two types of sera show very similar RIA inhibition curves (Fig. 4), thus supporting the use of RIA to compare SAA levels in various types of disease sera. DISCUSSION
While the structure of SAA has not yet been elucidated, certain aspects of its nature have become apparent. SAA appears to consist of 12,500 mol. wt. SAAL species arranged in a labile, easily dissociable structure of approximately 160,000 mol. wt. It would appear that all of the AA cross-reacting determinants contained in SAA are due to the low mol. wt. material, since SAA from the serum of a patient without amyloidosis was completely dissociated to SAAL by gel filtration on Sephadex G-100 in formic acid. The existence of an AA crossreacting polypeptide chain intermediate in mol. wt. to SAAL and SAA cannot be excluded in patients with secondary amyloidosis. However, since the proportion of SAA dissociating to SAAL has varied between individual separations of the same serum sample, from patients with or without amyloidosis, it seems likely that the immunoreactivity observed in the void volume fractions after gel filtration on Sephadex G-100 in guanidine hydrochloride or formic acid represents undissociated SAA or aggregated SAAL. The arrangement of SAAL polypep-
tides in SAA is unknown. It may be that one or two SAAL proteins are complexed to a high mol. wt. protein which does not cross-react with anti-AA antibodies, or alternatively, SAA may be a polymer of SAAL chains which contains in addition, low mol. wt. nonantigenic material such as peptides or lipid. It appears that SAAL is contained in SAA in a conformation such that it is soluble and can be dissociated. Dissociation of SAAL from SAA occurs upon storage or various experimental manipulations of serum, and during this process determinants which cross-react with anti-AA antibodies are exposed. It seems highly significant that SAAL aggregates easily and is then difficult to dissociate when it has a conformation such that antigenic determinants which cross-react with antibodies raised to the insoluble fibrils deposited in tissues are maximally exposed. We have observed that SAAL preparations from both serum PEK and serum AMB migrate poorly into sodium dodecyl sulphate polyacrylamide gels after lyophilization, and we have found it necessary to concentrate and store these proteins in the presence of 10% formic acid, removing water and acid under a stream of nitrogen immediately before electrophoresis is carried out. While denaturation of SAA-containing serum and tissue seems advantageous from the standpoint of increased sensitivity and more reproducible RIA determinations, the problem of determining with precision the SAAL content of a serum sample remains. Complete exposure of AA cross-reacting determinants would appear both to be difficult to achieve, and to ascertain when it had been achieved. When denaturation was carried out for 24 h at 370 at a ratio of serum-toformic-acid of 0.002, the observed AA cross-reactivity was approximately 90 ,tg/ml (Fig. 3). When it was carried out at a serum-to-acid ratio of 0*00025 (Fig. 4), the observed immunoreactivity was approximately 180 ,lig/ml of AA. The observation that reproducible inhibition
THE SERUM AMYLOID PRECURSOR SAA
curves can be constructed after treatment of serum dilutions with formic acid for 24 h at 370 supports the use of formic acid denaturation for comparing SAA levels in different serum samples. Thus it appears that SAA proteins in amyloid and nonamyloid sera share certain characteristics, one of which is a tendency to undergo conformational changes which result in the formation of heterogeneous mol. wt. SAA species and in an increased oxposure of antigenic determinants which are common with the tissue fibril proteins. Amyloid and nonamyloid SAA are also similar in that each can be dissociated to a low mol. wt. fraction, SAAL, and the two types of SAAL are identical in immunoreactivity (Fig. 4). However, there are differences between amyloid and nonamyloid SAA proteins, in that amyloid SAA has consistently been more resistant to dissociation to SAAL. Furthermore, amyloid and nonamyloid SAAL preparations show different mol. wt. distributions when examined by SDS polyacrylamide gel electrophoresis; the amyloid SAAL contains material which aggregates under strong dissociating conditions. There is a need, therefore, to examine SAAL preparations from the two types of sera thoroughly. Complete amino acid sequence determinations on both amyloid and nonamyloid SAAL may reveal sequence heterogeneity near the carboxyl portions of the proteins as being important in amyloidogenesis, despite the similarities in the amino terminal sequence. The degree of homogeneity of SAAL needs to be established also, since the presence of only small amounts of nonimmunoreactive proteins of similar mol. wt. to the SAAL protein would be difficult to detect, but such proteins might play an important role in the aggregation of SAAL. The present studies have employed antibodies raised to a 5300 mol. wt., 45 amino acid residue polypeptide AA which presumably arises by proteolytic digestion of the 12,500 mol. wt. SAAL, consisting of approximately 114 residues. If there are some determinants in SAAL which are
591
completely exposed in the native SAA species, use of 125SAAL and anti-SAAL, when available, may facilitate detection of SAA without denaturation. However, the use of antibodies raised to the AA fragment has provided considerable information about the flexibility of the SAA species. This flexibility may play a role in the deposition of insoluble P-pleated sheet amyloid fibrils in some patients with recurrent acute or chronic inflammatory conditions. We thank Miss Janice Ryan for capable assistance with all aspects of this work. K. M. was the recipient of a Medical Research Council (U.K.) Travelling Fellowship. REFERENCES ANDERS, R. F., NATVIG, J. B., MICHAELSEN, T. E. & HUSBY, G. (1975) Isolation and Characterization of Amyloid-related Serum Protein SAA as a Low Molecular Weight Protein. Scand. J. Immunol., 4, 397. ANDERS, R. F., NORDSTOGA, K., NATVIG, J. B. & HUSBY, G. (1976) Amyloid-related Serum Protein SAA in Endotoxin Induced Amyloidosis of the Mink. J. exp. Med., 143, 678. BEAVEN, G. H., GRATZER, W. B. & DAVIEs, H.G. (1969) Formation and structura of Gals and Fibrils from Glucaoon. Eur. J. Biochem., 11, 37. BENDITT, E. P., ERIKSEN, N., HERMODSON, M. A. & ERICSSON, L. H. (1971) The Major Proteins of Human and Monkey Amyloid Substance: Common Properties Including Unusual N-terminal Amino Acid Sequences. FEBS Lett., 19, 169. BENSON, M. D., ALDO-BENsoN, M. A., SHIRAHAMA, T., BOREL, Y. & COHEN, A.S. (1975) Suppression of in vitro Antibody Response by a Serum Factor (SAA) in Experimentally Induced Amyloidosis. J. exp. Med., 142, 236. BORNET, M. & EDELHOCH, H. (1971) Polypeptide Hormone Interaction. J. Biol. Chem., 246, 1785. DAVIES, D. R., PADLAN, E. A. and SEGAL, D. M. (1975) Three-Dimensional Structure of Immunoglobulins. Ann. Rev. Biochem., 44, 639. GLENNER, G. G., EANES, E. D., BLADEN, H. A., LINKE, R. P. & TERMINE, J. D. (1974) f-Pleated Sheet Fibrils: A Comparison of Native Amyloid with Synthetic Protein Fibrils. J. Histochem. Cytochem., 22, 1141. GLENNER, G. G., EIN, D., EANES, E. D., BLADEN, H. A., TERRY, W. D. & PAGE, D. (1971) The Creation of " Amyloid ' Fibrils from Bence Jones Proteins in vitro. Science, N. Y., 174, 712. GLENNER, G. G. & PAGE, D. L. (1976) Amyloid, Amyloidosis and Amyloidogenesis. Int. Rev. exp. Pathol., 15, 1. GLENNER, G. G., TERRY, W. D. & ISERSKY, C. (1973) Amyloidosis: Its Nature and Pathogenesis. Semin. Hematol., 10, 65.
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