Polymorphism in a kappa I primary (AL) amyloid protein (BAN)

Molecule Inzmunoli)g~, Vol. 23. No. I, pp. 13-78. 1986 Printed

in Great

Britain

C

POLYMORPHISM IN A KAPPA AMYLOID PROTEIN

I PRIMARY (BAN)

0161-5890186 $3.00 + 0.00 1986 Pergamon Press Ltd

(AL)

FRANCIS E. DWULET, TIMOTHY P. O’CONNOR and MERRILL D. BENSON Departments of Medicine and Biochemistry, Rheumatology Division, Indiana University Medical School, and Rheumatology Section, Richard L. Roudebush Veterans Administration Medical Center, Indianapolis, IN 46202, U.S.A. (Received 2 July 1985; accepted 26 July 1985) Abstract-In an attempt to understand the relationship of amino acid sequence to the formation of primary or multiple myeloma-related amyloid (AL amyloid), we have determined the complete amino acid sequence of amyloid protein BAN. This protein belongs to the kappa I immunoglobulin light chain subgroup and has a polypeptide chain length of 126 amino acids. It encompasses the entire variable region, the joining segment and the first tryptic peptide of the constant region. This protein has two unique features. First, the molecule is glycosylated. At position 61 the usual arginine residue has been replaced by an asparagine with the generation of the signal sequence Asn-Phe-Thr, to which a glucosaminecontaining carbohydrate unit is attached. Secondly, the protein is not monoclonal but consists of two chains which have the same variable region but different J-segments. Comparison of the BAN sequence with other amyloid and nonamyloid kappa I proteins reveals a systematic difference between the two groups. In the amyloid proteins, several hydrophilic framework residues have been replaced by hydrophobic residues. These substitutions may provide the nucleation sites for self-aggregation and fibril formation.

INTRODUCTION

Amyloidosis is the accumulation of extracellular protein deposits having certain physico-chemical properties which are a result of their unique structure (Glenner, 1980). These complexes are composed of a single protein organized into long unbranched strands of stacked subunits which form fibrils of indeterminate length with a beta-pleated sheet internal structure. There are several classes of systemic amyloidosis which can be identified by the subunit proteins from which they are assembled. Primary or (AL) amyloid is composed of monoclonal immunoglobulin light chains or segments originating from the variable (V) region of the molecule. These segments can be as small as half of the V-region or as large as the entire light chain (Glenner et al., 1971). Secondary or reactive amyloid (AA) is composed of a 76 amino acid polypeptide which is believed to be the degradation product of an acute-phase reactant protein (SAA) (Levin et al., 1972). This protein is found associated with high-density lipoprotein particles and serum concns are elevated in diseases characterized by chronic infection or inflammation (Parmelee et al., 1982; Benson and Cohen, 1979). The third type of systemic amyloidosis is a hereditary condition which is transmitted as an autosomal dominant trait. Although rare, the disease has a worldwide distribution and in all cases where the subunit protein has been identified it has been found to be a variant form of plasma prealbumin (Andrade et al., 1970; Dwulet and Benson, 1984; Pras et al., 1981; Saraiva et al., 1984; Tawara et al., 1983; Kametani et al., 1984).

In all of these conditions it is assumed that there is some property of the precursor molecule which induces it to self aggregate and form fibril complexes. For reactive amyloid (AA) this is believed to be related to high levels of the precursor protein (SAA) and its degradation products (Lavie et al., 1978). For the hereditary amyloids there is a point mutation in the prealbumin molecule which alters the proteins secondary structure and allows the molecule to self aggregate (Dwulet and Benson, 1984). However, for AL amyloid there is at present no known model to explain the biochemical mechanism of fibril formation. To this end we have begun a study of the amino acid structure of AL amyloid proteins for the purpose of elucidating any structural parameters relating to this condition. In this study we report the complete amino acid sequence of a kappa I AL amyloid protein and describe specific amino acid residues which may be unique to fibril-forming lightchain proteins.

MATERIALS AND METHODS Fibril isolation

Amyloid-laden tissue was obtained post morten from patient BAN, a 43-year-old man who died from rapidly progressive renal insufficiency. Fibrils were isolated from a 30-g sample of spleen by the method of Pras et al. (1968). The tissue sample was homogenized in a blender with 200ml of 0.01 M phosphate, 0.15 M sodium chloride, pH 7.4. The suspension was then separated in a Beckman 52-21 centrifuge with a JA20 rotor at 12,000 rpm for 30min. The super73

74

FRANCISE. DWULET et al.

natant was discarded and the pellet repeatedly homogenized and sedimented until the O.D. of the supernatant at 280 nm was less than 0.075. The pellet was then homogenized with water and the suspension centrifuged for 1 hr. This was repeated for a total of four cycles. The water wash supernatants and pellets were dialyzed exhaustively against water, then lyophilized and stored at -20°C. All four water wash preparations showed essentially pure amyloid fibrils when stained with Congo red. Subunit isolation

Amyloid fibrils (150 mg) were suspended in 8 ml of sample buffer which contained 6 M guanidine hydrochloride, 0.5 M Tris and 1 mg/ml EDTA, pH 8.5. After suspension the solution was deoxygenated with nitrogen gas and 80 mg of dithiothre~tol was added. The solution was stirred in the dark for 18 hr and then alkylated with 193 mg of iodoacetic acid which had been dissolved in 1 ml of 1 M sodium hydroxide. After 30 min the reaction was quenched with 200 ~1 of 2-mercaptoethanol. The sample was centrifuged to remove insoluble material and the supernatant applied to a column of Sepharose CL-6B (2.4 x 90 cm). Separated peaks as determined by absorption at 280 nm were pooled, dialyzed against distilled water and lyophilized. Tryptic digestion

Amyloid subunit protein BAN (6mg) was suspended in 2ml of water and a su~cient amount (usually SO-100 ~1) of 1 Nammonium hydroxide was added to solubilize the protein. The solution was blown with nitrogen gas to deoxygenate the sample and to remove excess ammonia. The sample was then made 0.1 M in ammonium bicarbonate by the addition of 0.2 ml of a 1 M stock solution. To this was added 0.12 mg of trypsin (2% by wt) dissolved in pH 3.0 water and the reaction allowed to progress for 12 hr at 37°C. The reaction was terminated by freezing and lyophilization. Peptide separation

The peptide mixture was suspended in 1 ml of 10% acetic acid and the insoluble material separated by centrifugation. The residue was then dissolved in O.Sml of 50% acetic acid. Peptides were separated by reverse-phase high-perfo~an~e liquid chromatography on a column (I x 25 cm) of Synchrom RPP. Contaminated pools were further purified on an Altex Ultrasphere C-18 column (1 x 25 cm). Both columns used 0.1% trifluoroacetic acid as the initial buffer and a gradient of acetonitrile to elute the peptides.

Tryptic peptide T-5 (40 nmoles) was dissolved in 1 ml of 0.1 M ammonium hydrogen carbonate and digested with 5 gg of chymotrypsin 12% (wtjwt)] for 12 hr at 37°C. The reaction was terminated by freez-

ing and lyophiliaation. Peptides were separated by HPLC on an Altex Ultrasphere C-18 column (1 x 25 cm) using 0.1% TFA as the initial buffer and a gradient of acetonitrile as the eluent. Amino acid analysis

Peptides were hydrolyzed under reduced pressure in 1 ml of redistilled 5.7 N HC1 containing 50 nl of a 5% phenol solution to reduce tyrosine oxidation. The samples were heated at 1lO”C for 20 hr and dried in vucuo over sodium hydroxide. The amino acid compositions were determined on a Beckman 119°C amino acid analyzer. Sequence analysis

All peptide and protein samples were sequentially degraded in a Beckman 890C sequenator using the 0.1 M quadrol program (121078). To all samples 3 mg of polybrene was added to prevent excessive peptide extraction from the reaction cup (Farr et al., 1978). Before degradation was begun the peptide and polybrene were subjected to one cycle in the sequenator in which no phenylisothiocyanate was added. This removed undesirable U.V. absorbing material so that identification of subnanomolar amounts of phenylthiohydantoin (PTH) derivatives could easily be accomplished. The PTH amino acids were identified by HPLC on an Altex Ultrasphere C-18 column (4.6 x 250 mm) using a minor modification of the procedure of ~imme~an et at. (1977). Those residues not clearly identified (usually threonine) were hydrolyzed under reduced pressure in 1 ml of 5.7 N HCl containing I mg of &Cl, at 150°C for 12 hr (Mendez and Lai, 1975). The liberated amino acid was then identified on a Beckman il9C amino acid analyzer. RESULTS

From the fibril preparation used to isolate the amyloid subunit protein a near quantitative recovery of the starting weight of material was obtained in the soluble fractions and the isolated amyloid subunit protein accounted for almost 60% of the recovered material. The complete amino acid sequence of the V-region of protein BAN is shown in Fig. 1, along with the peptides used to identify the entire structure. Advantage was taken of the sequenators ability to obtain long segments of structure. The intact BAN subunit protein was degraded for 48 cycles yielding a three-residue overlap with the long peptide T5. This segment of sequence clearly identified the protein as belonging to the kappa I subgroup. Peptide T5 was then degraded for 45 residues to position 90 with an unidenti~ed amino acid at position 61. Chymotrypsin subdigestion of this fragment yielded peptides which confirmed all the previously identified residues plus the two peptides from the carboxyl region to extend the sequence to residue 103. Peptide C3 was used to confirm the amino acid at position 61 as an Asx

Structure of amyloid protein BAN

,

C6

, h-7-f

-Co *

; ; +

*

*

*

*

CV +

I i

-a

-a

+

*

C'O_ +

t

*

105 Gly-'IW-Lym-Val-Gln-Ile-Ly.-Aq,izziii

Fig. 1. The complete amino acid sequence of the first 108 residues (variable region) of kappa I amyloid protein BAN. The arrows (+) designate residues identified by sequence analysis. Those directly under the amino acid indicate residues identified from degradation of the intact protein in the sequenator. The arrows under the lines indicate residues identified from peptides analyzed in the sequenator. Peptides identified with a T were isolated after tryptic digestion and those with a C were isolated after chymotrypsin cleavage of peptide T5. accounts for the carboxyl terminus of the BAN protein, contains the entire first tryptic peptide of the light-chain constant (C) region (Table 1). Amino acid composition and sequenator yield data from this peptide indicates that there is little if any heterogeneity at the carboxyl terminus of this peptide. No other peptides were isolated from any further into the C-region of the light chain.

residue and the presence of glucosamine on the amino acid analysis indicates the presence of an asparaginelinked carbohydrate. The sequence Asn-Phe-Thr (positions 61-63) is a signal sequence for the attachment of a carbonydrate chain to the asparagine (Robbins et al., 1977). Peptides T6a and T6b both accounted for residues 104-107 and represent a tworesidue polymorphism. Finally, peptide T8 which Table Residue CM-Cys ASP Tbr Ser GIU Pro GlY Ala Val Met Ile I .e,, TY~

Phe His LYS A@ TILl

Amyloid protein BAN I.5 (2) 9.3 (9) 10.1 (I I) 17.0(18) 15.8(15) 9.5 (9) 8.6 (8) 7.9 (8) 7.0 (7.5) 7.6 (8) 7.3 (7.5) -- is\-' 1-I 7.; 7.2 (7) 6.2 (6) 3.1 (3) 0.3 (I)

I. Amino Tl I-18

acid analysis T2 19-24

of protein

T3 2542

BAN peptides

T4 4345

~~.

0.6 (I) 2.1 (2) 0.9 (I) 4.5 (5) 2.1 (2) 1.0(l) I.1 (I) 1.0(l) I.1 (1) 0.9 (I) I .8 (2)

1.1 (I) I.7 (2)

I.1 (I)

I .8 (2) 3.1 (3) 0.9 (1) 1.2(l) 2.0 (2) 2.0 (2)

0.9 (I) 1.0(l)

I.1 (I)

I .9 (2) 1.0(l)

0.6 (I) 5.3 (5) 6.5 (7) 8.3 (9) 6. I (6) 3.3 (3) 6.1 (6) 2.0 (2) 1.0 (1) 2.8 3.6 5.5 4.3

I .7 (2) 1.0(l)

1.0 (I)

T5 46-103

I.1 (I)

(3) (4) (6) (4)

I.O(I)

(residues

per mole)

T6A 104107

0.9 (I)

T6B IO&l07

T7 108

I.1 (I) 0.9 (I) 1.9 (2) 2. I (2) 3.3 (3)

I.O(I)

2. I (2) 2.0 (2)

I.0 (I) I.1 (1)

1.0 (I) I.1 (I)

I.O(I) I.1 (I) 2.0 (2)

0.9 (I)

0.9 (I)

0.9(l) 1.0(l)

0.4 (I)

T8 109-126

FRANCISE. DWULET et al.

16 DISCUSSION

Within the BAN sequence there are a number of unique amino acid substitutions. The tyrosine at position 30 (CDRl) has not been previously observed and the serine at position 46 (FR2) has only been identified in a human DNA sequence (Bentley and Rabbits, 1980). Finally, in FR3 the asparagine-linked sugar at position 61 and the isoleucine at position 72 are also reported for the first time. The presence of multiple residues at positions 104 and 105 raises a question as to how such a polymorphism could have developed. This could result either from somatic mutations or the presence of two cell lines with different J-segment genes. While the Leu-Glu sequence is the product of one of the known five J-segment sequences, the Val-Gln sequence would require an as yet undescribed J-segment (Hieter et al., 1982). A review of other known kappa I sequences shows that there are a number of Jsegment sequences which are different from the five reported geonomic J-sequences (Kabat et al., 1983). Although the complete sequence data for kappa amyloid proteins are sparse, certain findings can be noted. It would appear that none of the different J-segment sequences has a predisposition for amyloid formation. Amyloid protein MEV (Eulitz and Linke, 1982) has a K-J3, while protein TEW (Putnam et al., 1973) has a K-J& and BAN has a K-J2. It is interesting to note that both the BAN and MEV proteins contain sequence changes which would require substitutions in their J-segment DNA. Thus it would seem that amyloid J-segments are mutationally quite active; however, from the present data it appears that the J-segment sequences of kappa amyloid proteins do not play a major role in fibril formation. The replacement at position 61 of an asparagine for an arginine is unexpected on two counts. First, the arginine at this position is highly conserved with only one change for a lysine being noted in 25 kappa light chain reported sequences (Kabat et al., 1983). Secondly, the change from arginine to asparagine would require two DNA base changes which is most unusual in an FR. The most probable intermediate is the single lysine residue seen at this position in protein BJ 26. The presence of carbohydrate on monoclonal immunoglobulin light chains is a relatively uncommon event. Studies of myeloma proteins show an incidence rate of 415% (Abel et al., 1968). The locations of these carbohydrate groups on a number of kappa and lambda light chains have been identified and they show a very asymmetrical distribution in the molecules (Sox and Hood, 1970; Spiegelberg et al., 1970; Garver et al., 1975; Kiefer et al., 1980). About two-thirds of the carbohydrate units are located around positions 26 and 92 which are parts of CDRl and CDR3 of the V-segment. It is not surprising that these portions of the molecule are glycosylated, because they are mutationally quite active and are in a random coil extended structure. This allows for easy incorporation of the carbo-

hydrate without disruption of the secondary structure of the light chain. The next most common site for carbohydrate attachment is at position 70 in kappa light chains, where the usual aspartic acid residue is replaced with an asparagine. This residue is located in the FR3 region and, based on the X-ray structure of protein REI, it is positioned on a beta turn on the same edge of the molecule as the CDR segments (Epp et al., 1975). Until now the only carbohydrate which was not attached to the CDR edge of the molecule was found in protein HOM where a mutation in the J-segment changed lysine 106 to an asparagine (Savvidou et al., 1981). This sugar chain would be located between the V- and C-regions and face away from the CDR. In protein BAN, position 61 is most amenable to accepting the large carbohydrate unit because it is located on the outer edge at a beta turn that faces toward the C-region and away from the active site. Thus, this bulky carbohydrate will face out into the solvent away from the interchain contact regions and the binding site. Since the BAN protein was isolated from amyloid fibrils, it is clear that the carbohydrate moiety at position 61 did not prevent subunit aggregation and fibril formation. There are two major mechanisms that may be involved in the formation of amyloid fibrils. (1) Abnormal degradation of monoclonal light chains might cause unusual intermediate polypeptide fiagments to be generated which could aggregate to form amyloid fibrils (Lavie et al., 1980). Thus amyloid fibril formation would result principally from the combination of specific proteases plus precursor protein concn. (2) Specific immunoglobulin light chains may be predisposed to amyloid formation by their secondary and tertiary structure (Soloman et al., 1982). Thus, specific amino acid substitutions which dictate secondary and tertiary structure of the light chain might be the determining factor in formation of the fibrillar deposits. To investigate the possiblity of secondary structure effecting amyloid formation, we have aligned the V-region sequences of kappa I proteins BAN, MEV (the only other kappa I amyloid protein which has been sequenced completely) and ROY (a proteotype kappa I myeloma sequence) as shown in Fig. 2. Several specific differences are observed. These involve the replacement of hydrophilic amino acids by hydrophobic ones at several framework residues. For the MEV protein, this involves the replacement of a threonine by an isoleucine at position 20 and a tyrosine by a phenylalanine at position 49. For the BAN protein there are two similar substitutions. A tyrosine has been replaced by a phenylalanine at position 36 and a threonine has been replaced by an isoleucine at position 72. In both proteins, two polar residues (threonine and tyrosine) have been replaced by nonpolar ones (isoleucine and phenylalanine). The substitutions at positions BAN 36 and MEV 49 (phenylalanine for tyrosine) and BAN 72 and MEV 20 (isoleucine for threonine) are all the result of

77

Structure of amyloid protein BAN ________~___________~~~~~“~~~~~-~~--~--~~

p*,

“_________Is__________________zo-----________,

+;__

10

5

~~X1~-Gln-Ibu-ThK-Glb-Sar-RrO-Sar-Serl-Gly-As~Arg-Val-Ihr-Ils-Thr-Cye-Arg-Alanet VA1 IlC net

Glfl

t________________________ pR2 _________;;_______________f5;__ 30 35 40 S4r-Cln-Sar-Val-Tyr-Alm-Tyr-Vsl-Ala-Trp-Phe-Gln-Gln-Ly~-P~n-Gly-Ly~-AlA-Pro-Ly~-Ser-Leu-Xle-T~-~pSer-W&l-Asp Leu-Am L@U Phe AspIle-Sar-Ile-Phe-mu-Asn LCU CDR,

_____“__“_

__________________,

__________________________-______________-__*___-______ PA3 _____________ 65 70 6G 75 CHO Ala-Scr-lh~-Le"-Gln-Ser-Gly-Val-Pro-S~r-~n-P~e-~r-Gly-Ser-Gly-Se~-Gly-~-~~Phe-Il~-~-~~-Il~Thr Aan S6f Gly-Arq Thr AW Glu-Ala mr SOI Thr-Phe LW ArV t

__________________________________________________, c______________- mS3 80 83 FO Ssr-Sar-~u-Gln-Pro-Glu-A~p-Phe-Al~-Thr-~r-~r-Cy~-Gl~-Gln-~r-ABn-S~r-~-PrO-

ASP Ile

GW

_c__*__

pR,

__________"___, f.______ 95 - -Tyr-Thr-Pha-ClySO??*-lhr-Alln Glu-VA1 Phs-Aep-Am-Lau

Lou

__________________,

105 Gln-Gly-Thr-Lys-Val-Gln-lla-tys-llrgf;i;zii Ihr-Val-Asp GIY Val-Aep+he GUY 100

Fig. 2. The complete amino acid sequence of the variable regions of kappa I proteins BAN, MEV and ROY. For simplicity, the full sequence of protein BAN is presented and only those positions which are different in the other proteins are shown. The location of the four framework regions and three complementarity-dete~ining regions are indicated above the BAN sequence.

single-base changes. They are unique in that they substitute hydrophobic amino acids for hydrophilic residues at surface positions (Epp et al., 1975). For both proteins, these substitutions occur near the ends of beta strands and any ~rturbation of structure will have its maximal effect at these positions. Also, in both proteins the substitutions occur on opposite faces of the molecule, thus providing hydrophobic residues at the two surfaces which are most likely to interact in fibril formation. Such replacements can lead to two possible effects. The first possibility is that the secondary structure wiI1 not be altered. In this case, the two hydrophobic amino acid residues will project into the solvent and act as hydrophobic sites where self-aggregation of the V-region can be initiated. The other possiblility is that the secondary structure will be altered to allow the placement of the hydrophobic amino acids in the interior of the molecule. Such an alteration occurred in the REI molecule where the three hydrophobic amino acid sidechains at positions 46-48 were all inserted into the interior with the resultant deformation of the beta structure in that portion of the molecule. If this occurred with these substitutions, one would expect a major change in the two beta-pleated sheets to accommodate the added bulk of the two additional sidechains. Such a light chain with highly deformed beta structures on the two major faces might also have the required characteristics for amyloid formation.

These findings lend themselves to the formation of a model for AL amyloid formation. It is proposed that a minimum of two amino acid substitutions which place hydrophobic amino acid residues at surface positions are needed to initiate fibril formation. In addition, these substitutions must be positioned near the ends of beta strands and must be located on opposite faces of the molecule for self aggregation to occur. Within this model can be found a number of explanations of some of the characteristics of AL amyloidosis. The requirement of two specific substitutions in the FRs explains the tow frequency of occurrence of this condition in relation to myeloma. Also the heterogeneity of cleavage sites in the C-region and the variability in the J-segment structure would indicate their apparent lack of importance for fibril formation. Finally, because of the large number of loci where amyloid-forming substitutions can take place, it is not surprising that amyloid-forming residue(s) common to all fibril proteins are not identifiable. Since this hypothesis is based on only two kappa I amyloid sequences, additional structures will have to be completed to test its validity. Acknowledgements-We would like to thank Marilyn E. Smith and Frank W. Kenny for their excellent technical assistance. Also, the suggestions of Dr Frank W. Putnam during the preparation of this manuscript are greatly appreciated. This work was supported by Veterans Adminis-

78

FRANCIS E. DWULET

tration Medical Research (MRIS 583-0888), and grants from RR-00750 (GCRC), United States Public Health Service. National Cancer Institute (1 ROl CA 22141), National Institute of Arthritis, Diabetks, and Digestive and Kidney Diseases (AM 20582 and AM 7448), the Arthritis Foundation, the Grace M. Showalter Trust and the Marion E. Jacobson Fund.

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.