characterization of amyloid deposits and p component from a patient with factor x deficiency reveals...

Characterization of Amyloid Deposits and P Component

From a Patient with Factor X Deficiency Reveals Proteins

Derived from a Lambda VI Light Chain

DANIEL COHEN, M.D.

New York, New York

MORDECHAI PRAS, M.D.

Tel-Aviv. Israel

EDWARD C. FRANKLIN, M.D.

BLAS FRANGIONE, M.D., Ph.D.

New York, New York

From the Irvington House Institute (DC, ECF, BF). and the Departments of Medicine and Pathology, New York Unjversity Medical Center (BF), New York, New York, and the Hefter lnstiiute of Medical Research, Chaim Sheba Medical Center at Tel- Hashomer, and Tel-Aviv Medical School, Tel-Aviv, Israel (MP). This work was supported in part by U.S. Public Health Service Research Grant AM 02594 and the Henri G&&erg Amyloid R&arch Grant. Requests for reprints shouM be addressed to Dr. Blas Frangione, Irvington House Institute, Department of Pathology, New York University Medical Center, 550 First Avenue, New York, New York 10016. Manuscript accepted June 3, 1982.

Amyloid fibrils were isolated from a spleen obtained at surgery from a 58-year-old white man wtth primary amyloidosis presenting with factor X deficiency and respondlng dramatically to splenectomy. Gel filtration on Ultragel ACA 54 in 5 M guanidine 1 M acetic acid yielded components with molecular weights between 17,000 and 13,000. Two of them (17K and 15K) were studied in detatl. Antigenic and amino acid sequence analysis showed that these proteins were related to lambda VI immunogkbulin IigfM chain. The predominant protein subunits of the amyloid fibril of the deposits (17K) was processed at the carboxy terminus in the same section of the con- stant region as the only other lambda VI amylotd protein previously reported. Amino terminal sequence of the 15K protetn revealed not only degradation at the C terminal, but also minor degradation at the amino terminal (three residues difference from the 17K species). P component was also isolated from the spleen and characterized. This represents the first antigenic and sequence analysis of tissue amyloid proteins and P component from a patient presenting with factor X defklency and anotfier example of amykfd proteks derived from the newly dkcovered amyloidogenic lambda VI light chain subgroup.

The amyloid diseases can now be divided immunochemically into several major fibril protein types. In the AL form associated with plasma cell proliferation, the fibrillar protein is composed mainly of amino terminal variable-region fragments of an immunoglobulin light chain. The deposits in acquired amyloidosis secondary to chronic underlying inflammatory infectious or neoplastic diseases and those in familial Mediterranean fever consist of the AA protein, which is a degradation product of an acute phase-reactant serum AA protein [ 11. A third type of amyloidosis, AF, associated with familial amyloid polyneuropathy syndromes, was recently shown to be related to prealbumin [ 2-51. The purpose of this paper is to report on the purification and primary structure of proteins isolated from the amyloid fibrils and the P component from the spleen of a patient with primary amyloidosis and factor X deficiency.

Factor X deficiency has not been reported to occur in secondary amyloidosis. Of the 30 reported cases of acquired factor X deficiency and systemic amyloidosis, serum or urinary paraproteins have been found in 13 and characterized as to light chain type by antigenic analysis in only nine [6-201. Eight of the 13 had IgG and three had IgA; two had only BenceJones proteins without intact monoclonal immunoglobulin. Of the nine light chains typed, five were kappa and four lambda [ 151. In no case has antigenic or sequence analysis of tissue

March 1983 The American Journal of Medicine Volume 74 513

LAMBDA VI AMYLOIDOSIS WITH FACTOR X DEFICIENCY-COHEN ET AL

amyloid proteins from such patients been reported, however. In fact, of the primary acquired amyloidoses unassociated with overt multiple myeloma, relatively few purified tissue proteins have been characterized; they have simply been assumed to be derived from immunoglobulin light chains. It remains important to substantiate the immunoglobulin origin of these primary amyloids. Their primary structure and cleavage patterns may yield clues as to the nature of the processing en- zymes involved. Furthermore, certain chemical types of light chains are degraded into fragments that are more likely to result in amyloidosis [21]. Elucidation of the basis of this phenomenon will require detailed bio- chemical studies of light chain-derived amyloid deposits.

CASE REPORT AND LABORATORY STUDIES

Clinical Data. A 58-year-old white man presented to Bel- levue Hospital with cachexia, massive splenomegaly, gross hematuria, and cutaneous bleeding. He had had surgery in the past without bleeding problems. He was afebrile, and physical examination revealed ecchymoses in periorbiil and weight-bearing areas, and a hard nontender spleen palpable to the level of the left iliac crest. Tine1 sign was negative, tendon reflexes were within normal limits, and the tongue was not enlarged. There were nc signs of congestive heart failure. Hematocrit was 16.0 percent, white blood cell count was 18,300 mm3, platelet count was 150,000, prothrombin time was 17111 seconds, and partial thromboplastin time was 60134 seconds. Urinalysis showed red blood cells and no significant proteinuria. Alkaline phosphatase level was 153, creatine phosphokinase level was 55 units/liter, lactic de- hydrogenase level was 268 IU/liter, albumin value was 2.8 g/dl, globulin level was 2.5 g/dl, bilirubin value was 0.9 mg/dl, serum glutamic-oxaloacetic transaminase value was 29 units/ml, and serum glutamic pyruvic transaminase value was 15 units/ml. Fibrinogen level was 270 mg/dl and factor X level was 10 percent of normal. Urine and serum immuno- electrophoresis revealed no paraproteins. Bone marrow biopsy findings and echocardiographic results were normal. A gingival biopsy specimen showed extensive amyloid de- position. Over five days in the hospital, the patient required 14 units of packed red blood cells because of hematuria. Massive infusions of fresh frozen plasma would not correct his clotting times. One thousand units of Konyne was required every four hours to ameliorate the bleeding. The patient un- derwent splenectomy; by the sixth postoperative day, bleeding had ceased and factor X level had returned to normal. Amyloid fibrils were extracted from the spleen, which was heavily infiltrated with amyloid deposits. Source and Purification of Amyloid Fibrils and Subunits. According to the method of Pras et al [22], 50 g of spleen tissue was homogenized five times in 0.15 Msaline solution, and the supernatants were discarded. The insoluble residue was homogenized in distilled water and subjected to ultra- centrifugation for one hour so that the amyloid fibrils became suspended as a mucoid mass at the upper layer. This upper layer was dialyzed against water and lyophilized. The yield

from 50 g of wet spleen tissue was 1.12 g of dry fibrils; 200 mg of lyophilized material was dissolved in 6 ml of 6 Mgua- nidine in Tris buffer, pH 10.2, and made 0.17 M in dithiothreitol for reduction overnight. Then, 2 ml of 2 M guanidine in 4 M acetic acid was added, and the solution was centrifuged at 100,000 X g, filtered, and fractionated on Ultragel ACA 54 columns equilibrated with 5 Mguanidine in 1 Macetic acid. Purity of protein fractions and molecular weight were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [23]. Amino Acid Analysis and Amlno Acid Sequence. Amino acid analysis was performed on a Durrum model P500 amino acid analyzer (Durrum Instrument, Sunnyvale, California). Samples were hydrolyzed in 2 ml of 6 N hydrochloride under vacuum for 24 hours at 11 OOC. To prevent degradation of tyrosine, 40 ~1 of a 10 percent aqueous solution of phenol was added.

Automated amino acid sequencing was performed on a Beckman 89OC sequencer (Beckman Instruments, Fullerton, California). Phenylthiohydantoin amino acids were identified by high-pressure liquid chromatography performed on a Waters HPLC model ALC/GPC-201 (Waters Instruments, Rochester, Minnesota) prepacked with a C18/~ Bondapak column (Waters Instuments) and eluted with a methanol-water gradient and by amino acid analysis after back hydrolysis by phenylthiohydantoin amino acids. Carboxy Terminus Determination. Purified amyloid protein was dialyzed exhaustively against distilled water, and 1 mg of lyophilized protein was added to 0.5 ml of 0.1 M ammo- nium bicarbonate, pH 8.0, with 20 jd of a 2 mg/ml suspension of either carboxy peptidase A or B (Sigma). The reaction mixture was maintained at 37’C. It was periodically centrifuged, and 40 ~1 aliquots of supernatant subjected to amino acid analysis at five, 10, 30, 60, and 120 minute intervals. Isolation of Amylold P Component from Spleen. The second to fourth saline supernatants of the spleen homogenate were mixed 1:l with acetone and allowed to precipitate in the cold overnight. This precipitate was exhaustively dialyzed against distilled water, lyophilized and fractionated on Ultragel ACA 54 in guanidine acetic acid as for the amyloid fibrils (except that the proteins were not reduced with dithiothreitol before gel filtration). Purity and molecular weight was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigenic Analysis of Amylold Proteins and P Component. Antigenic analysis of amyloid proteins was done by Oucht- erlony method in 1 percent agar, 0.1 percent sodium dodecyl sulfate in barbital buffer, using standard anti-kappa and anti-lambda antiserums or goat anti-human factor X. Goat anti-human factor X was provided by Dr. Sidones Morrison (State University of New York at Stony Brook), and purified human factor X was received courtesy of Dr. Bruce Furie (Tufts New England Medical Center). The goat antiserum gave a line of identity with fresh normal plasma and purified factor X.

Ouchterlony analysis with subgroup-specific antiserums was performed by Dr. Alan Solomon (University of Tennessee Medical Center). Antigenic analysis of the usual amyloid P component and washes of amyloid fibrils was performed with Ouchterlony plates identical to those used for the amyloid

514 March 1983 The American Journal of Medicine Volume 74


Figure 7. a, fractionation of amyloid fibrils from the patient’s spleen on 1 X 72 inch columns of Ultragel ACA 54 equilibrated in 7 M acetic acid/5 M guanidine. Fractions (2 ml/ tube) were collected. b,

sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 17 percent slab gel of purified components from Figure la. Markers: phosphorylase b 94,000 molecular weight, bovine serum albumin 67,000 molecular weight, ovalbumin 43,000 molecular weight, carbonic an- hydrase 30,000 molecular weight, soy- bean trypsin inhibitor 20,100 molecular weight, alpha la&albumin 14,400 molecular weight, AA protein 8,000 molecular weight.

2.5 - - la. lb.

-‘*

2.0 - IIIHP

5 2 1.5 -

2

1.0 -

0.5 -

0 :I’ ’ 1 70 80 90 100 110 120 130 1LO 150 160

Tube Number

proteins but without the addition of 0.1 percent sodium dodecyl sulfate to the agar.

RESULTS

Figure la shows the pattern obtained when the amyloid from the spleen was fractionated on Ultragel ACA 54. The tubes were pooled as indicated and are labeled as fractions I to V. Fraction 0 was not studied further. Fractions I to V contained the four low-molecular-weight amyloid protein subunits that ranged in size from 17,000 to 13,000 daltons. Their purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure lb). Only pooled fraction I (17K) and pooled fraction II (15K) were studied. The amino acid composition of pooled fractions I and II are shown in Table I, and their amino terminal sequences are compared with the lambda VI prototype AR [24] Figure 2. Twenty-one amino terminal residues of the 17K protein and 18 of the 15K were obtained and are identical to the previously reported lambda VI amyloid protein AR [24], with the exception of positions 9 and 20 where glutamic acid and isoleucine are substituted for serine and phenylal- anine, respectively. The 15K protein (Figure 2) starts at position 4. Its molecular weight and amino acid composition (Table I) indicate that it is degraded at the C terminal as well. Carboxy peptidase B digestion of the 17,000 molecular weight protein did not release any significant amino acids. On the other hand, carboxy peptidase A digestion of this protein released 2.1 nmol of alanine, 2.0 nmol of valine, and 1.1 nmol of threonine at two hours.

None of the four pooled fractions in Figure la reacted with standard anti-kappa or anti-lambda light chain antiserums. Pooled fraction of peak I (Figure la) reacted with subgroup-specific antiserums against lambda VI light chains. The other pooled fractions were not tested with the subgroup-specific antiserums. Neither the

TABLE I Amlno Acid Composition* of Amyloid Proteins from Patient

17K (Peak I, Figure 1A) 15K (Peak It, Ftgure 1A) (rsskfues per nlolscule) (retktues ~sr m&ails)

cyst 2.8 1.7 Asp 14.9 13.1 Thr 12.2 11.6 Glu 14.4 ld.1 Pro 9.01 8.9 GUY 12.5 11.0 Ala 10.9 9.3 Val 8.9 8.2 Met 1.0 0.9 Ile 5.4 4.8 Leu 8.45 7.6 Tyr 7.2 5.9 Phe 4.4 3.9 His 2.5 2.2 LYs 4.3 4.1 Ar9 6.1 4.1 Ser 18.9 17.8 Trpz

l Mean value of two runs each (not corrected). + Determined as cyst&c acid. 2 Not done.

March 1983 The American Journal of Medicine Volume 74 515


Protein

AR

5 10 15 20

Asp-Phe-Met-Leu-Thr-GIn-Pro-His-Ser-Val-Ser-GIu-Ser-Pro-Gly-Lys-Thr-Val-Thr-Phe-Ser

17K- GIU

15K Glu

Ile-

Ile-

Figure 2. Amino acid sequence of amyioid proteins (17K and 15K) obtained from the spleen of the patient in comparison to-lambda VI protein AR [2?4]. _

Figure 3. Top, sodium dodecyi sulfate-polyacrylamide ge, I electrophorMs; 17 percent slab gel of purified P component from the patient (lane IV), compared with P-rich tissue extracts previously characterized (lanes II, Ill). Bottom, Ou- chterlony plate showing line of identity between P component isolated from the patient and known usual amyloid P component. Center well contains rabbit anti-P component antiserum. Well I-P component of the patient; wells II and VI-known amyloid P component.

amyloid fibrils nor sequential saline solution or EDTA

washes of them gave any lines of reactivity with the goat anti-human factor X, despite concentration of the washes ten-fold (20 mg of amyloid fibril washed with 0.75 ml). Factor X was also not detectable in the initial saline solution extracts of the spleen once they were cleared of gross blood.

Figure 3, top, shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis of amyloid P component isolated from the patient’s spleen compared with a marker of known P component, and Figure 3, bottom, shows the reaction of identity between this P component and P component previously isolated from other tissue amyloids in our laboratory using antiserum raised in rabbits against known pure P component. The amino terminal sequence of our patient’s tissue P component is shown in Figure 4.

COMMENTS

On the basis of the partial amino acid sequences (Figure 2) and amino acid compositions, it would appear that the 17,000 molecular weight and 15,000 molecular weight amyloid proteins are derived from a light chain of the lambda VI subgroup [25]. Since the 17,000 molecular weight and 15,000 molecular weight species differ from each other by only three residues at the amino terminus, they therefore must have undergone the major processing at the carboxy terminus. The results of carboxy peptidase A and B digestions of the 17,000 protein are compatible with a C terminus with the sequence alanine-valine-threonine-valine-alanine (positions 148 to 152) ending very close to lysine 154, where the prototype lambda VI amyloid protein AR ends.

This case represents the first characterization of the tissue amyloid from a patient with factor X deficiency and substantiates the AL origin of the amyloid in such patients.

It seems unlikely that the lambda VI subgroup plays a role in the accumulation of factor X in the spleen, since, in other cases [6,11,12], kappa-type paraproteins were reported. Why factor X deficiency states

516 March 1983 The American Journal of Medicine Volume 74


5 IO 15 20 His-Thr-Asp-Leu-Ser-Gly-Lys-Val-Phe-Val-Phe-Pro-Arg-GIu-Ser-Val-Thr-Asp-His-Val

25 30 35 40

Asn-Leu-lie-Thr-Pro-Leu-Glu-Lys-Pro-Leu-Gln-( )-Phe-Thr-Leu-( I-Phe-Arg-Ala-Tyr

43 48 Ser-Asp-Leu-Ser-Arg-Ala-Tyr-Ser

Figure 4. Amino acid sequence of amyloid P (AP) component obtained from the spleen of the patient (Figure 3, top, lane IV); ( ) = negative.

arise only in association with primary amyloid deposits is not known.

Our study did not reveal evidence of factor X bound to isolated amyloid fibrils, as was indicated with a sensitive gamma carboxy glutamate assay in another study [26]. It remains to be determined whether or not all types of AL amyloid can be associated with factor X deficiency, if there is a large enough body burden with massive splenic involvement.

Our patient’s amyloid deposits were shown to contain large amounts of P component by antigenic analysis and amino terminal sequencing. It was identical to the 27 residues previously published for amyloid P component [27] ; we obtained 21 more residues up to position 46.

This case is particularly informative as it is another instance of lambda chain amyloidosis of the lambda VI subgroup that, in our experience, is overrepresented in primary light chain amyloid deposits [21] and may represent a particularly amyloidogenic molecule. Of the 19 known occurrences of the lambda VI subgroup, in only three cases were the patients not amyloidotic. In

these cases, no tissue was examined [25,26,29], and in only one [25] was the paraprotein a Bence-Jones protein. Of the three lambda VI amyloid proteins reported [24,30,31], the carboxyl terminus of only one (AR) is known. The amyloidogenicity of lambda VI could depend on its susceptibility to initial degradation in a vulnerable region between residues 150 and 154 as with AR and our protein. Cleavage at this point might be necessary for further processing, producing proteins of lower molecular weight than the predominant 17,000 molecular weight species. We have not excluded the presence of small amounts of intact lambda VI light chains in our patient nor the possibility that, in vivo, the predominant 17,000 molecular weight protein remained attached to the 66 carboxyl terminal residues from 14 1 to 217 [32] until the disulfide bridge between cysteine 139 and 198 was broken by reduction with dithiothreitol before gel filtration. If the disulfide bridge does not re- main intact in vivo, then initial processing of lambda VI would require both reduction and cleavage of peptide bonds.

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