Structural Identity of Bence Jones and Amyloid

Fibril Proteins in a Patient

with Plasma Cell Dyscrasia and Amyloidosis

WILLIAM D. TERRY, DAVID L. PAGE, SHIGERUKIMURA, TAKASHI ISOBE,ELLIOTr F. OSSERMAN,and GEORGEG. GLENNER

From the Immunology Branch, National Cancer Institute and Laboratory ofExperimental Pathology, National Institute of Arthritis and Metabolic Diseases,Bethesda, Maryland 20014, and the Institute of Cancer Research andDepartment of Medicine, Columbia University College of Physiciansand Surgeons, NewYork 10032

A B S T R A C T The partial amino acid sequence of theamyloid fibril protein isolated from the small intestineof a patient with plasma cell dyscrasia and associatedamyloidosis has been determined and compared with thesequence of the K-type Bence Jones protein isolated fromthe urine of the same patient. Identical sequences wereobserved for the 27 amino-terminal residues that could becompared. The C-terminal tryptic peptide of the amyloidprotein was identical with that of the Bence Jones pro-tein. Apparent molecular weights and amino acid com-positions of the Bence Jones and amyloid proteins weresimilar. It appears, therefore, that the predominant pro-tein present in the amyloid deposits in this patient wasan intact K-type light polypeptide chain that was identicalwith the urinary Bence. Jones protein.

INTRODUCTION

Considerable information concerning the nature andstructure of amyloid protein has been acquired in thepast few years. Available evidence indicates that in manyinstances, the major protein in amyloid fibrils is some

portion of an immunoglobulin polypeptide chain. Aminoacid sequence studies have shown that the amino-terminalsequence of some amyloid fibril proteins is homologouswith that of light polypeptide chains (1, 2). Moreover, it

This work was presented in part at the 20th Colloquiumon Peptides in Biologic Fluids, Brugges, Belgium, 3-4 May1972.

Dr. Osserman is an American Cancer Society ResearchProfessor of Medicine.

Received for publication 15 August 1972 and in revisedform 16 January 1973.

is possible to create fibrils having the tinctorial, ultra-structural, and crystallographic properties of amyloidfibrils by proteolytic digestion of certain Bence Jonesproteins (3).

Immunoglobulin-related amyloid fibril proteins havebeen found in a number of clinical settings, includingso-called "primary" amyloidosis (2, 4, 5), amyloidosissecondary to epidermolysis bullosa and tuberculosis (2,4) and isolated nodular pulmonary amyloidosis (6, 18).Information concerning the sequence of amyloid fibrilprotein from patients with plasma cell dyscrasia andamyloidosis has, however, not been available. It is ofsome importance to establish that, in a patient withplasma cell dyscrasia, circulating homogeneous immuno-globulin, and amyloidosis, the immunoglobulin of theamyloid fibril is identical with the circulating homo-geneous immunoglobulin or at least a portion of it. Fail-ure to demonstrate such a relationship would have im-portant implications for the pathogenesis of amyloid de-posits (7).

It was possible to obtain sequence information for theBence Jones protein and the amyloid protein of the samepatient since one of us (Dr. Osserman) had studied apatient who died in 1958 with plasma cell dyscrasia,K-type Bence Jones proteinuria, and amyloidosis (case14 in reference 8). The sequence of the Bence Jonesprotein from this patient has been determined in anotherlaboratory (9).1 When the patient died, organs con-taining amyloid deposits were removed at autopsy andstored in the frozen state. This paper will present evi-

'Whitley, E. J., and F. W. Putnam. Personal communi-cation.

1276 The Journal of Clinical Investigation Volume 52 May 1973 -1276-1281

lence indicating the identity of the urinary Bence Jonesprotein with the major protein isolated from a concen-trate of the patients' amyloid fibrils.

METHODSIsolation of Bence Jones protein. The Bence Jones pro-

tein was precipitated from whole urine by 2.8 M ammoniumsulfate and stored as a paste at - 200C. At the time of theseexperiments, the precipitate was dialyzed against water andlyophilized. Lyophilized material was dissolved in 0.2 MTris-HCl pH 8.0 buffer and subjected to gel filtration onSephadex G-200. Eluted material was tested by double dif-fusion in agar with specific antisera, and the fractions con-taining K-type light chains were pooled, dialyzed againstwater, and lyophilized.

Isolation of amyloid fibril protein. A segment of smallintestine measuring 20 cm in length was the source of theamyloid fibril concentrate. Previous microscopic examinationrevealed extensive replacement of the subserosa by amyloiddeposits as defined by green polarization birefringence afterCongo red staining (10). The frozen small intestine washydrated in saline buffered to pH 7.1 with 0.1 M sodiumphosphate (phosphate-buffered saline, PBS).2 The amyloid-laden subserosal tissue was dissected from the muscularis,homogenized in PBS, and a preparation having the typicaltinctorial (10), crystallographic, and electron microscopic(3) characteristics of an amyloid fibril-rich concentrate wasobtained as previously described (4). The amyloid fibrilconcentrate was lyophilized and completely dissolved in 6 Mguanidine-HCl containing 0.1 N Tris-HCI, pH 8.0 and made0.01 M in dithiothreitol with agitation under nitrogen at250C for 3 h (11). The solution was diluted to 5 M guani-dine-HCl in 1 N acetic acid, and gel filtration chroma-tography was performed sequentially using Sepharose 4Band Sephadex G-100 columns equilibrated with 5 M guani-dine-HCl in 1 N acetic acid (4). The major protein frac-tions obtained were dialyzed exhaustively with distilledwater and lyophilized. The yield of purified amyloid fibrilprotein was calculated on the basis of dry weight and Lowryprotein determinations of the lyophilized crude amyloidfibril concentrates, and of the lyophilized purified amyloidfibril protein.

Peptide maps. The purified amyloid and Bence Jonesproteins were reduced with 0.001 M dithiothreitol and alky-lated with 0.01 M iodoacetamide. Reduced, alkylated proteinswere digested with trypsin (Worthington Biochemical Corp.,Freehold, N. J., TPCK trypsin) at a protein to enzyme ratioof 100: 1 at pH 8.7 in 0.1 N ammonium bicarbonate buffer.Analytic maps were prepared by spotting 2 mg of thedigest on Whatman 3 M paper. Descending chromatog-raphy was in water, butanol, and acetic acid (40: 27: 8) for20 h, and was followed by electrophoresis in pH 3.5 bufferat 3000 V for 50 min. Papers were stained with collidin-ninhydrin. Preparative peptide maps were prepared by spot-ting 5 mg of digest on Whatman 3 M paper. Chromatog-raphy and electrophoresis were as above. Peptides werelocated by lightly spraying the paper with a mixture of 75cm3 of 99%o ethanol and 25 cm' of 2 M acetic acid, to whichwas added 50 mg of ninhydrin. Peptides were cut out andeluted with water in a moist chamber.

Polyacrylamide disc gel electrophoresis. Studies were per-formed with proteins reduced and alkylated by the proce-

2 Abbreviations used in this paper: PBS, phosphate-buf-fered saline; PTH, phenylthiohydantoin; SDS, sodiumdodecyl sulfate.

FIGURE 1 Acrylamide gel electrophoresis on 12% gels, in0.1% SDS, 4 M urea buffered at pH 7.1. Left, Tew amyloidfibril concentrate before gel filtration; middle, purified Tewamyloid; right, purified Tew Bence Jones protein. Anode isto the bottom.

dure described above (peptide maps), as well as with non-reduced and alkylated preparations. Samples of the amyloidfibril concentrate, major amyloid fibril protein, and BenceJones protein were dissolved at a concentration of 2-8mg/ml of 8 M urea buffered to pH 8.8 with 0.1 N Tris-HCI containing 50 mMdithiothreitol and incubated at 370Cfor 30 min. An equal volume of 8 M urea buffered to pH7.1 with 0.1 N sodium phosphate containing 50 mMdithio-threitol and 2% sodium dodecyl sulfate (SDS) was added.Gels were prepared with 12% polyacrylamide with a 37:1monomer to bis ratio, 0.1%AN, N, N', N'-tetramethylenedia-mine in a buffer containing 0.1%o SDS in 4 M urea adjustedto pH 7.1 with 0.1 M sodium phosphate, and polymerizationwas performed using 0.05 vol of a 1% ammonium per-sulfate solution. Upper and lower electrode compartmentscontained the gel buffer made 1%o in 8-mercaptoethanol.Bromphenol blue was used as a tracking dye. A 30-40 1.Isample was applied and electrophoresis performed at 3 mAper tube for 16 h. The gels were washed with 50%o methanolin 10% acetic acid for 3 h and stained with Coomassie blue.

Amino acid composition. Samples of the purified amy-loid fibril and Bence Jones proteins were hydrolyzed in 6N HCl under vacuum at 110'C for 24 h. Hydrolyzed sam-ples were flash evaporated and analyzed on an amino acidanalyzer (Beckman model 120C, Beckman Instruments, Inc.,Fullerton, Calif.).

Amino acid sequence analysis. The amino-terminal se-quence of the purified amyloid fibril protein was obtainedwith a protein sequencer (Beckman model 890). Reagentswere sequencer-grade chemicals used as obtained from Beck-man Instruments, Inc., except that ethyl acetate solvent wasmade 0.1%o with aldehyde-free acetic acid, and 10 mg ofdithioerythritol was added to each 950 ml of butyl chloridesolvent. The thiazolinone derivatives obtained from the se-quencer were converted to phenylthiohydantoin (PTH)amino acids with 1 N HCl at 80'C for 10 min. PTH aminoacids were identified on a gas chromatograph (Varianmodel 1840, Varian Associates, Walnut Creek, Calif.) (12).

Amyloid Fibril Protein Structure 1277

FIGURE 2 Peptide maps of Tew amyloid and Bence Jones proteins. Reduced and alkylatedproteins were digested with trypsin, and subjected to descending paper chromatography fol-lowed by electrophoresis at pH 3.5. a, amyloid; b, Bence Jones. Streak in lower left is chroma-tography marker. Arrow shows direction of chromatography.

A portion of each PTH amino acid sample was convertedto the free amino acid by hydrolysis with 6 N HCOat 1500Cfor 20 h. Free amino acids were identified by analysis on theamino acid analyzer.

RESULTS

The amyloid fibril concentrate consisted of a number ofproteins before gel filtration, as evidenced by the eightor more protein-staining bands seen on SDS acrylamidegel electrophoresis (Fig. 1, left). The most heavilystaining of these bands, as well as two lighter stainingbands of higher molecular weight, were also detected inthe purified amyloid preparation (Fig. 1, middle). Theseresults are consistent with the conclusion that the majorprotein component of the amyloid fibril concentrate ispresent in the purified amyloid preparation.

Yields were calculated, both on the basis of dry weightand as protein assayed by the Lowry method. The puri-fied major protein fraction represented 42.5% by dryweight and 49.2% by protein determination of the crudeamyloid fibril concentrate. Allowing for losses in dialysisand handling, it is therefore estimated that the finalproduct represents at least 60% of the protein presentin the amyloid fibril concentrate. These data are alsoconsistent with the conclusion that the purified prepara-tion represents the major protein component of theamyloid fibril concentrate.

The isolated, native Bence Jones protein gave threebands on SDS acrylamide gel electrophoresis. Thesebands had mobilities identical with those of the purifiedamyloid protein (Fig. 1, middle and right), indicatingthat proteins of similar molecular weights were presentin these two preparations. It should be noted that duringthe isolation procedure, amyloid fibril protein was notalkylated after reduction by dithiothreitol. Alkylationwas avoided in order that performic acid oxidations andidentification of the C-terminal tryptic peptide could becarried out. However, after reduction and alkylation ofthe amyloid and Bence Jones proteins, SDS acrylamidegel electrophoresis showed only a single band corre-sponding in mobility to that of the lowest molecularweight protein, and electrophoresis of a mixture of theamyloid fibril and Bence Jones proteins also showedonly a single band. This indicates that the higher mo-lecular weight constituents in the nonreduced and alky-lated preparations were disulfide-linked polymers of ap-proximately the same size in both the amyloid and BenceJones preparations.

Additional studies were performed to compare thestructural characteristics of the purified amyloid fibriland Bence Jones proteins. Both preparations were re-duced and alkylated, digested with trypsin, and studiedby peptide mapping at pH 3.5. The maps show minorvariation in the intensity of the spots, but within the lim-

1278 Terry, Page, Kimura, Isobe, Osserman, and Glenner

its of the technique, the two maps appear to he identical(Fig. 2).

The purified anmyloid fibril and Bence Jones proteinswere hlvdrolvzed in 6 N HCl and amino acid comiposi-tions determined. The compositions are quite similar(Table I), again indicating the close structural rela-tionship between the amyloid fibril and Bence Jonesproteins.

The amino acid sequence of the purified amy-loid protein was determined for the amino-terminal 27residues and is compared with the sequence of the BenceJones protein (Fig. 3), which was determined by Doc-tors Whitley and Putnam (9).' As can be seen, the amy-loid fibril protein is homogeneous, and the sequence ofthe amyloid and Bence Jones proteins are identical. Theyield of the first amino acid was 40% of theoretical, ayield similar to that obtained wtih known purified pro-teins treated in the same manner, indicating that theprotein being sequenced was the major protein presentin the purified amyloid fibril preparation. Repeated at-tempts to extend the sequence past position 27 were un-successful, suggesting that the amino group of the gluta-mine at position 27 probably becomes blocked during thechemical reactions, resulting in a blocked N-terminuswhich is unreactive with phenylisothiocyanate. Similarproblems were encountered in sequencing the BenceJones protein.3 These results indicate that the amyloid

3Whitley, E. J., and F. WV. Putnam. Personal communi-cation.

TABLE IAmino Acid Composition of Tew Amyloid and

Bence Jones Proteins

Bence Jones Amyloid

LysHisArgAspThrSerGluProGlyAlaValMetIleLeuTyrPhe

mol/100 mol5.21.43.99.06.7

12.312.7

6.26.76.47.50.973.2

10.03.44.4

5.01.43.89.06.7

11.513.0

6.17.16.67.50.993.39.93.64.5

5 10Tew B. J. Asp-lle-Val-MIet-Thr-Glni-Ser-Pro-Leu-Ser-1Few Amvloid Asp-I)-le-Val-M\Iet-TI hr-Glni-Ser-Pro-Leu-Ser-

15 20B. J. Leu-Pro-Val-Thr-Pro-Gly-Glui-Pro-Ala-Ser-Amyloid Leu-Pro-Val-Thr-Pro-Gly-GlI-Pro-Ala-Ser-

25B. J.Amyloid

I le-Ser-Cys-Arg-Ser-Ser-GlnIle-Ser-Cys-[ ]-Ser-Ser-Glx

FIGURE 3 Amino-terminal sequence of Tew amyloid proteincompared with the sequence of Tew Bence Jones protein(Whitley, E. J., and F. WV. Putnam, personal communica-tion). Sequence was obtained with an automatic protein se-quencer. Brackets [ ] indicate residue not identified.

fibril and Bence Jones proteins are identical in theiramino-terminal portions.

The results of the acrylamide gel and peptide mapstudies suggested that the amyloid protein was an in-tact light polypeptide chain. Additional studies were per-formed to determine whether the carboxy-terminal tryp-tic tripeptide expected for K-type light polypeptide chainscould be isolated from the amyloid protein. Preparativetryptic peptide maps of the amyloid and Bence Jonesproteins were prepared and sprayed lightly with ninhy-drin to locate peptides in the area of the map where theC-terminal peptide should migrate (Fig. 2). As ex-pected, only one peptide was found in this region (be-low and to the left of the arrow indicating direction ofchromatography) and the compositions of the amyloidand Bence Jones peptides were compared with that ex-pected for the K-chain C-terminal peptide (Table II).These results indicate that both the amyloid fibril pro-tein and the Bence Jones protein contain a peptide iden-tical with that expected for the carboxy-terminus of aK-light chain and provide further evidence that the Tewamyloid protein is an intact K-chain.

DISCUSSION

The major constituent of the amyloid fibril protein iso-lated from the tissue of this patient with plasma cell

TABLE I IMolar Ratios Relative to Glu

Tew TewK-Chain Bence Jones amyloid

expected obtained obtained

Gly 1 0.87 0.86Glu 1 1.00 1.00Cy SO3H 1 1.34 1.17

Amino acid compositions of tryptic tripeptides eluted fromTew amyloid and Bence Jones maps, compared with ex-pected composition for C-terminal tripeptide of K-type lightpolypeptide chains.

Amyloid Fibril Protein Structure 1279

Proteins hydrolyzed in 6 N HCI at 110'C for 24 h. Resultsexpressed as mol/100 mol of amino acid.

TABLE IIISummary of some Chemically Characterized Major Amyloid Fibril Proteins

Amyloid fibrilClinical Organ N-terminal

Patient reference analyzed sequence Reference

5 (Ale) 10 Amyloidosis (4, 5) liver K-chain (2)

4 (Art) Amyloidosis with epidermolysis bullosa and tuberculosis (4) spleen K-chain (2)

14 (Tew) Amyloidosis with plasma cell dyscrasia (8) intestine K-chain This paper

(LuA) Isolated nodular pulmonary amyloidosis (*) lung X-chain (6)

A-4 Amyloidosis with tuberculosis (13) liver Arg-Ser-Phe-Phe- (14)

2 (Med) Amyloidosis with rheumatoid arthritis (4) spleen Arg-Ser-Phe-Phe- (15)

Paa 1° Amyloidosis thyroid Ser-Phe-Phe §

Pab Amyloidosis with bronchiectasis t thyroid Ser-Phe-Phe §

* Page, D. L., C. Isersky, M. Harada, and G. G. Glenner. 1972. Immunoglobulin origin of localized nodular pulmonary amyloido-sis (18).$ Anders, R. Personal communication.§ Glenner, G. G., R. Anders, and W. D. Terry. Unpublished data.

dyscrasia appears to be an intact K-polypeptide chainidentical with the Bence Jones protein isolated from thepatient's urine. The evidence for identity is as follows:(a) The reduced and alkylated purified amyloid fibrilprotein gave acrylamide gel electrophoresis patternsidentical with those obtained with the Bence Jones pro-tein. (b) The sequence of the amino-terminal 27 aminoacids was identical for both proteins. (c) Both proteinshad a carboxy-terminal tripeptide (defined by the ab-sence of lysine or arginine) of appropriate peptide maplocation and amino acid composition for the C-terminalpeptide of a K-chain. (d) Amino acid compositions were

essentially identical and peptide maps of the two proteinswere indistinguishable, indicating that no major inser-tions or deletions are present in the portion of the amy-loid fibril protein molecule not directly sequenced. Thedata concerning the yield of protein extracted from amy-loid tissue, as well as the yield of the N-terminal aminoacids in the sequencer, indicate that the amyloid proteinbeing sequenced is indeed the major protein constituentof the amyloid fibrils, and it seems reasonable to con-

clude, therefore, that the major protein constituent of the

amyloid fibrils is the complete K-Bence Jones protein that

was present in the patient's circulation and urine.Previous studies have shown that the amyloid fibril

proteins from a case of primary amyloidosis (2, 4, 5)and a case of amyloidosis secondary to epidermolysisbullosa and pulmonary tuberculosis (2, 4) had amino

acid sequences homologous with those of K-chains (Ta-ble III). The amyloid protein from a patient with iso-lated nodular pulmonary amyloidosis was shown to have

an amino-terminal tetrapeptide homologous with that of

X-chains (6, 18). The present work adds amyloid associ-ated with plasma cell dyscrasia to the group of diseasestates with immunoglobulin proteins in the amyloiddeposits.

The pathogenic significance of these results are ob-scure, but certain possibilities deserve consideration. Aspreviously reported, the plasma cell dyscrasia and as-sociated amyloidosis in this patient were preceded bymany years of chronic colitis (8). It was further shownthat fluorescein-labeled Tew Bence Jones protein had anunusual binding affinity for small intestine, which couldbe specifically blocked by pretreatment with the unlabeledBence Jones protein. It was therefore suggested that theTew Bence Jones protein might have been part of anautoantibody, i.e., the light chain of an anti-intestine an-tibody, produced in great excess as a consequence of thedevelopment of a plasma cell dyscrasia after long-termintestinal inflammatory disease. This implies that thedeposition of the Bence Jones protein, at least initially,is a specific immunologic phenomenon. The continuedaccumulation of the Bence Jones would then be the proxi-mate cause of the amyloidosis, perhaps due to a combina-tion of immunologic and physicochemical properties.

Whereas the above speculations are consistent with theclinical and experimental data, there is no proof for thishypothesis. Alternative speculations can be generated andfurther experiments will be required to achieve an un-derstanding of the pathogenesis of this disease.

The finding that the major protein component of cer-tain amyloid deposits does not appear to be an immuno-globulin (13-17) is obviously important to any specula-tions concerning pathogenesis. It is possible that theseamyloid fibril proteins of unknown origin are related toimmunoglobulins and that they are: (a) a new variableregion subgroup of either light or heavy chains, (b) afragment of a constant region of a light or heavy chainthat has not yet been sequenced, or (c) a portion ofS-piece, J-piece or of some other immunoglobulin as-sociated polypeptide chain that is as yet unsequenced.Alternatively, either of the two different types of amyloid

1280 Terry, Page, Kimura, Isobe, Osserman, and Glenner

fibril protein may be deposited in tissue as the endprocess of two entirely different pathogenetic mecha-nisms. This seems unlikely, since one patient with amy-loid fibrils of immunoglobulin type had the clinical pic-ture of chronic disease that is stated to be more frequentlyassociated with amyloid fibril protein of unknown ori-gin (2, 4), and, conversely, the thyroid of at least onepatient with the clinical picture of "primary" amyloido-sis without known chronic disease contained amyloidfibril protein of unknown origin.4 The overlapping clini-cal pictures asociated with tissue deposition of each typeof amyloid fibril protein suggests that they both may re-sult from similar pathogenic mechanisms.

The work presented here demonstrates that when a pa-tient with plasma cell dyscrasia produces a monoclonalprotein and develops amyloidosis, the major protein inthe amyloid may be that monoclonal protein. It will beimportant to investigate other patients of this type todetermine whether this is an invariable finding, orwhether in some patients with this clinical picture, the"nonimmunoglobulin" form of amyloid fibril proteincan predominate in the tissues.

ACKNOWLEDGMENTSThe superb technical help of Mr. Robert Guyer and Mr.Matthew Miller is gratefully acknowledged. We thank Doc-tors Whitley and Putnam for permitting us to review andcite their unpublished work.

This work was supported by grant CA-02332 from theNational Cancer Institute.

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2. Glenner, G. G., W. D. Terry, M. Harada, C. Isersky,and D. L. Page. 1971. Amyloid fibril proteins: proofof homology with immunoglobulin light chains by se-quence analyses. Science (Wash. D. C.). 172: 1150.

3. Glenner, G. G., D. Ein, E. D. Eanes, H. A. Bladen,W. D. Terry, and D. L. Page. 1971. Creation of "amy-

'Glenner, G. G., R. Anders, and W. D. Terry. Unpub-lished data.

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11. Glenner, G. G., M. Harada, C. Isersky, P. Cuatrecasas,D. L. Page, and H. Keiser. 1970. Human amyloid pro-tein: diversity and unformity. Biochem. Biophys. Res.Commnun. 41: 1013.

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Amyloid Fibril Protein Structure 1281