Partial amino acid sequence of an amyloid fibril protein from unusual cutaneous cystic lesions in myeloma-associated amyloidosis

ARTICLE

Systemic amyloidosis are generally classified into primary, myeloma-associated, secondary, haemodialysis-associated and heredofamilial amyloidosis [1]. In primary and myeloma-associated amyloidosis, the fibrils are composed of amyloid L protein (AL) and appear to be a consequence of plasma cell dyscrasia. Amino acid sequence analysis has demonstrated that AL consists of fragments of an immunoglobulin polypeptide light chain, particularly the variable (N-terminal) region, or of an intact immunoglobulin light chain, or both [2, 3]. AL is usually associated with a similar abnormal immunoglobulin light chain, which is commonly lambda class [4, 5], in serum, except a rare case of immunoglobulin heavy-chain-associated amyloidosis [6]. It has been reported that amyloidosis develops in about 15% of myeloma patients [7]. Kappa and lambda amyloid fibril proteins have been studied in materials from various origins such as liver [8], spleen [9], and urine [5]. However, amyloid fibrils in skin lesions from myeloma-associated amyloidosis have not been fully examined, except nodular primary cutaneous amyloidosis [10-12]. Although it is well known that amyloidosis shows various clinical manifestations including petechia, purpura, papules, lichen, nodules, tumors, plaques, scleroderma-like changes, poikiloderma, alopecia, xanthoma and bullous lesion [1, 7], cystic nodules are uncommon. We present here a patient with cystic nodules in systemic amyloidosis associated with multiple myeloma. We isolated water-extracted materials, which were confirmed as amyloid fibrils by electron microscopy, and were characterized by immunoblot and amino acid sequence analyses.

Case report

A 71 year-old Japanese woman presented with proteinuria in 1989. She was diagnosed as having multiple myeloma because of an increased monoclonal immunoglobulin light chain of kappa type in the serum (Bence-Jones proteinemia) and urine (Bence-Jones proteinuria), and the finding of more than 50% of atypical plasma cells in the myelogram. Although she received treatment with melphalan and prednisolone, and high-dose prednisolone (80 mg/day), it was ineffective. Bone absorption in the lumbar vertebra was found in 1993 by X-ray examination, suggesting the infiltration of myeloma cells to the bones. In 1995, she received a surgical operation for left carpal tunnel syndrome. She developed subcutaneous cystic nodules with tenderness on the sacral region (Fig. 1), the flexural sites of bilateral elbow joints and the dorsal regions of bilateral wrist joints in April of 1996. They were skin-colored, soft, fluctuated nodules which were approximately 3 cm in diameter. Purpura, petechia, macroglossia and oral lesions were not observed. Laboratory examination in April of 1996 showed 24.5 x 10⁵/l of erythrocyte, 7.6 g/dl of hemoglobin, 24.1% of hematocrit, 4,200/l of leukocyte, 11% of monocyte, 301 IU/dl of ALP, 555 mg/dl of IgG, 28 mg/dl of IgA, 10 mg/dl of IgM, 1.74 mg/dl of CRP and 100 mg/dl of urine protein. This multiple myeloma was judged as stage 3b. By incision of the cystic nodule on the right wrist, several red granules with a diameter of 2-3 mm were found in glutinous gray-white liquid in the cystic nodule. Histological findings of the nodule on the wrist showed normal epidermis and a big cavity between the deep dermis and subcutaneous tissue (Fig. 2A). Eosinophilic depositions on the wall of the cavity (Fig. 2B) and red granules were positively stained by periodic acid Schiff (PAS) and Congo red stains, and showed apple-green birefringence with a polarizing light microscope. They were positive for antibody to kappa light chain of immunoglobulin by immunostaining, but not for antibody to lambda light chain. We diagnosed this case as systemic amyloidosis associated with multiple myeloma.

From June 1996, her anemia and renal dysfunction worsened. She died of renal dysfunction on November 21, 1996.

Materials and methods

Extraction and purification of amyloid fibril protein

The extraction of amyloid fibrils from glutinous gray-white liquid in the cavity between deep dermis and subcutaneous tissue was performed according to the methods of Pras [13] and Glenner [14]. The liquid material (6 ml of total volume) obtained by incision of the nodule were homogenized in 30 ml of physiological saline. The homogenized material was sonicated for 15 sec three times with a sonicator. Then it was centrifuged at 12,000 g at 4° C for 30 min and the supernatant was removed. We repeated this centrifugation until absorbance value of the supernatant decreased to less than 0.05 at 280 nm. The final precipitate was resuspended with 30 ml of distilled water, homogenized, and sonicated. The homogenate was centrifuged at 12,000 g at 4° C for 30 min and this procedure was repeated 5 times. The first supernatant was discarded and the second to fifth supernatants were collected (approximately 120 ml of total volume) and recentrifuged at 108,000 g at 4° C for 2 hrs. The precipitate was stored at ­ 80° C until used. The material extracted with distilled water was used for the following studies; electron microscopy, amino acid sequence, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining, periodic acid-Schiff (PAS) reaction, and immunoblotting with antibodies to kappa and lambda light chains.

Electron microscopy

Amyloid fibrils were observed by the transmission electron microscope (Model 100S, JEOL, Tokyo, Japan) under negative staining with 2% phosphotungstic acid.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

To determine molecular weights of peptides in the extracted material, SDS-PAGE was carried out. Aliquots of the samples were mixed with SDS-PAGE sample buffer (10% 2-mercaptoethanol, 2% SDS, 30% glycerol, 0.25 M Tris, pH 6.8), heated at 98° C for 3 min (10, 30 or 50 mg of protein per lane), and subjected to reducing SDS-PAGE in 20% polyacrylamide gel by the method of Laemmli [15]. The gel was stained with PAS reaction according to the method of Felgenhauer et al. [16]. After completing the PAS reaction, the gel was stained by CBB. Protein standards for molecular weights (Bio-Rad, Richmond, CA) were as follows: lysozyme, 20.5 kDa; soybean trypsin inhibitor, 28.4 kDa; carbonic anhydrase, 34.2 kDa; ovalbumin, 48 kDa; bovine serum albumin, 77 kDa; phosphorylase B, 103 kDa.

An aliquot of distilled water-extracted material was subjected to immunoblotting with antibodies to kappa and lambda light chains. Proteins after SDS-PAGE were electroblotted onto Hybond-ECL (Amersham, Buckinghamshire, UK) by using a semi-dry transfer apparatus (Bio-Rad) for 30 min at 10 V. The membrane was preincubated with a blocking solution containing 5% skim milk and 0.2% Tween 20 in PBS for 1 hr at room temperature. The membrane was then incubated overnight at 4° C with diluted monoclonal antibodies to human kappa light chain (HP6156) and to human lambda light chain (HP6054) (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). After incubation with horseradish peroxidase-conjugated anti-mouse IgG (0.2 ml per ml) in the blocking solution for 2 hrs, the membrane was washed and reacted with ECL detection reagents (Amersham) and exposed to hyperfilm.

Amino acid sequence analysis

Isolated amyloid materials in the sample buffer for SDS-PAGE were heated at 98° C for 3 min and subjected to SDS-PAGE using 20% polyacrylamide gel. Separated proteins were electrophoretically transferred onto a polyvinylden difluoride membrane (Millipore Corp. Bedford, MA) and stained with CBB. The band on the membrane strip stained with CBB was cut and applied to an amino acid sequence analysis [17-18] by a gas-phase protein sequenator connected to Amino Acid Analyzer, PI-2020 (Toso Corp., Hiroshima, Japan).

Results

Characterization of water-extracted material

The isolated material contained fibrils as shown by negative staining electron microscopy (Fig. 3). These fibrils are staight and non-branching with a diameter of 8 to 10 nm. The material appears to be composed of amyloid fibrils alone from its morphology.

A polypeptide with molecular weight of 20kDa was detected by PAS (data not shown) and CBB staining (Fig. 4) after SDS-PAGE. In immunoblot analysis, an anti-kappa light chain antibody reacted with two polypeptides with molecular weights of 20 and 29 kDa, whereas the anti-lambda light chain antibody did not (Fig. 4).

The purified 20 kDa protein was subjected to amino acid sequence analysis as described in Materials and methods. The amino acid sequence of the N-terminal 19 residues was determined as shown in Table I. These residues have a sequence homology to kappa I light chain of 82%, kappa II of 59%, kappa III of 47% and kappa IV of 59% [19], respectively.

Discussion

Approximately 15 to 20% of Bence Jones proteins appear to be amyloidogenic in that they have the property of precipitating as amyloid fibrillar material in vitro proteolytic digestion [20-23]. This amyloidogenic property is associated with the light chain variable region (V_L) and is more commonly observed with lambda than with kappa monoclonal light chains. The human lambda and kappa light chains have been divided into six lambda (lambda I, lambdaII, lambdaIII, lambdaIV, lambdaV, lambdaVI) and four kappa (kappa I, kappaII, kappaIII, kappaIV) subgroups on the basis of the characteristic amino acid sequence [24]. Subgroup lambda VI has been shown to be most commonly found in association with primary systemic or myeloma-associated amyloidosis [24, 25]. There is little information about amino acid sequences of amyloid proteins from skin lesions except the report showing that amyloid protein from nodular primary cutaneous amyloidosis has a sequence closely homologous to lambdaIII subgroup of immunoglobulin light chain [11]. The sequence of the N-terminal region of 20 kDa protein of the present case revealed a 82% homology to that of subgroup kappaI of the immunoglobulin light chain, and less than 60% to the three other subgroups. Amyloid fibrils in the cystic nodules may be derived from kappaI light chain. The cutaneous cystic nodules in the present case are rare in myeloma-associated amyloidosis. It is unknown whether the uncommon clinical features are associated with amyloid fibrils derived from kappaI light chain.

The distilled water-extracted materials appeared to be amyloid fibrils in electron microscopy, and a single band with a molecular weight of 20 kDa was detected by SDS-PAGE and immunoblotting with antibody to kappa light chain of immunoglobulin. However, another band with a molecular weight of 29 kDa was also positive with antibody to kappa light chain, although this band was not detected by CBB staining. Although the reason for this contradictory observation is unknown, there is a possibility that a co-purified trace component with a molecular weight of 29 kDa may be kappa light chain of immunoglobulin, because it strongly reacted with antibody to kappa light chain. This may be due to a high antigenicity of intact kappa light chain of immunoglobulin than kappa type of AL, which is a denatured peptide of kappa light chain. Since unfortunately we have no serum and urine of this patient for further study, it is unknown whether the amyloid protein in the cutaneous lesion is identical to the Bence Jones protein in this patient.

REFERENCES

1. Black MM. Amyloid and the amyloidoses of the skin. In: Champion RH, Burton JL, Ebling FJG, eds. Textbook of Dermatology. 5th ed. London: Blackwell Scientific Publications, 1992: 2333-44.

2. Glenner GG, Terry W, Harada M, Iserky C. Amyloid fibril proteins: proof of homology with immunoglobulin light chain by sequence analyses. Science 1971; 172: 1150-1.

3. Terry WD, Page DL, Kimura S, Isobe T, Osserman EF, Glenner GG. Structural identity of Bence Jones and amyloid fibril proteins in a patient with plasma cell dyscrasia and amyloidosis. J Clin Invest 1973; 52: 1276-81.

4. Buxbaum J. Aberrant immunoglobulin synthesis in light chain amyloidosis. Free light chain and light chain fragment production by human bone marrow cells in short-term tissue culture. J Clin Invest 1986; 78: 798-806.

5. Bellotti V, Merlini G, Bucciarelli E, Perfetti V, Quaglini S, Ascari E. Relevance of class, molecular weight and isoelectric point in predicting human light chain amyloidogenicity. Br J Haematol 1990; 74: 65-9.

6. Eulitz M, Weiss DT, Solomon A. Immunoglobulin heavy-chain-associated amyloidosis. Proc Natl Acad Sci USA 1990; 87: 6542-6.

7. Breathnach SM. Amyloid and amyloidosis. J Am Acad Dermatol 1988; 18: 1-16.

8. Gertz MA, Skinner M, Cohen AS, Conners LH, Kyle RA. Structural and immunologic studies of a kappa amyloid fibril protein. In: Glenner GG, Osserman EF, Benditt EP, Cohen AS, Zucker-Franklin D, eds. Amyloidosis. New York, NY: Plenum Press, 1986: 517-24.

9. Dwulet FE, O'Conner TP, Benson MD. Polymorphism in a kappa I primary (AL) amyloid protein (BAN). In: Glenner GG, Osserman EF, Benditt EP, Cohen AS, Zucker-Franklin D, eds. Amyloidosis. New York, NY: Plenum Press, 1986: 503-7.

10. Kitajima Y, Seno J, Aoki S, Tade S, Yasita H. Nodular primary cutaneous amyloidosis. Arch Dermatol 1986; 122: 1425-30.

11. Kitajima Y, Hirata H, Kagawa Y, Yasita H. Partial amino acid sequence of an amyloid fibril protein from nodular primary cutaneous amyloidosis showing homology to lambda immunoglobulin light chain of variable subgroup III (AlambdaIII). J Invest Dermatol 1990; 95: 301-3.

12. Inazumi T, Hakuno M, Yamada H, Tanaka M, Naka W. Characterization of the amyloid fibril from primary localized cutaneous nodular amyloidosis associated with Sjögren's syndrome. Dermatology 1994; 189: 125-8.

13. Pras M, Schubert M, Zucker-Franklin D, Tajima S, Horada T, Nishikawa T. The characterization of soluble amyloid prepared in water. J Clin Invest 1968; 47: 924-33.

14. Glenner GG, Page D, Isersky C, Rimon A, Franklin EC. Murine amyloid fibril protein: Isolation, purification and characterization. J Histochem Cytochem 1971; 19: 16-28.

15. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.

16. Felgenhauer K, Weis A, Glenner GG. The demonstration of tryptophan, tyrosine and carbohydrate-containing proteins in disc electrophoresis gels. J Chromatog 1970; 46: 116-9.

17. Schroeder WA. Degradation of peptides by the Edman method with direct identification of the PTH-amino acid. Methods in Enzymology 1967; 11: 445-61.

18. Hunkapiller MW, Hood LE. Protein sequence analysis: automated microsequencing. Science 1983; 219: 650-9.

19. Solomon A, Kyle RA, Frangione B. Light chain variable region subgroups of monoclonal immunoglobulins in amyloidosis AL. In: Glenner GG, Osserman EF, Benditt EP, Cohen AS, Zucker-Franklin D, eds. Amyloidosis. New York, NY: Plenum Press, 1986: 449-62.

20. Glenner GG, Ein D, Eanes ED, Bladen HA, Terry W, Page DL. Creation of " amyloid " fibrils from Bence Jones proteins in vitro. Science 1971; 174: 712-4.

21. Glenner GG. Amyloid deposits and amyloidosis. The ß-fibriloses. N Engl J Med 1980; 302: 1283-92.

22. Linke RP, Zucker-Franklin D, Franklin EC. Morphologic, chemical, and immunologic studies of amyloid-like fibrils formed from Bence Jones proteins by proteolysis. J Immunol 1973; 111: 10-23.

23. Linke RP, Tischendorf FW, Zucker-Franklin D, Franklin EC. The formation of amyloid-like fibrils in vitro from Bence Jones proteins of the V lambdaI subclass. J Immunol 1973; 111: 24-6.

24. Solomon A, Frangione B, Franklin EC. Bence Jones proteins and light chains of immunoglobulins. Preferential association of the V lambda VI subgroup of human light chains with amyloidosis AL(lambda). J Clin Invest 1982; 70: 453-60.

25. Sletten K, Westermark P, Husby G. Structural studies of the variable region of immunoglobulin light-chain-type amyloid fibril proteins. In: Glenner GG, Osserman EF, Benditt EP, Cohen AS, Zucker-Franklin D, eds. Amyloidosis. New York, NY: Plenum Press, 1986: 463-75.