Acta Pathol. Jpn. 31(1): 27-33, 1981

PURIFICATION OF AMYLOID FIBRILS AND PROTEIN AA FROM MOUSE AMYLOID DEPOSITS INDUCED BY

CASEINATE AND M. BUTYRICUM

Yohei TAKAHASHI, Hiroyuki MURO, Masahiro GOTO, and Haruyuki SHIRASAWA

Department of Pathology, Harnamateu Univereity School of Medicine, Hamamatsu

Amyloidosis was induced in mice by the simultaneous injection of sodium caseinate and heat-killed M. butyricum. Amyloid fibrils were isolated by collagenase digestion, 1 M NaCl extraction and repeated washing with 0.15 M NaC1. The amyloid fibril fraction was practically free of contaminations such as collagen, chromatin and membranes as judged by electron micro- scopic morphometry. The protein AA was purified from the isolated fibrils to an apparent homogeniety as judged by sodium dodecyl sulphate polyacry- lamide gel electrophoresis using one step gel filtration from Sephacryl S- 200 in the presence of 6 M guanidine-HC1 and 50 mM dithiothreitol. The molecular weight of the peptides of the protein AA were 8,500 and 10,000. ACTA PATHOL. JPN. 31: 27-33, 1981.

Ilztroductirm

A fine fibrillar structure of amyloid is well accepted in various ~ys tems.4)~P Experimental amyloidosis has been widely studied and its similarity to human secondary amyloidosis is well recognized.2P The main component of amyloid fibrils in secondary amyloidosis and experimental amyloidosis was designated as protein AA.7 PRAS et al. developed a simple method for the isolation of amyloid fibrils based upon the facts that the fibrils are insoluble in physiological saline while i t is soluble in distilled water.14 However, the fibrils isolated from the experimentally produced animal amyloidosis contained some impure materials such as DNA, collagen, and membranes. We report here a modified method of isolation of amyloid fibrils from experimental mouse amyloidosis. The isolated fibrils by the method are practically free of contaminations. Furthermore, we will add the one step purification of protein AA from the isolated fibrils.

Materials and Methods Induo th of m e arnyloidoek: Six to seven weeks old ICR/SLC male mice were obtained from the Shizuoka Agricultural Cooperative Association for Laborcttory Animals. Five to 6 mice were caged together and maintained on laboratory chow diet and water ad libitum. Each mouse

Accepted for publication January 17, 1980.

Yohei TAKAHASHI, M.D., Department of Pathology, Hamamatsu University School of Mediaine, 3800 Handa-cho, Hamamatsu-shi 431-31, JAPAN.

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28 PURIFICATION OF AMYLOID PROTEINS Acta Pathol. Jpn.

received subcutaneous injections of 0.2 ml of 8% Na-caseinate (Hammersten grade, E. Merck Co., Darmstadt, W. Germany) containing heat-killed M . butyricum (0.6 mg/ml, Difco Lab., Detroit, Mich.) for two weeks followed by injection of Na-caseinate alone every day. After 8 to 10 weeks, the animals were sacrificed and small pieces of various organs were fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, and alkaline Congo red. The Congo red stained sections were examined in polarized light. The remaining tissues of the spleen, liver and kidney were kept forzen a t -80°C until used. Electron microscopy: Amyloid materials were examined electron microscopically by the negative staining method a t several steps during purification procedure. A small drop of suspension of amyloid material was placed on a carbon-coated collodion film on a copper grid. Excess liquid was withdrawn with filter paper and the grid was allowed t o dry for one minute. Then a drop of 1% phosphotungstate was placed, and the grid was immediately drained against filter paper. The specimens were examined and photographed in a JEM-100C electron microscope with 80 KV accelerating voltage. Sodium dodecyl sulphnte (BUS) polyac ylnmide gel electrophoresis: Electrophoresis and molecular weight determination were carried out on 10% polyacrylamide gels in 0.1% SDS a t pH 7.2 by the method of Weber and Osbornlo. Cytochrome c (M.W. 12,500), chymotrypsinogen A (25,000), egg albumin (45,000), and bovine serum albumin (67,000) (Combithek, Boehringer, Mannheim, W. Germany) were used as standard proteins for calibration of molecular weight.

Results

Mouse amyloidosis: Most animals receiving combined injections of Na-caseinate and M . butyricum showed amyloid deposits in the spleen, liver, and kidneys, although the degree of deposition varied. Fig. 1 represents amyloidosis of the spleen and the liver stained with hematoxylin and eosin. The most extensive amyloidosis was seen in the perifollicular zone of the spleen. In the liver, a moderate amount of amyloid was recognized in the portal areas and the space of Disse. The kidneys showed a small amount of deposits in the glomeruli and arterioles. Congo red stained sections exhibited green birefringence under polarized light corresponding to the deposited areas.

Isolation of nmyloid jibrils : The collected organs from 20 to 30 animals were processed together. Amyloid laden tissues of spleen, liver, and kidney were thawed and homo- genized in 500 ml of 0.15 M NaCl and centrifuged a t 10,000 rpm for 30 min at 4". The precipitate was homogenized and centrifuged as described above. After repeating the steps several times, pH was adjusted to 7.4 with Tris-C1 and CaCl, was added to a

Fig. 1. Experimentally induced mouse amyloidosis. (la) spleen, (lb) liver, hematoxylin and eosin stain, x 100.

31(1): 1981 Y. TAKAHASHI U al. 29

final concentration of 1 mM. To this solution collagenase (1 mg/6&100 mg protein, extracted from Clostridium histolymm, grade 11, Boehringer Mannheim, W. Germany) was added and stirred slowly for 12 hours a t 30". After the collagenase digestion, homo- genization and centrifugation in 0.15 M NaCl were repeated again until the supernatant became clear. At this step the absorbance a t 280 nm of the supernatant was around O.l/cm. Then the precipitate was homogenized with 2M) ml of 1 M NaCl and stirred for 12 hours a t 4". At this step, the supernatant showed absorbance of about 0.5/cm a t 280 nm. By this procedure, the bulk of DNA, nucleoproteins were removed. The homogenization and centrifugation in 260 ml of 0.16 M NaCl were repeated again until the absorbance of the supernatant became less than 0.06. The following serial extraction with distilled water was performed in the same manner as the original method. The final suspensions of amyloid fibrils showed opalescence and fine reflexion to light. From the ultraviolet spectrum of the purified amyloid, the ratio of 280/260 nm was approximately 1.45, so the contamination of DNA into the preparation was estimated to be less than 0.760/12. Electron microscopic examination revealed that the preparations obtained from the above procedures are free of contaminations such as collagen, chromatin, and membranes. Amyloid fibrils consisted of long double filaments, which were twisted with each other with a periodicity of about 150 nm. Each filament measured about 6 nm in width. The length of fibrils varied, among which longer ones measured more than 1 pm. No branching of fibrils was observed (Pig. 2). These results quite agree with the past o b s e r ~ a t i o n s . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ SDS polyacrylamide gel electrophoresis revealed two major bands, the molecular weight of which were 9,100 and lO,OOO, respectively (Fig. 4. A).

Pur@mtion of amyloidfibril protein AA: Amyloid fibrils were solublized in a solution of 6 M guanidine-HCl, 60 mM dithiothreitol, and 60 mM Tris-C1, pH 8.0, a t a con- centration of 10 mg/ml. The suspensions were stirred for two hours a t room tem- perature under N,. After solubilization the pH was adjusted to 4.0 with acetate buffer (20 mM) and centrifuged at 10,000 rpm for 30 min. The supernatant was applied on a Sephacryl5-200 superfine column (2.6 x 90 cm) equilibrated with 20 mM acetate buffer (pH 4.0) containing 6 M guanidine-HC1 and 1 mM dithiothreitol. The amyloid proteins were then eluted with the equilibration buffer. The elution profile of proteins from the gel is as presented in Fig. 3. Three major peaks appeared and were designated peak I, peak 11, and peak 111. Peak I contains large molecular weight material. Peak I11 contains degraded materials of lower molecular weights and was dialyzable. Peak I1 was further divided into two parts, named peak II--1 and peak 11-2. Peak 11-1 and 11-2 had absorption peak a t 278 nm and a shoulder a t 290 nm and was as same as that of purified amyloid fibrils. Each fraction was dialyzed against distilled water and lyophilized. Then proteins were analyzed by SDS gel electrophoresis as described under Materials and Methods. The SDS gel electrophoretic pattern of the purified amyloid fibrils and the fractions are as shown in Fig. 4. Peak 11-1 and 11-2 fractions showed identical two bands and molecular weights of which were 8,600 and lO,OOO, respectively. In peak 11-1 (Fig. 4, C) the slow band was main, whereas the fast

30 PURIFIC!ATION OF AMYLOID PROTEINS Acta Pathol. J p w

Fig. 2. Electron micrograph of the purified amyloid fibrils isolated by our modified method, negatively stained with 1% phosphotungstate. x 181, 250.

31(1): 1981 Y. TAKAHASHI d d. 31

Fraction number

Fig. 3. Gel chromatography of purified amyloid fibrils through a 25x90 om column of Sephacryl S-200 superfine. Elution waa carried out with 6 M guddine-HC1, 1 mM dithiothreitol, and 20 mM acetate buffer, pH 4.0. Fraction volume waa 6 ml. Cytoohrome c (M.W. 12,500) was eluted at the point indicated by the arrow.

1 67,000

46,666

26,000

12,600

A B C D E Fig. 4. SDS disc electrophoresis of amyloid proteina in 10% polyacrylamide gels. (A)

purified amyloid fibrils, (B) peak I fraotion, (C) peak 11-I fraction, (D) peak II-2’ fraction, and (E) control emzymes: cytochrome c (M.W. 12,MH)), chymotripainogen A (26,000), e& albumin (45,000), and bovine aerum albumin (67,000).

band was main in the peak 11-2 (Fig. 4, D). The peak I material showed no bands but stayed at the top of the gels (Fig. 4, B). Comparing the data of these fractions with that of native amyloid fibrils, it is concluded that the main components of amyloid protein were collected in the peak I1 fractions, and these are the protein AA which consists of two heterogenous subunits. The origin and the nature of the peak’I material remains to be elucidated.

Discussion

Among the many methods to induce experimental amyloidosis, repeated parenteral injections of casein have been most widely used8. Injections of Freund’s adjuvant with

32 PURmUATION OE AMYLAOID PROTEINS Acta Pathl . Jpn.

addition of extra M. butyricum were found to induce amyloidosis in mice in short in- duction time.16 In the present study, combined injections of casein and M . butyricum were used to induce amyloid deposits as abundant as possible. When we isolate amyloid fibrils from the experimentaly induced animal amyloid-laden tissue according to the original method by PRAS et al., contamination of chromatin and collagen fiber is inevitable. Chromatin is not soluble in 0.15 M NaC1, while i t is soluble in water or high concentration of salt.lP By extraction with 1 M NaCl chromatin was removed. As reported by COHEN and CALINS,~ amyloid fibrils were stable against collagenase digestion. In the 6nal preparation obtained by our procedure with collagenase diges- tion, collagen fibers could not be identified under the electron microscope. Thus it is possible to extract pure amyloid fibrils even from tissues, which contain relatively small amount of amyloid by 1 M NaCl extraction and collagenase digestion.

Fractionation of amyloid protein after 6 M guanidine-HCl treatment was carried out through gel filtration of Sephacryl 5-200 superfine. This one step gel filtration was satisfactory to separate protein AA fraction from others. The protein AA purified by us consisted of two subfractions, and the molecular weights were 8,500 and 10,000 from gel electrophoresis.

Studies on the induction of amyloidosis are now in progress in our laboratory using anti-protein AA antibody, which reacts only with amyloid immunohistochemically verifying conversely the purity of our protein AA.

Our data quite agree with other reports.%17

Ackunourledgementa: The authors are indebted to Mr. Hideki HATTORI, Department of Forensic Medicine, Mr. Masaaki KANETA, Department of Pathology, for their technical help and to Dr. Masanori FUKUSHIMA, Department of Internal Medicine, Aichi Cancer Center, for his critical reading and helpful suggestions during the preparation of the manuscript.

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