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A Protein AA-Variant Derived from a Novel Serum AA Protein, SAA1 δ,in an Individual from Papua New Guinea
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 320–323 (1996)
0892
A Protein AA-Variant Derived from a Novel Serum AA Protein, SAA1 d, in an Individual from Papua New Guinea Per Westermark,* Knut Sletten,† Gunilla T. Westermark,* John Raynes,‡ and Keith P. W. J. McAdam‡,§ *Department of Pathology I, University Hospital, Linköping, Sweden; †Department of Biochemistry/Biotechnology Center of Oslo, University of Oslo, 0316 Oslo, Norway; ‡Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; and §Medical Research Council Laboratories, Fajara, Banjul, Gambia Received April 21, 1996 A major protein AA amyloid protein was purified and characterised from a Papua New Guinean individual. This AA protein differed from all previously characterised SAA variants by the combination of Ala52, Val57, Asn60, Phe68, Phe69 and Gly72. Since the prevalence of AA-amyloidosis is unusually high in Papua New Guinea this AAd must originate from a novel SAAd which may represent a particularly amyloidogenic variant. © 1996 Academic Press, Inc.
AA- (secondary or reactive) systemic amyloidosis occurs as a usually late sequel of several chronic inflammatory diseases, in developed countries most commonly arthritides and in other countries certain infectious diseases such as tuberculosis, leprosy and malaria (1, 2). The precursor protein of the fibril in AA amyloidosis is serum amyloid A protein (SAA) which is a normal apolipoprotein of the high density lipoprotein in plasma. SAA increases in plasma concentration by up to 1000 fold following severe acute inflammation. SAA constitutes a family of proteins and in man two major forms, SAA1 and SAA2, with some distinctive allelic variants are found in plasma as acute phase reactants (for review, see (3)) produced by the liver. Healthy individuals have low levels of SAA and this includes a high proportion of constitutively synthesised SAA (cSAA) which is coded for by a third SAA gene. In the pathogenesis of AA-amyloidosis, long term high plasma levels of SAA are needed but since not all individuals fulfilling this criterion develop amyloidosis, other factors are also necessary. In other forms of amyloidosis, e.g. familial transthyretin amyloidosis, a variant protein of higher fibrillogeneity exists. A particular isoform in the mouse (SAA2) is preferentially deposited into fibrils but there is no similar preference in human AA amyloid. Although the human AA fibril protein is largely derived from SAA1a, this may reflect the fact that this SAA variant is present in higher concentrations than other SAA forms. In Papua New Guinea there is a high frequency of expression of SAA2b isoforms (4) but there is no indication that this is associated with a risk of AA-amyloidosis. In Familial Mediterranean Fever (FMF), amyloid AA derived from both SAA1 and SAA2 has been described (5, 6). Three alleles of SAA1 (SAA1a, b, g) and 2 of SAA2 (SAA2a, b) have been described (3). In the present paper we describe a protein AA with an amino acid sequence clearly showing that it must be derived from an until now undescribed SAA variant, SAA1d, which may be associated with a high prevalence of AA-amyloidosis. In Papua New Guinea 7% of over 1000 autopsies revealed amyloidosis on histology (7) and in some isolated areas 10% of the population were found to have positive biopsies (1). The biochemical basis of this AA amyloidosis (8) has not previously been elucidated. MATERIALS AND METHODS Amyloid fibrils were extracted as described (9) from the amyloid-laden kidney of patient Kop who died with systemic amyloidosis. Kop lived in Asaro, near Goroka in the Eastern Highlands of Papua New Guinea and presented with nephrotic 320 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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syndrome aged about 40. He had an enlarged thyroid and when he died in renal failure, post mortem examination revealed extensive replacement of renal and thyroid tissue with amyloid. Fibrils were defatted in chloroform-methanol (2:1), dissolved in 6 M guanidine HCl in 0.1 M Tris HCl, pH 8.0, containing 0.1 M EDTA and 0.1 M dithiothreitol and gelfiltered through a Sepharose 6B-CL column, equilibrated with 5 M guanidine HCl in distilled water. Elution was performed with this solution and monitored at 280 nm. Pooled fractions were precipitated with saturated ammonium sulphate, dialyzed against deionized water, lyophilized and subjected to a second gel filtration through a Sephacryl S300 HR column, other conditions being as above. Fractions were precipitated, dialyzed and lyophilized as above. Analytic procedures. Sodium dodecyl sulphate 10–20% gradient polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Blobel and Dobberstein (10). Western blot analysis (11) was performed as described (12) with the use of two different rabbit antisera against synthetic peptides, corresponding to human SAA, residues 24–34 and 91–104, respectively. Anti SAA24–34 recognises all human protein AA variants while anti SAA91–104 only reacts with amyloid containing full-length SAA. Protein cleavage. Cyanogen bromide cleavage (13) and cleavage with BNPS-skatole (14) was performed as described earlier. The protein was also digested with protease V-8 for 21 hours at +37°C followed by reversed phase high performance liquid chromatography on a Pep.S. C2/C18 column (15). Amino acid sequence analysis. N-terminal amino acid sequence analysis was performed with an automatic protein sequencer (477 A, Applied Biosystems, Perkin Elmer, Foster City, CA) coupled to a PTH amino acid analyser (120A, Applied Biosystems).
RESULTS SDS-PAGE of extracted amyloid fibrils showed one strongly predominating band corresponding to a Mr of less than 10 kDa preceded by a weaker band. Gel filtration of dissolved amyloid fibrils revealed the usual AA-pattern with one major retarded peak preceded by two small peaks. Nterminal amino acid sequence analysis of the major peak protein revealed a sequence typical of protein AA but lacking the N-terminal arginine residue of SAA. The same material was cleaved by cyanogen bromide and run for 17 steps before cleavage with BNPS-skatole and sequenced for another 17 steps. The results confirmed earlier data for positions 1 to 40 and established the sequence of positions 54 to 70 (Fig. 1). N-terminal sequence of a more retarded protein fraction just preceding the major protein AApeak at gel filtration, was run for 23 steps which confirmed the sequence from above. The same material was then used for cyanogen bromide cleavage and sequenced for another 30 steps which established the sequence of positions 24 to 53. The same material was then used for cleavage with BNPS-skatole and put back in the sequencer for another 11 steps establishing the sequence of positions 53 to 63. In order to reveal the complete sequence, protein AA was digested with protease V-8 and the
FIG. 1. Amino acid sequences of positions 1–76 of the different human SAA variants that have been described compared to the primary structure of the AA-protein described in this study. 321
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
derived peptides were separated on a reverse phase column. Four peptide fractions covering positions 1 to 8, 9 to 25, 26 to 55 and 63 to 75 were purified and characterised by Edman degradation. Two other fractions were found to contain a mixture of peptides starting in positions 9, 26 and 63 and positions 16 and 26, respectively. The data established the complete sequence corresponding to position 2–76 of SAA1 and SAA2 (Fig. 1). Western blot analysis of amyloid fibrillar material revealed reaction with anti SAA24–34 but not with anti SAA91–104 consistent with the complete lack of a C-terminal part of SAA in the amyloid fibrils. As is seen in Fig. 1 there are phenylalanine residues at positions 68–69, indicating an origin from SAA1 since SAA2 has Leu-Thr at these positions. There are three known allelic forms of SAA1, differing from each other by variations at positions 52, 57, 60 and 72. The combination of Ala52, Val57, Asn60 and Gly72 is not seen in any of these (Fig. 1). Protein AA resembles most closely SAA1b and SAA1g from both of which it differs by two amino acid residues. DISCUSSION The results of the present study have identified a novel protein AA-variant in a patient from an area in Papua New Guinea with an unusually high prevalence of AA-amyloidosis (16). This AA-variant differs from other known AA/SAA-proteins at positions previously known to vary between SAA forms. This Papuan protein AA comes from a fourth SAA1 form, SAA1d. Each SAA and protein AA variant occurs in two forms, one with an N-terminal arginine residue and one des-arginine form. In protein AA, both forms are usually found, except for the long AA-variants associated with vascular amyloidosis (17). In the present material with an AA-protein of the most common length, only des-arg AA was identified. It is known from certain familial amyloidoses, e.g. familial transthyretin amyloidosis that single amino acid substitutions render amyloidogenic properties to the fibril protein. In the mouse, two major SAA variants (SAA 1 and 2) are synthesised by the liver as acute phase reactants and at approximately equal amounts, but only SAA2 is deposited as amyloid (18, 19). In humans, all of the known SAA variants are shown to give rise to AA-protein and amyloid (3, 13, 20) and, as a matter of fact, protein AA obviously derived from a mixture of SAA isoforms has been demonstrated at several times in amyloid deposits (21, 22). However, also in humans particularly amyloidogenic AA-variants may occur since expression of SAA1g is associated with an increased risk for AA-amyloidosis (23) although protein AA derived from this species has only rarely been demonstrated in amyloid deposits (21, 22). Given the unusually high prevalence for AAamyloidosis in the Papua New Guinean population to which patient Kop belonged, SAA1d may represent an especially amyloidogenic variant. This finding therefore offers one possible explanation for the susceptibility of certain Papua New Guinean populations for systemic AA-amyloidosis. The finding in the present study therefore is a strong argument for further studies of AA-protein and SAA variants in this population. ACKNOWLEDGMENTS K.P.W.J. McAdam worked at the Papua New Guinea Institute of Medical Research in Goroka. Further studies were supported by the American Kidney Fund, the Mae J. Drielsma Memorial Fund, the Swedish Medical Research Council, the Swedish Medical Society, and the Research Council of Norway.
REFERENCES 1. McAdam, K. P. W. J. (1978) Papua New Guinea Med. J. 21, 69–78. 2. McAdam, K. P. W. J., Raynes, J. G., Alpers, M. P., Westermark, G. T., and Westermark, P. Papua New Guinea Med. J. (in press). 3. Husby, G., Marhaug, G., Dowton, B., Sletten, K., and Sipe, J. D. (1994) Amyloid: Int. J. Exp. Clin. Invest. 1, 119–137. 4. Raynes, J. G., Hultquist, R., Molyneux, M., Griffin, G., and McAdam, K. P. W. J. (1991) in Amyloid and Amyloidosis (Natvig, J. B., Førre, Ø., Husby, G., Husebekk, A., Skogen, B., Sletten, K., and Westermark, P., Eds.), pp. 119–123. Kluwer Academic, Dordrecht. 322
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Vol. 223, No. 2, 1996 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
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Levin, M., Franklin, E. C., Frangione, B., and Pras, M. (1972) J. Clin. Invest. 51, 2773–2776. Prelli, F., Pras, M., Shtrasburg, S., and Frangione, B. (1991) Scand. J. Immunol. 33, 783–786. Cooke, R. A., and Champness, L. T. (1970) Med. J. Austral. 2, 1177–1184. Anders, R. F., Price, M. A., Wilkey, I. S., Husby, G., Takitaki, F., Natvig, J. B., and McAdam, K. P. W. J. (1976) Clin. Exp. Immunol. 24, 49–53. Pras, M., Schubert, M., Zucker-Franklin, D., Rimon, A., and Franklin, E. C. (1968) J. Clin. Invest. 47, 924–933. Blobel, G., and Dobberstein, B. (1975) J. Biol. Chem. 67, 835–851. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. Gustavsson, Å., Engström, U., and Westermark, P. (1994) Am. J. Pathol. 144, 1301–1311. Sletten, K., and Husby, G. (1974) Eur. J. Biochem. 41, 117–125. Fontana, A. (1972) Meth. Enzymol. 25, 419–423. Syversen, P. V., Juul, J., Marhaug, G., Husby, G., and Sletten, K. (1994) Scand. J. Immunol. 39, 88–94. Anders, R. F., Price, M. A., and Wilkey, I. S. (1976) Trop. Geogr. Med. 28, 273–282. Westermark, G. T., Sletten, K., and Westermark, P. (1989) Scand. J. Immunol. 30, 605–613. Meek, R. L., Hoffman, J. S., and Benditt, E. P. (1986) J. Exp. Med. 163, 499–510. Shiroo, M., Kawahara, E., Nakanishi, I., and Migita, S. (1987) Scand. J. Immunol. 26, 709–716. Westermark, G. T., Sletten, K., Grubb, A., and Westermark, P. (1990) Am. J. Pathol. 137, 377–383. Møyner, K., Sletten, K., Husby, G., and Natvig, J. B. (1980) Scand. J. Immunol. 11, 549–554. Baba, S., Takahashi, T., Kasama, T., and Shirasawa, H. (1992) Biochim. Biophys. Acta 1180, 195–200. Baba, S., Masago, S. A., Takahashi, T., Kasama, T., Sugimura, H., Tsugane, S., Tsutsui, Y., and Shirasawa, H. (1995) Hum. Mol. Genet. 4, 1083–1087. Dwulet, F. E., Wallace, D. K., and Benson, M. D. (1988) Biochemistry 27, 1677–1682. Beach, C. M., de Beer, M. C., Sipe, J. D., Loose, L. D., and de Beer, F. C. (1992) Biochem. J. 282, 615–620. Kluve-Beckerman, B., Dwulet, F. E., and Benson, M. D. (1988) J. Clin. Invest. 82, 1670–1675.
323
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 320–323 (1996)
0892
A Protein AA-Variant Derived from a Novel Serum AA Protein, SAA1 d, in an Individual from Papua New Guinea Per Westermark,* Knut Sletten,† Gunilla T. Westermark,* John Raynes,‡ and Keith P. W. J. McAdam‡,§ *Department of Pathology I, University Hospital, Linköping, Sweden; †Department of Biochemistry/Biotechnology Center of Oslo, University of Oslo, 0316 Oslo, Norway; ‡Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; and §Medical Research Council Laboratories, Fajara, Banjul, Gambia Received April 21, 1996 A major protein AA amyloid protein was purified and characterised from a Papua New Guinean individual. This AA protein differed from all previously characterised SAA variants by the combination of Ala52, Val57, Asn60, Phe68, Phe69 and Gly72. Since the prevalence of AA-amyloidosis is unusually high in Papua New Guinea this AAd must originate from a novel SAAd which may represent a particularly amyloidogenic variant. © 1996 Academic Press, Inc.
AA- (secondary or reactive) systemic amyloidosis occurs as a usually late sequel of several chronic inflammatory diseases, in developed countries most commonly arthritides and in other countries certain infectious diseases such as tuberculosis, leprosy and malaria (1, 2). The precursor protein of the fibril in AA amyloidosis is serum amyloid A protein (SAA) which is a normal apolipoprotein of the high density lipoprotein in plasma. SAA increases in plasma concentration by up to 1000 fold following severe acute inflammation. SAA constitutes a family of proteins and in man two major forms, SAA1 and SAA2, with some distinctive allelic variants are found in plasma as acute phase reactants (for review, see (3)) produced by the liver. Healthy individuals have low levels of SAA and this includes a high proportion of constitutively synthesised SAA (cSAA) which is coded for by a third SAA gene. In the pathogenesis of AA-amyloidosis, long term high plasma levels of SAA are needed but since not all individuals fulfilling this criterion develop amyloidosis, other factors are also necessary. In other forms of amyloidosis, e.g. familial transthyretin amyloidosis, a variant protein of higher fibrillogeneity exists. A particular isoform in the mouse (SAA2) is preferentially deposited into fibrils but there is no similar preference in human AA amyloid. Although the human AA fibril protein is largely derived from SAA1a, this may reflect the fact that this SAA variant is present in higher concentrations than other SAA forms. In Papua New Guinea there is a high frequency of expression of SAA2b isoforms (4) but there is no indication that this is associated with a risk of AA-amyloidosis. In Familial Mediterranean Fever (FMF), amyloid AA derived from both SAA1 and SAA2 has been described (5, 6). Three alleles of SAA1 (SAA1a, b, g) and 2 of SAA2 (SAA2a, b) have been described (3). In the present paper we describe a protein AA with an amino acid sequence clearly showing that it must be derived from an until now undescribed SAA variant, SAA1d, which may be associated with a high prevalence of AA-amyloidosis. In Papua New Guinea 7% of over 1000 autopsies revealed amyloidosis on histology (7) and in some isolated areas 10% of the population were found to have positive biopsies (1). The biochemical basis of this AA amyloidosis (8) has not previously been elucidated. MATERIALS AND METHODS Amyloid fibrils were extracted as described (9) from the amyloid-laden kidney of patient Kop who died with systemic amyloidosis. Kop lived in Asaro, near Goroka in the Eastern Highlands of Papua New Guinea and presented with nephrotic 320 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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syndrome aged about 40. He had an enlarged thyroid and when he died in renal failure, post mortem examination revealed extensive replacement of renal and thyroid tissue with amyloid. Fibrils were defatted in chloroform-methanol (2:1), dissolved in 6 M guanidine HCl in 0.1 M Tris HCl, pH 8.0, containing 0.1 M EDTA and 0.1 M dithiothreitol and gelfiltered through a Sepharose 6B-CL column, equilibrated with 5 M guanidine HCl in distilled water. Elution was performed with this solution and monitored at 280 nm. Pooled fractions were precipitated with saturated ammonium sulphate, dialyzed against deionized water, lyophilized and subjected to a second gel filtration through a Sephacryl S300 HR column, other conditions being as above. Fractions were precipitated, dialyzed and lyophilized as above. Analytic procedures. Sodium dodecyl sulphate 10–20% gradient polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Blobel and Dobberstein (10). Western blot analysis (11) was performed as described (12) with the use of two different rabbit antisera against synthetic peptides, corresponding to human SAA, residues 24–34 and 91–104, respectively. Anti SAA24–34 recognises all human protein AA variants while anti SAA91–104 only reacts with amyloid containing full-length SAA. Protein cleavage. Cyanogen bromide cleavage (13) and cleavage with BNPS-skatole (14) was performed as described earlier. The protein was also digested with protease V-8 for 21 hours at +37°C followed by reversed phase high performance liquid chromatography on a Pep.S. C2/C18 column (15). Amino acid sequence analysis. N-terminal amino acid sequence analysis was performed with an automatic protein sequencer (477 A, Applied Biosystems, Perkin Elmer, Foster City, CA) coupled to a PTH amino acid analyser (120A, Applied Biosystems).
RESULTS SDS-PAGE of extracted amyloid fibrils showed one strongly predominating band corresponding to a Mr of less than 10 kDa preceded by a weaker band. Gel filtration of dissolved amyloid fibrils revealed the usual AA-pattern with one major retarded peak preceded by two small peaks. Nterminal amino acid sequence analysis of the major peak protein revealed a sequence typical of protein AA but lacking the N-terminal arginine residue of SAA. The same material was cleaved by cyanogen bromide and run for 17 steps before cleavage with BNPS-skatole and sequenced for another 17 steps. The results confirmed earlier data for positions 1 to 40 and established the sequence of positions 54 to 70 (Fig. 1). N-terminal sequence of a more retarded protein fraction just preceding the major protein AApeak at gel filtration, was run for 23 steps which confirmed the sequence from above. The same material was then used for cyanogen bromide cleavage and sequenced for another 30 steps which established the sequence of positions 24 to 53. The same material was then used for cleavage with BNPS-skatole and put back in the sequencer for another 11 steps establishing the sequence of positions 53 to 63. In order to reveal the complete sequence, protein AA was digested with protease V-8 and the
FIG. 1. Amino acid sequences of positions 1–76 of the different human SAA variants that have been described compared to the primary structure of the AA-protein described in this study. 321
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
derived peptides were separated on a reverse phase column. Four peptide fractions covering positions 1 to 8, 9 to 25, 26 to 55 and 63 to 75 were purified and characterised by Edman degradation. Two other fractions were found to contain a mixture of peptides starting in positions 9, 26 and 63 and positions 16 and 26, respectively. The data established the complete sequence corresponding to position 2–76 of SAA1 and SAA2 (Fig. 1). Western blot analysis of amyloid fibrillar material revealed reaction with anti SAA24–34 but not with anti SAA91–104 consistent with the complete lack of a C-terminal part of SAA in the amyloid fibrils. As is seen in Fig. 1 there are phenylalanine residues at positions 68–69, indicating an origin from SAA1 since SAA2 has Leu-Thr at these positions. There are three known allelic forms of SAA1, differing from each other by variations at positions 52, 57, 60 and 72. The combination of Ala52, Val57, Asn60 and Gly72 is not seen in any of these (Fig. 1). Protein AA resembles most closely SAA1b and SAA1g from both of which it differs by two amino acid residues. DISCUSSION The results of the present study have identified a novel protein AA-variant in a patient from an area in Papua New Guinea with an unusually high prevalence of AA-amyloidosis (16). This AA-variant differs from other known AA/SAA-proteins at positions previously known to vary between SAA forms. This Papuan protein AA comes from a fourth SAA1 form, SAA1d. Each SAA and protein AA variant occurs in two forms, one with an N-terminal arginine residue and one des-arginine form. In protein AA, both forms are usually found, except for the long AA-variants associated with vascular amyloidosis (17). In the present material with an AA-protein of the most common length, only des-arg AA was identified. It is known from certain familial amyloidoses, e.g. familial transthyretin amyloidosis that single amino acid substitutions render amyloidogenic properties to the fibril protein. In the mouse, two major SAA variants (SAA 1 and 2) are synthesised by the liver as acute phase reactants and at approximately equal amounts, but only SAA2 is deposited as amyloid (18, 19). In humans, all of the known SAA variants are shown to give rise to AA-protein and amyloid (3, 13, 20) and, as a matter of fact, protein AA obviously derived from a mixture of SAA isoforms has been demonstrated at several times in amyloid deposits (21, 22). However, also in humans particularly amyloidogenic AA-variants may occur since expression of SAA1g is associated with an increased risk for AA-amyloidosis (23) although protein AA derived from this species has only rarely been demonstrated in amyloid deposits (21, 22). Given the unusually high prevalence for AAamyloidosis in the Papua New Guinean population to which patient Kop belonged, SAA1d may represent an especially amyloidogenic variant. This finding therefore offers one possible explanation for the susceptibility of certain Papua New Guinean populations for systemic AA-amyloidosis. The finding in the present study therefore is a strong argument for further studies of AA-protein and SAA variants in this population. ACKNOWLEDGMENTS K.P.W.J. McAdam worked at the Papua New Guinea Institute of Medical Research in Goroka. Further studies were supported by the American Kidney Fund, the Mae J. Drielsma Memorial Fund, the Swedish Medical Research Council, the Swedish Medical Society, and the Research Council of Norway.
REFERENCES 1. McAdam, K. P. W. J. (1978) Papua New Guinea Med. J. 21, 69–78. 2. McAdam, K. P. W. J., Raynes, J. G., Alpers, M. P., Westermark, G. T., and Westermark, P. Papua New Guinea Med. J. (in press). 3. Husby, G., Marhaug, G., Dowton, B., Sletten, K., and Sipe, J. D. (1994) Amyloid: Int. J. Exp. Clin. Invest. 1, 119–137. 4. Raynes, J. G., Hultquist, R., Molyneux, M., Griffin, G., and McAdam, K. P. W. J. (1991) in Amyloid and Amyloidosis (Natvig, J. B., Førre, Ø., Husby, G., Husebekk, A., Skogen, B., Sletten, K., and Westermark, P., Eds.), pp. 119–123. Kluwer Academic, Dordrecht. 322
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Levin, M., Franklin, E. C., Frangione, B., and Pras, M. (1972) J. Clin. Invest. 51, 2773–2776. Prelli, F., Pras, M., Shtrasburg, S., and Frangione, B. (1991) Scand. J. Immunol. 33, 783–786. Cooke, R. A., and Champness, L. T. (1970) Med. J. Austral. 2, 1177–1184. Anders, R. F., Price, M. A., Wilkey, I. S., Husby, G., Takitaki, F., Natvig, J. B., and McAdam, K. P. W. J. (1976) Clin. Exp. Immunol. 24, 49–53. Pras, M., Schubert, M., Zucker-Franklin, D., Rimon, A., and Franklin, E. C. (1968) J. Clin. Invest. 47, 924–933. Blobel, G., and Dobberstein, B. (1975) J. Biol. Chem. 67, 835–851. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. Gustavsson, Å., Engström, U., and Westermark, P. (1994) Am. J. Pathol. 144, 1301–1311. Sletten, K., and Husby, G. (1974) Eur. J. Biochem. 41, 117–125. Fontana, A. (1972) Meth. Enzymol. 25, 419–423. Syversen, P. V., Juul, J., Marhaug, G., Husby, G., and Sletten, K. (1994) Scand. J. Immunol. 39, 88–94. Anders, R. F., Price, M. A., and Wilkey, I. S. (1976) Trop. Geogr. Med. 28, 273–282. Westermark, G. T., Sletten, K., and Westermark, P. (1989) Scand. J. Immunol. 30, 605–613. Meek, R. L., Hoffman, J. S., and Benditt, E. P. (1986) J. Exp. Med. 163, 499–510. Shiroo, M., Kawahara, E., Nakanishi, I., and Migita, S. (1987) Scand. J. Immunol. 26, 709–716. Westermark, G. T., Sletten, K., Grubb, A., and Westermark, P. (1990) Am. J. Pathol. 137, 377–383. Møyner, K., Sletten, K., Husby, G., and Natvig, J. B. (1980) Scand. J. Immunol. 11, 549–554. Baba, S., Takahashi, T., Kasama, T., and Shirasawa, H. (1992) Biochim. Biophys. Acta 1180, 195–200. Baba, S., Masago, S. A., Takahashi, T., Kasama, T., Sugimura, H., Tsugane, S., Tsutsui, Y., and Shirasawa, H. (1995) Hum. Mol. Genet. 4, 1083–1087. Dwulet, F. E., Wallace, D. K., and Benson, M. D. (1988) Biochemistry 27, 1677–1682. Beach, C. M., de Beer, M. C., Sipe, J. D., Loose, L. D., and de Beer, F. C. (1992) Biochem. J. 282, 615–620. Kluve-Beckerman, B., Dwulet, F. E., and Benson, M. D. (1988) J. Clin. Invest. 82, 1670–1675.
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