Biclonal Systemic AL-Amyloidosis with One Glycosylated and One Nonglycosylated AL-Protein*
The work was supported by The Research Council of Norway and the Swedish Medical Research Council (Project No. 5941).
Abstract
The amyloid fibril protein AL was isolated from the spleen of a patient with systemic amyloidosis. Size-exclusion chromatography of the solubilized amyloid fibrils revealed a distinct, retarded asymmetric peak. The symmetrical part of the peak showed on SDS-PAGE two positive periodic acid Schiff-staining bands at 14 and 16 kDa. Staining with Coomassie Brilliant Blue revealed in addition two proteins with masses of 13 and 20 kDa. The 14 and 16 kDa bands were the strongest ones. N-Terminal analyses of the four blotted bands showed that the N-termini were the same in all cases. Elucidation of the amino acid sequence established an AL-chain of 157 residues as well as a fragment covering positions 188–207 of the constant region. Two tryptic peptides derived from the same region, positions 25–46, showed an identical sequence, except for position 34 where both alanine and threonine residues occurred. Monosaccharide compositional analysis of the threonine-containing peptide revealed an oligosaccharide in the N-glycosylation site, position 32–34. Mass analysis of the glycopeptide verified the oligosaccharide. The AL-chains belong to the kappa 3a germline gene and verifies that the glycosylated chain is a mutated form. The AL-chains differ from that of the germline in 14 positions. The J-segment is of JκIII and is mutated in position 106.
Introduction
Systemic AL-amyloidosis is a life-threatening disease with generally poor prognosis. Amyloid deposits are found in virtually all tissues with the exception of the central nervous system. Involvement of the heart, kidneys or peripheral nerves often dominates the clinical picture [1].
The amyloid fibril protein AL is derived from a monoclonal immunoglobulin light chain, and usually the amyloid protein lacks a large piece of the C-terminus of the constant region. However, in some cases, the amyloid fibril protein consists almost entirely of the constant region fragment, but its contribution to the fibril formation is unknown [2–4]. Both kappa and lambda chains occur as AL-proteins and although lambda-VI seems to be more amyloidogenic than other light chains, there is no specific amyloid-forming subgroup.
Normally, immunoglobulin light chains do not aggregate into fibrils. However, because of some kind of instability of the protein, a partial unfolding seems to occur that results in amyloid fibril formation. In an effort to explore why certain immunoglobulin light chains aggregate into amyloid fibrils while others do not, Raffen et al. [5] have compared the amino acid sequences of light chains from patients known to suffer from amyloidosis with those of silent light chains. In this way, they have been able to identify certain positions which seem to be important for fibrillogenesis. Thus, these data indicate that the amino acid sequence may be an important denominator of amyloid formation. Another interesting fact is that many AL-proteins are glycosylated, usually in the variable part [6, 7]. Although not yet proven, glycosylation may cause some immunoglobulin light chains to adopt an alternative and amyloid-prone conformation [8].
Materials and methods
Source of amyloid fibrils A 69-year-old man with known enlarged liver, cardiac fibrillation and a suspicion of gastrointestinal malignancy developed hemiparesis, and computer tomography revealed cerebral infarction. He died suddenly after 2 weeks. Autopsy revealed a recent cerebral infarction probably because of emboli from thrombi in the left auricle. An unexpected systemic amyloidosis with enlarged liver, spleen and heart was found. Histologic analysis revealed very pronounced amyloid infiltration in the liver parenchyma (amyloid >50%), lardaceous spleen (amyloid >90%) and severe, diffuse amyloid infiltration in the thyroid and adrenal cortex. The myocardium contained moderate spotty amyloid deposits in addition to a more severe vascular amyloidosis. Renal arteries contained large amounts of amyloid while the glomeruli were almost completely spared. The bone marrow exhibited vascular amyloid but no myeloma.
Splenic material was stored frozen at −20 °C until used. Amyloid fibrils were extracted using a modified water extraction method. Lyophilized amyloid fibrils were defatted, dissolved overnight in 6 m guanidine HCl in 0.1 m Tris–HCl, pH 8.0, containing 0.1 m ethylenediaminetetraacetic acid (EDTA) and dithiothreitol and gel-filtered through a Sepharose 6B-CL column with 5 m guanidine HCl in distilled water. Material from the main retarded peak (at 280 nm) was used for structural studies.
Structural studies Protein samples were hydrolysed and analysed as described [9]. Automatic Edman degradation was performed using two different sequencers, namely Model 477A connected to a 120A analyser from Applied Biosystems (Foster City, CA, USA) and Model 241 from Hewlett Packard (Waldbronn, Germany). The carboxymethylated amyloid fibril protein was digested with trypsin, protease aureus and chymotrypsin and was cleaved by cyanogen bromide and 2-(2-nitrophenylsulphonyl) 3-methyl-3-bromoindolenine (BNPS) skatole [2]. The derived peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Pep-S, C2/C18 column (Pharmacia LKB, Uppsala, Sweden) using two different elution systems [10]. All peptides were purity-checked by capillary electrophoresis [10]. Monosaccharide compositional analyses were performed on a purified tryptic glycopeptide [2]. The molecular mass of the glycopeptide was determined using a MALDI TOF instrument (Voyager-DE, PE Biosystems, Foster City, CA USA). SDS-PAGE was performed according to Laemmlie [11], and the gels were stained with PAS (periodic acid Schiff) followed by Coomassie Brilliant Blue [12]. Retarded fractions from the size-exclusion chromatography were screened by enzyme-linked immunosorbent assay (ELISA) with an antibody against the C-terminal part of a kappa constant region [3]. The fractions reacting with the antibody were purified by a C8 column in an RP-HPLC system, as described previously [3].
Results
The amyloid fibril protein was eluted from a size-exclusion column as an asymmetric peak. The symmetrical part of the peak was collected and used for most of the sequence studies. The void volume peak was, in this case, very small. SDS-PAGE of the main fraction revealed, after staining with PAS, two bands corresponding to molecular masses of 14 and 16 kDa. Staining with Coomassie Brilliant Blue resulted in four rather distinct bands with molecular masses of 13, 14, 16 and 20 kDa. The 14 and 16 kDa bands were the strongest. N-Terminal analysis of the amyloid fibril protein fraction revealed the amino acid sequence of positions 1–39 which showed that the AL-chain was derived from a kappa III (Fig. 1). Position 34 revealed two residues, namely threonine (Thr) and alanine (Ala). N-Terminal analyses of transferred protein bands from the polyacrylamide gel revealed that all bands contained the N-terminal sequence of the AL-chain. The data also showed that the protein was ragged from three to five residues.
The primary structure of the three polypeptides from AL 142/85 (V-ås). Line marked with xxxxxxxx shows the data obtained from N-terminal analyses of AL 142/85 fraction. Peptides derived from digestions with trypsin, protease aureus and chymotrypsin are marked with a T, Pa and Ch, respectively. Peptides derived after cleavage with BNPS skatole is marked with BNPS. ◆, N-glycosylation site.
The complete amino acid sequence of positions 1–157 was established from isolation and characterization of peptides derived from digesting the AL-protein with trypsin, protease aureus and chymotrypsin (Fig. 1). Additional data were also obtained from cleavage of the protein with cyanogen bromide and BNPS skatole (Fig. 1). Overlapping peptides for positions 126–127 and 142–143 could not be obtained. The yield of peptides from this region dropped gradually, indicating a ragged C-terminus. Peptides covering positions 157 and 188 could not be found, and accordingly, BNPS-4, starting in position 149, could not be detected (Fig. 1). A short C-terminal fragment covering positions 188–207 was, however, obtained. Some of the more retarded fractions from the size-exclusion chromatography (eluted after the main peak) were tested with an antibody specific for the C-terminus [3]. Immunoreactive fractions were further purified by RP-HPLC. Selective fractions were analysed by Edman degradation and found to start in positions 108, 111, 127, 150 and 159.
Two tryptic peptides were obtained for positions 25–46 (Fig. 1). Tryptic peptide T-3 gave no amino acid residue in cycle 8, only the general background, and as a Thr residue was verified in cycle 10 (position 34), the results indicated an N-glycosylation (Fig. 1). The other peptide, T-3*, revealed asparagine and Ala residues in these cycles, whereas the other cycles were identical (Fig. 1).
Tryptic peptide T-3 was also taken for monosaccharide compositional analysis and for mass analysis. The monosaccharide analysis revealed six to seven residues of glucosamine, three residues of mannose, two residues of galactose and one residue each of fucose and sialic acid. Mass analyses of the tryptic glycopeptide, positions 25–45, revealed four major peaks corresponding to masses of 4325.34, 4179.35, 4122.44 and 3976.6. These data correspond to the composition of the glycopeptide, except that the N-acetylglucosamine number should be 5 instead of 6–7. The other peaks correspond to masses where some of the terminal fucose and N-acetylglucosamine residues of the oligosaccharide have been cleaved off.
As the amyloid fibril material consisted of two almost identical immunoglobulin chains, of which only one was glycosylated, we questioned whether the two chains occurred as a homogeneous mixture or may have different tissue affinity, PAS staining was performed on sections from the spleen, kidney, liver and adrenal gland. Amyloid at all locations was clearly PAS-positive. However, in all tissue samples, amyloid deposits within larger arteries exhibited a much stronger PAS positivity (Fig. 2).
Amyloid in a liver section of patient AL142, stained with PAS. There is an apparent stronger PAS (periodic acid Schiff) reaction of the amyloid in the vessel wall (centre) compared with the diffuse amyloid infiltration in the liver parenchyma.
Discussion
Two of the four main bands detected by SDS-PAGE were found positive for PAS staining. Edman degradation of the four transferred protein bands verified the same N-terminal sequence and showed that the AL-protein was derived from a κIII immunoglobulin light chain. N-Terminal analysis of the amyloid fibril protein fraction gave the sequence of positions 1–39 and revealed heterogeneity in position 34. From the tryptic digest, two peptides were isolated with identical sequences to that of positions 25–45, except for the Ala/Thr exchange in position 34. This exchange results in an N-glycosylation site at this region. A monosaccharide compositional analysis as well as mass analysis of the tryptic peptide verified an oligosaccharide with a mass of about 2 kDa.
To our knowledge, this would be the first case where the amyloid fibril protein consists of two different AL-chains. Considering the conformational differences between these two AL-chains, where an oligosaccharide with a mass of about 2 kDa covers a large part of the light chain surface, it is really astonishing that both chains contain the fibrillogenic properties. The result suggests that glycosylation alone, is not the only factor involved in the fibril formation. However, could this indicate that glycosylation enhances fibrillogenesis and starts the self-perpetuating protein conformation chain reaction, also involving the nonglycosylated immunoglobulin light chain?
The composition of the oligosaccharide was similar to that found in AL MS [7], AL 366 [9] and the amyloid-associated Bence Jones protein MH and suggests a similar biantennary structure [7]. The heterogeneity would be in agreement with our earlier data and could in part be explained by an in situ degradation [7].
The amino acid sequence study established a polypeptide length of about 157 residues, which corresponds to a higher molecular mass than the protein bands positively stained with PAS. The yield of the peptides covering the last 30 residues gradually decreased, which in part could explain this discrepancy. Characterization of several retarded fractions from the size-exclusion chromatography showed that the light chain was heavily fragmented and that the smallest one was composed of residues 188–207. No peptides or any amino acid sequence data could be found for positions 158–188. These data confirm what we already have observed in other cases, namely that the amyloid fibrils are composed of fragments of the variable region as well as fragments of the C-terminal part of the constant region [3]. It is still a question how these fragments influence the fibril formation or whether they are degradation fragments derived after the fibrils have been formed.
The elucidated amino acid sequence shows that AL V-ås 142 belongs to the kappa 3a germline gene and differs from that in 14 positions. Six of them are between positions 50 and 61, corresponding to CDR2 and part of FR3. Besides the glycosylation, the most dramatic exchanges are found in positions 53, V/T, 55, D/A and 59, Q/P. It is worth mentioning that the Thr in position 34 is a mutated form of the kappa 3a germline gene. The J-segment is of JκIII with a mutation in position 106 where a phenylalanine residue has replaced isoleucine.
Amongst the 31 cases of AL-type of amyloidosis we have analysed, six of them have been of the κIII subgroup, which is in contrast to that found for the 60 patients with systemic amyloidosis in a recent study, where none of them belonged to this subgroup[13].
One of the yet unsolved questions in AL-amyloidosis is why the pattern of amyloid deposition varies so immensely between individuals. Differences in interaction between the deposited light chains and tissue factors have been suggested as one possible explanation. A glycosylated protein must have other properties than one without glycosylation. However, in patient V-ås 142, the amyloid fibrils obtained from the spleen contained a glycosylated AL-chain as well as a nonglycosylated AL-chain derived from the same germline gene. This could indicate that the glycosylated protein was conformationally changed, triggering fibrillogenesis. Unfortunately, we do not have markers that are specific for the separate AL-proteins in AL V-ås 142, but it was interesting to note that PAS positivity was much stronger in vessel amyloid compared with other amyloid depositions.
Acknowledgments
The technical assistance of Jessie Juul is gratefully acknowledged. We thank Madeleine Jarild for the PAS-stained sections.
