The Occurrence in Sera with M-Componentsof Different Proteins Related to AmyloidFibrils
C;, HUSBY. J. B, NATVIG & M, HARBOEInstitute of Immunolog)' and Rheumatology, Rikshospitalet University Hospital,md University of Oslo, Institute of Experimental Medicai Research, O.slo, Norway
Husby, G., Natvig, J. B. & Harboe, M, The Occurrence in Sera with M-Compo-nents of Different Proteins Related to Amyloid Fibrils. Scand. / , Immunol. 3.391-396, 1974,
A component (protein ARC) antigenicaliy related to the uncommon amyloid fibrilprotein AR was detected in 16 of 352 sera with M-components. Another serumcomponent (protein ASC), related to the commoner nonimmunoglobulin amyloidfibril subunit protein AS, was found in l49 of the same 352 sera. However, onlyone of the 16 sera with protein ARC contained protein ASC, giving evidence for ahigh degree of mutual exclusion between the two amyloid fibril-related componentsin serum. A considerable deviation of the normal x/A. ratio towards a predominanceof the X type of light chain was seen among the sera with monoclonal IgG or IgAand protein ARC. No similar deviation of the X/A ratio could be observed amongthe sera with protein ASC.
G. Husby, M.D., Institute of Immiir.olagy and Rheumatology, F. Qi'a?ns gt. 1,Oslo 1, Nor-way
During recent years many new data on theantigenic and chemical composition of amyloidfibrils have been reported (5, 14). Some am-yloid fibrils appeared to consist of light-chainvariable fragments from homogeneous immuno-globulins (7), while another type of fibril hadthe nonimmunoglobulin protein AS as a majorcomponent (1, 3, 4, 10, 15, l6, 23).
Recently, a second component, protein AR,which composed 66% of the total protein ofthe amyloid fibrils derived from a patient(A.R,) with primary amyloidosis, was describedby Husby et al. (14). It was suggested thatprotein AR represented a major component of athird type of amyloid fibril.
In previous reports (10, 12, 13, 17) we havedescribed a component found in human sera,called protein ASC, that was structurally relatedto amyloid protein AS. This semm protein maybe a precursor of certain amyloid fibrils (12).
It is the aim of the present communication toreport another serum component related to theamyloid protein AR. This component, ARC,was first detected in the serum of Patient A.R.,who was the source of this particular amyloidfibril preparation (14). Since patients withlymphoproliferative disorders frequently have ahigh concentration of protein ASC in serum(12, 13) and are prone to amyloidosis, serafrom such patients were chosen for the searchfor the amyloid fibril protein AR-related com-ponent.
MATERIALS AND METHODS
Amyloid fibril subunits. Preparation of amyloidfibril subunits, protein AS from amyloid prep-arations I (T.H.) and XVIII (J.B.), and pro-tein AR from amyloid fibril preparation XXII
392 C. Husby, J. B. Natvig & AI. Harhoe
Fig, 1. Double immunndiffusion in gel. Anti-proteinAR in centra! well tested against degraded amyloidA.R. {well 1), serum A.R. (2) . purified amyloidprotein AR (3). two sera with M-components con-taining protein ARC (4, 5), and a normal controlserum (6),
(A,R.) was carried out as previously described(11, l4, l6, 17), using extraction of amyloidfibrils with water, followed by degradation ofthe fibrils with 6 M guanidine--0.1 M dithio-threitoi (DTT), or with 0.1 N NaOH andsubsequent gel filtration on Sephadex G-lOO(8, 21).
P-component. The P-component of amyloid(2, 6) was prepared from an amyloid-ladenliver of a patient with secondary amyloidosis,by the method described by Glenner et al. (6),
Sera and seri/m proteins. Sera from 352 pa-tients were tested for the presence of amyloidfibril proteins AR- and AS-related components.The main criterion for selection was that thesera had shown a significant monoclonal bandwhen subjected to agar gel electrophoresis, per-formed as a routine laboratory procedure at theInstitute for Experimental Medical Research,Oslo City Hospital. The M-components werethereafter typed for heavy-chain class and also,in all cases of IgG and IgA, for light-chaintype, by immunoelectrophoresis. Some cases of
IgM were typed for y, and A after densitygradient ultracentrifugation (9). For IgG andIgA approximately equal numbers of sera withV. and A monoclonal proteins were tested. Apanel of individual and pooled normal seraser\'ed as controls. Isolated serum proteins, in-cluding various immunoglobulins and chainsand fragments thereof (heavy and light chains,F(ab')2, and Fc), were also included for con-trol.
Antisera. Antisera to protein AS and to pro-tein AR were obtained by immunizing rabbitswith crude amyloid preparations treated with0.1 N NaOH (DAM), The antiserum to pro-tein AS (anti-amyloid J.B.) has been describedpreviously (13, 17). After absorption withthree times its volume of pooled normal humanserum, it was monospecific to protein AS in thetest systems used.
When tested against protein AR, the anti-protein AR antisemm showed a single precipi-tation line fusing with the line of the crudedegraded amyloid used for immunization. Noprecipitation reaction was obtained with theother main, high molecular weight component(the void volume material) of amyloid A.R.,with 21 other amyloid preparations or subfrag-ments, or with normal human plasma or plasmaproteins, when tested in double immunodiffu-sion (14). Yet a moderate absorption withnormal human serum was carried out by adding0.2 ml human serum to I ml of the antiserum.
An antiserum to the P-component of amyloidwas kindly given to us by Dr. A, S. Cohen.
Immunologic methods. Double immunodiffu-sion was carried out in 1% agarose in barbitalbuffer, pH 8.6, ionic strength 0,025. The thick-ness of the gel was 1 mm; the diameter of thewells, 3 mm; and the center-to-center distanceof the wells, 7.5 mm. The plates were kept ina moist chamber at room temperature for 48hr and then photographed.
RESULTS
Amyloid prolein AR-related component (ARC)in human sera
The semm of Patient A.R. reacted stronglywith anti-protein AR in double immunodiffu-
Serum Proteins Related lo Amyloiil 395
Table I, Amyloid protein AR-related component in sera with monoclonal immunoglobulins
M-component
IgGIgAIgM
Total no.tested
15580
117
Total no,positive
736
Precipitationreaction
202
Inhibitionreaction
534
Light-chain type {no.
X
1/700/393/10*
positive/no, tested)
A
6/853/412/6*
Only 16 of the 117 IgM M-components were typed for light chains.
,sion (Fig. 1). In addition, of the 352 sera withM-components, 16 reacted with the antiserum(Table I). Four of these sera and the serumfrom Patient A.R, gave a precipitation line fus-ing with that obtained with purified proteinAR or with crude NaOH-treated amyloid A.R.(DAM XXII). A reaction of antigenic identitywith protein AR was thus obtained with thesesera, as far as the antiserum and the methodcould detect. The precipitation reaction obtainedwith the positive sera was completely inhibitedby adding protein AR or crude degraded amy-loid A.R. (DAM XXII) to the antiserum. Inaddition, 12 sera clearly inhibited the precip-itation reartion between protein AR and anti-protein AR when applied in neighboring wellsof protein AR (Fig. 2), The sera that gavea visible precipitation line fusing with that ofprotein AR were also tested in dilutions notgiving direct precipitation, and in these casesa similar inhibition reaction was obtained. Thisshowed that the sera that gave only a reactionof inhibition even in an undiluted state hadsimilar antigenic determinants, but in lowerconcentrations than those giving a precipitationreaction with anti-protein AR.
Protein AR did not react with an antiserumto the P-component or with anti-protein AS.When sera with protein ARC were testedagainst anti-P-component, a precipitation linewas obtained, but this line showed a reactionof nonidentity with that obtained with anti-protein AR. Furthermore, no precipitation re-action was obtained between anti-protein ARand the P-component, and tbe latter componentdid not even inhibit the reaction between anti-protein AR and protein AR or a serum contain-
ing protein ARC. No antigenic similarity be-tween the P-component and the amyloid fibrilprotein AR or the related protein ARC in serumcould, therefore, be observed.
No precipitation or inhibition reaction wasobtained when a panel of individual and poolednormal sera and various isolated serum proteins,including immunoglobulins and their chains orfragments, were tested with anti-protein AR.
Among the sera with protein ARC, the M-component was of the IgG type in seven cases,one of these being of kappa and the other sixof lambda type. Three positive sera had mono-clonal IgA, and all these were of lambda type.Five sera contained monoclonal IgM, three ofK and two of A type, while one serum (serumSe, 9), contained didonal IgM (IgM, jc plusIgM,A). Thus, II of the \6 positive sera(69%) contained monoclonal Ig with A Hghtchains, four sera had monoclonal Ig with xlight chains, while one serum contained diclonalIg with both x and \ light chains. A con-siderable deviation of the normal K/A ratiotowards predominance of Ig with A type wasthus observed among the sera containing theamyloid protein AR-related material (Table I).
Amyloid protein AS-related serum compo-nent (ASC). Out of the 352 sera tested, 149(42%) contained protein ASC in sufficientamounts to be detected by the double immuno-diffusion test system (Table II), A precipitationreaction of antigenic identity with that of puri-fied protein AS was always obtained with thepositive sera. The frequency of protein ASCwas similar in sera witb M-components of IgGand IgA class (47%), whereas the sera withmonoclonal IgM contained protein ASC with
394 C. Hushy. } . B. Nati'ig & M. Harboe
Fig. 2. Anti-protein AR in lower well testtd ayainsta serum with M-coniponent (well 1), degraded amy-loid A,R. (2), and a normal control scrum (3). Theserum in well 1 clearly inhibited the precipitationreaction between amyloid A.R. and anti-protein AR.
a significantly lower frequency (32%). No sig-nificant deviation of the expected yj\ ratio wasobserved among the protein ASC-containingsera (Table II).
Mutual exclusion of protein ARC and proteinASC in serum. Only one serum (with M-com-ponent IgA,A,) of the \6 sera reactive withanti-protein AR showed a reaction with anti-protein AS. A highly significant state of mutualexclusion between the two serum componentsantigenicaliy related to amyloid protein AR andamyloid protein AS, respectively, was thus ob-served (P < 0.05).
DISCUSSION
Recent characterization of amyloid fibril proteinfrom different individuals and ditferent clinicaltypes of amyloidosis has provided evidence fordifferent structural classes of amyloid fibrilsand their subunits (1, 5, 6, 14). A search forserum material related to the different types ofamyloid fibril protein was a natural consequenceof these findings.
Putnam et al. (22), using amino acid se-quence analysis, showed that the x Bence Jonesprotein from a patient (Tew) with multiplemyeloma and amyloidosis was chemically iden-tical with the amyloid fibril protein isolatedfrom the small intestine of the same patient.The sequence obtained for the 27 amino-ter-minal residues of the x chains was identicalwith the amyloid fibril protein previously stud-ied by Terry et al. (24). Thus, these amyloidfibrils were probably generated from homol-ogous immunoglobulin light chains present inexcess amounts in the organism.
Furthermore, a serum component (proteinASCI) that is antigenicaliy identical with anothermain class of amyloid fibril protein, protein AS,was described by Levin et al. (18) and byHusby et al. (10, 12, 13, 17). It was suggestedthat the protein ASC detected in serum was acirailating precursor of the amyloid fibril pro-tein AS (12).
In the present study, a third serum compo-nent (protein ARC), related to a third anduncommon amyloid fibril protein, protein AR(14), is described. Protein ARC was detectedwith a low frequency (5%) in a panel of 352
Table II, Amyloid protein AS-related component in sera with monoclonal immunoglobulins*
M-Component
IgGIgA
Total no.tested
15580
Total no.positive
7338
positive
4747
Light-chain type (no.
X
3 1 /TO9/595/10
positive/no, tested)
A
42/8510/41
2/6
* The sera with monoclonal Ij{G and IgA contained protein AS with a significantly higher frequency thanthe sera with monoclonal IgM (P <^ 0.01, Fisher-Irwin test).
Serum Proteins Related to Amyloid 395
-.era with 'M-components'. By analogy with pro-[tin ASC it is, therefore, possible that theprotein ARC in serum is a precursor for theamyloid fibrils that have the uncommon pro-tein AR as a major component. The low fre-i-juency of protein ARC in serum correlates wellwith the rare occurrence of amyloid protein ARitself (14), However, the finding of proteinARC in the serum of patients other than Pa-tient A.R, (14) showed that the amyloid pro-tein AR is not unique in its antigenicity.
The present results also showed that there isno antigenic relationship of protein AR andprotein AS to the P-coniponent of amyloid.This was expected, since the P-component is not;i constituent of the amyloid fibrils (2, 6, 12).It has also been shown to be antigenicaliy iden-tical with a normal serum protein (2) butchemically different from both protein AR andprotein AS (12, 14).
The amyloid protein AR was isolated froma patient with primary amyloidosis (14). Aclose relationship of primary amyloidosis andmultiple myeloma has previously been suggested(19, 20). The finding of the related proteinARC in some sera with M-components was,therefore, not unexpected.
The frequency of prolein ASC in the pres-ently studied sera (42%) was somewhat lowerthan reported in a previous study, in whichthe same antiserum to protein AS and the sameimmunological method were used (12). Thisdiscrepancy was most probably due to differ-ences in the criteria for selection of sera withM-components. In the previous study, only serafrom patients with clinically established mye-lomatosis and macroglobulinemia, with orwithout complicating amyloidosis, were exam-ined (12). In contrast, a monoclonal band atagar gel electrophoresis was the only criterionfor selection of sera in the present study, andmany of the patients had benign monoclonalgammopathy.
Another notable observation was the positivecorrelation of protein ARC-con tain ing sera withM-coniponents of the A type. This was so forboth IgG and IgA myeloma proteins, whereasthe monoclonal macroglobulins present in thesera with protein ARC showed no significant
selection of light-chain type. In this connection,a reversion of the usual x/A. ratio towards apredominance of the \ type has been observedpreviously in patients with plasma cell dyscrasiascomplicated by amyloidosis (20). Even if themajor amyloid fibril protein, protein AR, inthis particular case of primary amyloidosis didnot appear to be a light-chain fragment, thisdoes not exclude the possibilit)' that light chainsor other immunoglobulin chains or fragmentsby some mechanism may influence the forma-tion of such amyloid fibrils. However, no simi-lar deviation in the x/A ratio was observedamong the sera that contain protein ASC.
The high degree of mutual exclusion betweenprotein ARC and protein ASC in serum wasstriking and fits well with the recent observationthat amyloid fibrils containing protein AR donot contain detectable amounts of protein AS(14). There is evidence that protein AR cansubstitute for protein AS in the formation ofamyloid fibrils, in interaction with the common,high molecular weight protein component ofthe fibrils (14). This seems to be the case forthe related materials found in serum—proteinARC and protein ASC, respectively-—which aresuggested to be circulating precursors foramyloid fibrils.
ACKNOWLEDGEMENTS
This work was supported by the NorwegianResearch Council for Science and the Humani-ties, the Norwegian Rheumatism Research Coun-cil, and Anders Jahre's Eund for the Promotionof Science. The skillful technical assistance ofMrs. J. Deverill and Mrs. Vibeke Skou isgratefully acknowledged.
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Received 8 February 1974
