178
Journal of the Neurological Sciences, 108 (1992) 178-183 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
JNS 03725
Characterization of a transthyretin-related amyloid fibril protein from cerebral amyloid angiopathy in type I familial amyloid polyneuropathy Fuyuki K a m e t a n i 1, Shu-ichi Ikeda
2, N o b u o
Yanagisawa
2, Tsuyoshi
Ishi 3 a n d Norinao H a n y u 4
Department of Molecular Biology and 3 Department of Ultrastructure, Psychiatric Research Institute of Tokyo, Tokyo 156 (Japan) 2 Department of Medicine (Neurology), Shinshu University School of Medicine, Matsumoto 390 (Japan) and ¢ Department of Neurology, Nagao Red Cross Hospital, Nagano 380 (Japan)
(Received 28 June, 1991) (Revised, received 14 October, 1991) (Accepted 14 October, 1991) Key words: Familial amyloid polyneuropathy; Cerebral amyloid angiopathy; Amyloid; Transthyretin; Amyloid fibril protein; Amino acid sequence
Summary Recently, it has been reported that transthyretin (TrR)-immunoreactive amyloid deposition with cerebral amyloid angiopathy in central nervous system is a common pathological finding in type I familial amyloid polyneuropathy (FAP). In the present study, we performed isolation and sequence analysis of TrR-related amyloid fibril protein from the meninges of a patient with type I FAP. Purified major amyloid fibril protein had a molecular weight of 15 kDa. Complete sequence analysis revealed that this amyloid fibril protein was a variant T r R with a single amino acid substitution of methionine for valine at position 30. This variant T r R is a previously unrecognized as cerebrovascular amyloid fibril protein. Furthermore, the patients with type I FAP are well known to have the variant TTR in the serum. These suggest that cerebrovascular amyloid fibril protein in type I FAP may derive from a serum precursor.
Introduction
Cerebral amyloid angiopathy (CAA) is a pathological condition characterized by amyloid deposition in the walls of leptomeningeal and cortical blood vessels (Tomonaga 1981; Vinters and Gilbert 1983; Vinters 1987). This condition has been observed in the brains of patients with Alzheimer's disease (AD), adult Down syndrome (DS) (Castafio and Frangione 1988), Icelandic-type hereditary cerebral hemorrhage with amyioidosis (HCHWA-I) (Gudmundsson et el. 1972), Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) (Luyendijk et al. 1988), spongiform encephalopathy seen in sheep scrapie (Allsop et al. 1985) and Creutzfeldt-Jakob disease (CJD) (Keohane et al. 1985), and also the brains of non-demented aged persons (Castafio and Frangione 1988).
Correspondence to: Shu-ichi lkeda, Department of Medicine (Neumlosy), Shinshu University School of Medicine, Matsumoto 390, Japan. Tel.: 0263(35)4600. Fax: 0263(34)0929.
To date, studies of cerebrovascular amyloids have revealed the biochemical and immunohistochemical features of three different amyloid fibril proteins:/3/A4 protein in AD, DS, HCHWA-D and non-demented aged persons, cystatin-C in HCHWA-I, and prion protein (PrP 27-30) in CJD and scrapie. However, the exact mechanisms involved in the pathogenesis of amyloid deposition are still unknown. Recently, one of us (S.I.) has reported that transthyretin (TrR)-immunoreactive amyloid deposition with CAA in the central nervous system is a common pathological finding in type I familial amyioid polyneuropathy (FAP) which is one form of hereditary generalized amyloidosis (Ushiyama et al. 1991). To clarify the pathogenic mechanisms of amyloid deposits in the central nervous system of this disease, it is necessary to biochemically analyze the TTR-immunoreactive amyloid fibril protein in CAP, of the patients. In the present study, we performed isolation and sequence analysis of amyloid fibril protein seen in the central nervous system in type I FAP. Complete sequence analysis revealed that the amyloid fibril protein was a variant TrR with a single amino acid
179 substitution of methionine for valine at position 30 and so is identical to amyloid fibril protein isolated from visceral organs in other cases with this disorder (Tawara et al. 1983; Kametani et al. 1984).
Superose 12 column (Pharmacia LKB Biotechnology) equilibrated with 6 M guanidine-HCl/0.1 M TrisHCI/0.4 mM EDTA, pH 8.6. The fractions containing the amyloid fibril protein were purified by reverse phase HPLC using an Aquapore RP-300 (Applied Biosystem) column.
Materials and methods
Analysis of transthyretin gene Total genomic DNA was isolated from collected leukocytes, and an 852 bp subsequence of the TTRgene including exon 2 with the nucleotide substitution (corresponding to the valine to methionine change at position 30) was amplified using the polymerase chain reaction method. The amplified DNA was digested with restriction endonuclease Ball or Nsil (Bethesda Research Labs.) and then all DNA samples were electropboresed through an agarose gel. The details of this DNA analysis technique have been reported elsewhere (lkeda et al. 1991).
Immunohistochemistry of cerebral amyloid angiopathy Paraffin-embedded sections were prepare~ from temporal cortex. Immunostaining of cerebrovascular amyloid deposits was carried out using the streptavidin-biotin-horseradish peroxidase method described previously (Ikeda et al. 1989), and the primary antibody used in this study was 1:500 diluted antiserum to human TI'R (Dako Japan Co., Ltd.).
Extraction of amyloid fibril protein Amyloid fibrils were isolated by the procedure of Glenner and Wong (1984). The meninges (ca. 2.5 g) was cut into small pieces and homogenized in 25 ml 10 mM phosphate-buffered saline (PBS) containing 0.01% sodium azide. After centrifugation at 47 400 x g for 60 min at 4°C, the supernatant was discarded. The pellet was washed repeatedly as described above. The brownish top layer of the resultant pellet was enriched in amyloid fibrils as monitored by polarizing microscopy after Congo red staining. The amyloid enriched top layer (wet weight ca. 350 rag) was homogenized in 8.75 ml 50 mM Tris-HCl/0.01 mM CaCl2/3 mM NaN 3, pH 7.6. Solid coilagenase (EC 3.4.24.3, Sigma Chemical Co., type I) was added in a 1 : 100 ratio (w/w) and the mixture incubated at 37°C for 16 h. After the digestion, the mixture was centrifuged at 47 400 x g for 60 min. The supernatant was discarded and the resultant pellet (crude amyloid fraction) was washed in PBS.
Separation and purification of amyloid fibril protein The crude amyloid fraction (wet weight ca. 60 mg) was dissolved in 240 /zi 6 M guanidine-HCl/0.1 M Tris-HCl/0.4 mM EDTA/34 mM DTr, pH 8.6, and incubated overnight at room temperature. After centrifugation the supernatant fraction was applied to a
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting of amyloidfibril protein The fractions obtained from Superose 12 chromatography were dialyzed, lyophilized, and dissolved in sample buffer for SDS-PAGE (Laemmli 1970). After electrophoresis, the separated proteins were transferred to PVDF membrane sheets (Millipore Ltd.) and were detected by an immunochemical staining method (Towbin et al. 1979). The primary antibodies used in the immunochemical assay were an antiserum raised against a synthetic peptide consisting of residues 1-24 of /3/A4 (Ishii et al. 1989), a monoclonal antibody (4D12/2/6) raised against a synthetic peptide consisting of residues 8-17 of fl/A4 (Allsop et al. 1986), an affinity-purified rabbit antiserum (AG8206) to human cystatin C (L/Sfberg et al. 1987) and an affinity-purified antibody to human TTR (Dako Japan Co., Ltd.). The specimens were immunostained with the 1:500 to 1 : 1000 diluted antisera or ascites fluid described above.
Sequence analysis of amyloid fibril protein Purified protein was digested with TPCK-trypsin (EC 3.4.21.4, Worthington, E / S = 1/20) in 50 mM Tris-HCl, pH 8.0 at 37°C for 16 h. The digests were separated and purified by reverse-phase HPLC as described above. The purified peptides were sequenced with an automatic sequencer (Applied Biosystems 477A and 120A).
Results
The autopsy case aged 51 originated from a Japanese FAP family, and a detailed clinical picture of this family has been reported as atypical FAP showing cerebellar ataxia and pyramidal tract signs in addition to a sensorimotor and autonomic peripheral neuropathy (lkeda et ai. 1987). Using the radioimmunoassay method described by Nakazato et al. (1984b), a variant TTR with a methionine for valine substitution at position 30 was detected in his serum (serum concentration: 9.47 mg/dl), and the mutation of the TI'R gene responsible for this amino acid substitution was also demonstrated (Fig. 1). Histopatbological examinations revealed severe deposits of TTR-immunoreactive amyloid in systemic organs including peripheral nerves, and in the brain this amyloid deposition was mainly seen on the leptomeningeal vessels and pia-arachnoid membranes (Fig. 2). The detailed neuropathological find-
180
M1
2 3 o
0.32¢::
o
8 .~ 5(
Fig. 1. TTR gene analysis. Lane M: DNA molecular weight pHY marker; 1: non-digested DNA; 2: DNA after digestion with Bail; 3: DNA after digestion with NsiI. Two abnormal bands (consisting of 505 and 347 bp fragments after digestion with Bali and of 498 and 347 bp fragments after digestion with NsiI) are seen in addition to the normal 852 band. This DNA analysis pattern is specific for the heterozygousgene abnormality of type I FAP (Sakaki et al, 1989).
ings of this case and their relation to the central nervous system dysfunctions will be reported elsewhere (Ikeda et al., in preparation). The crude amyloid preparation was separated by Superose 12 chromatography into 4 fractions as shown in Fig. 3. Each fraction was analyzed by SDS-PAGE and immunoblotting. All 4 fractions contained antiTI'R immunoreactive materials, but the immunoreactivities to fraction IV were weaker than the other fractions as shown in Fig. 4. The reason of this phe-
I
o
-'--T
T-
20
80 Time (min)
Fig. 3. Elution profile of crude amyloid fraction on a Superose 12 column. Each of the fractions indicated by horizontal bars was pooled and examined further.
nomenon is not clear. Fraction III (MW 35 kDa) and IV (MW 15 kDa) which represented major protein components seemed to be dimeric and monomeric forms of TTR, respectively. Antibodies t o / ~ / A 4 protein and cystatin C did not react with these materials (data not shown). Fraction IV (MW 15 kDa) was
k
Fig. 2. Immunohistochemicalfindings of cerebral amyloidangiopathy. TTR-immunoreactive amyloiddeposits are seen on many subarachnoidal vesselsand leptomeninges.Bar ffi I00/~m.
181 1
2
3
4
5
6
7
8
9
10 GP
I0 20 T G T G E S K C P L M V K V L D A V R G S ............................>
30 NVAMHV
PAl
(v)
I. . . . . . . . . . . I . . . . . . . . . . . t. . . . . . . . . . . . . . . . . . . . .
M
T2
(k
T3
T4
40 50 60 F R K A A D D T W E P F A S G K T S E S G E L H G L T T E -->I
...........................
i
@e
E EF
I ...............................
T5-6
T?
70 80 90 I YKVEI D T K S Y W K A L G I S P F H E H A E V V F T .......... • I .......... > I ...... > I ............................... T8 T9 TI0
VEG
100 A N D S G P R R Y T .............
110 IAALL
S PYSYS
I .....................
T11-12a
120 TTAVVTNPK
I .........................
E I
T12b-13
Fig. 6. Complete amino acid sequence of purified amyloid fibril protein. The primary structure was established by direct sequencing of the purified amyloid protein and its individual tryptic peptides. Since the amino terminal sequence of the protein is heterogeneous, the data obtained for the major sequence starting at position 4 are
shown. Fig. 4. SDS-PAGE and immunoblotting of the separated amyloid fractions. Lanes 1-5: CBB staining; lanes 6-10: immunoblotting with anti-Tl'R antibody. Lanes 1 and 6, crude amyloid fraction; lanes 2 and 7, fraction I; lanes 3 and 8, fraction lI; lanes 4 and 9, fraction Ill; lanes 5 and 10, fraction IV.
E co
8
°
2
i
0.32 -
further purified by reverse phase HPLC and sequenced. Purified 15-kDa amyloid fibril protein displayed ragged N-terminal amino acid sequences. However, the main sequence (37%) was GTGESKxPLMVKVLDand was identical with the sequence of TCR starting at position 4. As shown in Figs. 5 and 6, tryptic peptides obtained from the purified 15-kDa amyloid protein were analyzed by automated Edman degradation and placed by homology to normal TTR (Kanda et al. 1974). Amino acid sequences of all of the tryptic peptides except for the T4 peptide corresponded to subsequences of normal TTR as shown in Fig. 6. The T4 peptide was GSPAINVAMHVFR and had a substitution of methionine for valine at position 9 of this peptide as shown in Fig. 6. Thus, this amyloid fibril protein had an identical sequence to normal TTR except for a methionine for valine substitution at position 30.
Discussion
0
60
Time (rain) Fig. 5. Elution profile of tryptic digests of purified amyloid fibril protein. Separation was achieved on an Aquarpare RP-300 column (2.1 x 100 ram) with a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid at 0.2 ml/min flow rate. Peak 1, T8; peak 2, 3"9; peak 3, T2 and T3; peak 4, T12b-13; peak 5, T5-6; peak 6, T4; peak 7, TI0; peak 8, T7; peak 9, Tll-12a.
Type I FAP is a systemic amyloidosis presumably caused by a variant TYR due to a gene mutation product responsible for a valine to methionine ~ubstitution at position 30 (Sasaki et al. 1984; Sakaki et al. 1989). In this disease, severe amyloid deposits composed of this variant TI'R are seen in the peripheral n e r v e s and various visceral organs (Andrade 1952; Ikeda et al. 1987; Hanyu et al. 1989). Recently, one of us (S.I.) has found that CAA is a common pathological finding in the brain of patients with type I FAP. The amyloid fibril protein comprising this form of CAA was demonstrated to be antigenically related to T r R , and any immunohistochemicai reactions for anti-/3/A4 protein antibody or anti-cystatin C antiserum were not seen, eve.,., a~cr formic acid pretreatment (Ushiyama et aL 1991). In the present study, to confirm this observa-
182 tion, we characterized the amyloid fibril protein obtained from the meninges in a type I FAP patient for the first time. It was found that crude amyloid fibril proteins reacted with only anti-TrR antibody and did not react with antibodies to well-known cerebral amyloid fibril proteins, /3/A4 protein and cystatin C. The major protein component had a molecular weight of 15 kDa, the same as that of normal TI'R. Complete sequence analysis revealed that this protein was a variant TTR with a substitution of methionine for valine at position of 30. Additionally, we also found minor components with molecular weights of 5-8 kDa and their N-terminal sequences were LTI'EEEFVE and IYKVEIDTK corresponded to positions at 58 and 68 of TTR, respectively (data not shown). It is well known that a variant TI'R with a methionine for valine substitution at position 30 is present in the serum of patients with type I FAP (Nakazato et al. 1984a; Suzuki et al. 1987). Therefore, these results suggest that the amyloid fibril protein in CAA of type I FAP is derived from a serum variant TI'R. CAA is observed in a variety of brain degenerative diseases (Castafio and Frangione 1988) and this type of amyloid vascular change selectively involves the leptomeningea! and cortical vessels of the brain (Tomonaga 1981; Vinters and Gilbert 1983; Vinters 1987). In the central nervous system of type 1 FAP patients, leptomeningeal vessels and pia-arachnoid membranes are the principal site of TTR-related amyloid deposition and this amyloid does not significantly affect the brain parenchyma (Ushiyama et al. 1991). Amyloid fibril protein isolated from meninges of a patient with type I FAP was identified with the variant TTR as described above. Similar pattern of amyloid deposits but more heavy involvement is seen in familial oculoleptomeningeal amyloidosis (Hamburg 1971; Okayama et al. 1978; Goren et aL 1980; Uitti et al. 1988), and amyloid fibril protein in the disease was immunohistochemically shown to be TTR (Uitti et al. 1988). However, this unusual form of hereditary amyloidosis mainly involves the central nervous system, and the patients with this disorder usually lack distinct clinical manifestations ascribable to amyloidotic polyneuropathy, autonomic disorder or visceral lesions. Accordingly, the clinical pictures of familial oculoleptomeningeal amyloidosis differ from those of type l FAP. The pathological mechanism of amyloid deposition on the cerebral vessels is incompletely understood, and the origin of cerebrovascular amyloid (/~/A4 protein, cystatin C and PrP protein) still remains controversial. In considering the morphological similarity of CAA seen in diverse disorders (consisting of biochemically different amyloid fibril proteins), TI'R-type CAA observed in patients with type I FAP is likely to be a
valuable model for the study of cerebrovascular amyloid deposits derived from an abnormal serum protein, the causative gene abnormality of which has been clearly demonstrated. Further studies are required to clarify the exact pathogenesis of CAA. Acknowledgements We thank Dr. M. Nakazato for the testing of serum variant TTR, Dr. D. AIIsop for the 4D12/2/6 antibody and critical reading of the manuscript, Dr. A.O. Grubb for the AG8206 antibody, Miss. K. Tanaka for her excellent technical support, Mr. K. Kato and Mr. A. Kagiwada for preparing the photographs. This research was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture and grants from Kanae Foundation of New Medicine and from the Primary Amyloidosis Research Committee of the Ministry of Health and Welfare, Japan.
References AIIsop, D., M. Landon, M. Kidd, J.S.l.owe, G.P. Reynolds and A. Gardner (1986) Monoclonal antibodies raised against a subseuence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer's disease. Neurosci. Lett., 68: 252-256. Allsop, D., S. Ikeda, M. Bruce and G.G. Glenner (1988) Cerebrovascular amyloid in scrapie-affected sheep reacts with antibodies to priori protein. Neurosci. Lett., 92:234-239. Andrade, C. (1952) A peculiar form of peripheral neuropathy: familial atypical generalized amyloidosis with special involvement of the peripheral nerves. Brain, 75: 408-427. Castafio, E.M. and B. Frangione (1988) Human amyloidosis, Alzheimer disease and related disorders. Lab. Invest., 58: 122132. Glenner, G.G. and C.W. Wong (1984) Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Comman., 120: 885-891. Goren, H., M.C. Steinberg and H.F. Gholam (1980) Familial oeuIoleptomeningeal amyloidosis. Brain, 103: 473-495. Gudmundsson, G., J. Hallgrlmsson, T.A. Jdnasson and O. Bjarnason (1972) Hereditary cerebral haemorrhage with amyloidosis. Brain, 95: 387-404. Hamburg, A. (1971) Unusual cause of vitreous opacities. Primary familial amyloidosis.Ophthalmologica, 162: 173-177. Hanyu, N., S. Ikeda, A. Nakadai, N. Yanagisawa and H.C. Powell (1989) Peripheral nerve pathological findings in familial amyloid polyneuropathy: a correlative study of proximal sdatic nerve and sural nerve lesions. Ann. Neurol., 25: 340-350. lshii, T., F. Kametani, S. Haga and M. Sato (1989) The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease. Neuropathol. Appl. Neurobiol., 15: 135-147. Ikeda, S., N. Hanyu, M. Hongo, J. Yoshioka, H. Oguchi, N. Yagagisawa, T. Kobayashi, H. Tsukagoshi, N. Ito and T. Yokota (1987) Hereditary generalized amyloidosis with polyneuropathy. Clinicopathological study of 65 Japanese patients. Brain, 110: 315-337. Ikeda, S., D. AIIsop and G.G. Glenner (1989) Morphology and distribution of plaque and related deposits in the brains of Alzheimer's disease and control cases. An immunohistochemieal study using amyloid/3-protein antibody. Lab. Invest., 60: 113-122. lkeda, S., T. Nakano, N. Yanagisawa, T. Suzuki, N. Hanyu and Y. Sakaki (1991) Diagnosis of familial amyloid polyneuropathy: gene analysis with primer-directed enzymatic amplification of DNA, isolation of plasma variant prealbumin and immunohistochemical
183 identification of tissue amyloid protein. Clin. Neurol. (Tokyo), 31: 363-371. Kametani, F., H. Tonoike, A. Hoshi, T. Shinoda and S. Kito (1984) A variant prealbumin-related low molecular weight amyloid fibril protein in familial amyloid polyneuropatby of Japanese origin. Biochem. Biophys. Res. Commun., 125: 622-628. Kanda, Y., D.S. Goodman, R.E. Ganfleld and F.J. Morgan (1974) The amino acid sequence of human plasma prealbumin. J. Biol. Chem., 249: 6796-6805. Keohane, C., R. Peatfield and L.M. Duchen (198.~)Subcc~te spongiform encephalopathy (Creutzfeldt-Jakob d~sease; with amyloid angiopathy. J. Neurol. Neurosurg. Psychiat., 48: 1175-1178. Laemmli, U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. L6foerg, H., A.O. Grubb, E.K. Nilsson, O. Jensson, G. Gudmundsson, H. BI6ndal, A. Arnason and L. Thorsteinsson (1987) Immunohistechemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis. Stroke, 18: 431-440. Luyendijk, W., G.T.A.M. Bots, M. Vegter-van der Vlis, L.N. Went and B. Frangione (1988) Hereditary cerebral haemorrhage caused by cortical amyloid angiopatby. J. Neurol. Sci., 85: 267-280. Nakazato, M., IL Kangawa, N. Minamino, S. Tawara, H. Matsuo and S. Araki (1984a) Identification of a prealbumin variant in the serum of a patient with familial amyloidotie polyneuropatby. Biochem. Biophys. Res. Commun. 122, 712-718. Nakazato, M., K. Kangawa, N. Minamino, S. Tawara, H. Matsuo and S. Araki (1984b) Radioimmunoassay for detecting abnormal prealbumin in the serum for diagnosis of familial amyloidotic polyneuropatby (Japanese type). Biechem. Biopbys. Res. Commun., 122: 719-725. Okayama, M., !. Goto, J. Ogata, T. Omae, I. Yoshida and H. Inomata (1978) Primary amyloidosis with familial vitreous opacities. Arch. Intern. Med., 138: 105-111.
Sakaki, Y., H. Sasaki, IL Yoshioka and H. Furuya (1989) Genetic analysis of familial amyloidotic polyneuropathy, an autosomal dominant disease. Clin. Chim. Acta, 185: 291-298. Sasaki, H., Y. Sakaki, H. Matsuo, T. Goto, Y. Kuruiwa, I. Sahashi, A. Takahashi, T. Shinoda, T. Isobe and Y. Takagi (1984) Diagnosis of familial amyloidotic polyneuropatby by recombinant DNA techniques. Biochem. Biophys. Res. Commun., 125: 636-642. Suzuki, T., T. Azuma, S. Tsujino, R. Mizuno, S. Kishimoto, Y. Wada, A. Hayashi, S. Ikeda and N. Yanagisawa (1987) Diagnosis of familial amyloidotic polyneuropathy: isolation of variant prealbumin. Neurology, 37: 708-711. Tawara, S., M. Nakazato, K. Kangawa, H. Matsuo and S. Araki (1983) Identification of amyloid prealbnmin variant in familial amyloidotic polyneuropatby (Japanese type). Biechem. Biophys. Res. Commun., 116: 880-888. Tomonaga, M. (1981) Cerebral amyloid angiopathy in the elderly. J. Am. Geriatr. Soc., 29: 151-157. Towbin, H., T. Staehelin and J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Uitti, R.J., J.R. Donat, B. Rozdilsky, R.J. Schneider and A.H. Koeppen (1988) Familial oculoleptomeningeal amyloidosis. Report of a new family with unusual features. Arch. Neurol., 45: 1118-1122. Ushiyama, M., S. lkeda and N. Yanagisawa (1991) Transthyretin-type cerebral amyloid angiopatby in type I familial amyloid polyneuropatby. Acta Neuropathol., 81: 524-528. Vinters, H.V. (1987) Cerebral amyloid angiopathy: a critical review. Stroke, 18: 311-324. Vinters, H.V. and J.J. Gilbert (1983) Cerebral amyloid angiopatby: incidence and complicatkms in the aging brain. II. The distribution of amyloid vascular changes. Stroke, 14:924-928.
Journal of the Neurological Sciences, 108 (1992) 178-183 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
JNS 03725
Characterization of a transthyretin-related amyloid fibril protein from cerebral amyloid angiopathy in type I familial amyloid polyneuropathy Fuyuki K a m e t a n i 1, Shu-ichi Ikeda
2, N o b u o
Yanagisawa
2, Tsuyoshi
Ishi 3 a n d Norinao H a n y u 4
Department of Molecular Biology and 3 Department of Ultrastructure, Psychiatric Research Institute of Tokyo, Tokyo 156 (Japan) 2 Department of Medicine (Neurology), Shinshu University School of Medicine, Matsumoto 390 (Japan) and ¢ Department of Neurology, Nagao Red Cross Hospital, Nagano 380 (Japan)
(Received 28 June, 1991) (Revised, received 14 October, 1991) (Accepted 14 October, 1991) Key words: Familial amyloid polyneuropathy; Cerebral amyloid angiopathy; Amyloid; Transthyretin; Amyloid fibril protein; Amino acid sequence
Summary Recently, it has been reported that transthyretin (TrR)-immunoreactive amyloid deposition with cerebral amyloid angiopathy in central nervous system is a common pathological finding in type I familial amyloid polyneuropathy (FAP). In the present study, we performed isolation and sequence analysis of TrR-related amyloid fibril protein from the meninges of a patient with type I FAP. Purified major amyloid fibril protein had a molecular weight of 15 kDa. Complete sequence analysis revealed that this amyloid fibril protein was a variant T r R with a single amino acid substitution of methionine for valine at position 30. This variant T r R is a previously unrecognized as cerebrovascular amyloid fibril protein. Furthermore, the patients with type I FAP are well known to have the variant TTR in the serum. These suggest that cerebrovascular amyloid fibril protein in type I FAP may derive from a serum precursor.
Introduction
Cerebral amyloid angiopathy (CAA) is a pathological condition characterized by amyloid deposition in the walls of leptomeningeal and cortical blood vessels (Tomonaga 1981; Vinters and Gilbert 1983; Vinters 1987). This condition has been observed in the brains of patients with Alzheimer's disease (AD), adult Down syndrome (DS) (Castafio and Frangione 1988), Icelandic-type hereditary cerebral hemorrhage with amyioidosis (HCHWA-I) (Gudmundsson et el. 1972), Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) (Luyendijk et al. 1988), spongiform encephalopathy seen in sheep scrapie (Allsop et al. 1985) and Creutzfeldt-Jakob disease (CJD) (Keohane et al. 1985), and also the brains of non-demented aged persons (Castafio and Frangione 1988).
Correspondence to: Shu-ichi lkeda, Department of Medicine (Neumlosy), Shinshu University School of Medicine, Matsumoto 390, Japan. Tel.: 0263(35)4600. Fax: 0263(34)0929.
To date, studies of cerebrovascular amyloids have revealed the biochemical and immunohistochemical features of three different amyloid fibril proteins:/3/A4 protein in AD, DS, HCHWA-D and non-demented aged persons, cystatin-C in HCHWA-I, and prion protein (PrP 27-30) in CJD and scrapie. However, the exact mechanisms involved in the pathogenesis of amyloid deposition are still unknown. Recently, one of us (S.I.) has reported that transthyretin (TrR)-immunoreactive amyloid deposition with CAA in the central nervous system is a common pathological finding in type I familial amyioid polyneuropathy (FAP) which is one form of hereditary generalized amyloidosis (Ushiyama et al. 1991). To clarify the pathogenic mechanisms of amyloid deposits in the central nervous system of this disease, it is necessary to biochemically analyze the TTR-immunoreactive amyloid fibril protein in CAP, of the patients. In the present study, we performed isolation and sequence analysis of amyloid fibril protein seen in the central nervous system in type I FAP. Complete sequence analysis revealed that the amyloid fibril protein was a variant TrR with a single amino acid
179 substitution of methionine for valine at position 30 and so is identical to amyloid fibril protein isolated from visceral organs in other cases with this disorder (Tawara et al. 1983; Kametani et al. 1984).
Superose 12 column (Pharmacia LKB Biotechnology) equilibrated with 6 M guanidine-HCl/0.1 M TrisHCI/0.4 mM EDTA, pH 8.6. The fractions containing the amyloid fibril protein were purified by reverse phase HPLC using an Aquapore RP-300 (Applied Biosystem) column.
Materials and methods
Analysis of transthyretin gene Total genomic DNA was isolated from collected leukocytes, and an 852 bp subsequence of the TTRgene including exon 2 with the nucleotide substitution (corresponding to the valine to methionine change at position 30) was amplified using the polymerase chain reaction method. The amplified DNA was digested with restriction endonuclease Ball or Nsil (Bethesda Research Labs.) and then all DNA samples were electropboresed through an agarose gel. The details of this DNA analysis technique have been reported elsewhere (lkeda et al. 1991).
Immunohistochemistry of cerebral amyloid angiopathy Paraffin-embedded sections were prepare~ from temporal cortex. Immunostaining of cerebrovascular amyloid deposits was carried out using the streptavidin-biotin-horseradish peroxidase method described previously (Ikeda et al. 1989), and the primary antibody used in this study was 1:500 diluted antiserum to human TI'R (Dako Japan Co., Ltd.).
Extraction of amyloid fibril protein Amyloid fibrils were isolated by the procedure of Glenner and Wong (1984). The meninges (ca. 2.5 g) was cut into small pieces and homogenized in 25 ml 10 mM phosphate-buffered saline (PBS) containing 0.01% sodium azide. After centrifugation at 47 400 x g for 60 min at 4°C, the supernatant was discarded. The pellet was washed repeatedly as described above. The brownish top layer of the resultant pellet was enriched in amyloid fibrils as monitored by polarizing microscopy after Congo red staining. The amyloid enriched top layer (wet weight ca. 350 rag) was homogenized in 8.75 ml 50 mM Tris-HCl/0.01 mM CaCl2/3 mM NaN 3, pH 7.6. Solid coilagenase (EC 3.4.24.3, Sigma Chemical Co., type I) was added in a 1 : 100 ratio (w/w) and the mixture incubated at 37°C for 16 h. After the digestion, the mixture was centrifuged at 47 400 x g for 60 min. The supernatant was discarded and the resultant pellet (crude amyloid fraction) was washed in PBS.
Separation and purification of amyloid fibril protein The crude amyloid fraction (wet weight ca. 60 mg) was dissolved in 240 /zi 6 M guanidine-HCl/0.1 M Tris-HCl/0.4 mM EDTA/34 mM DTr, pH 8.6, and incubated overnight at room temperature. After centrifugation the supernatant fraction was applied to a
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting of amyloidfibril protein The fractions obtained from Superose 12 chromatography were dialyzed, lyophilized, and dissolved in sample buffer for SDS-PAGE (Laemmli 1970). After electrophoresis, the separated proteins were transferred to PVDF membrane sheets (Millipore Ltd.) and were detected by an immunochemical staining method (Towbin et al. 1979). The primary antibodies used in the immunochemical assay were an antiserum raised against a synthetic peptide consisting of residues 1-24 of /3/A4 (Ishii et al. 1989), a monoclonal antibody (4D12/2/6) raised against a synthetic peptide consisting of residues 8-17 of fl/A4 (Allsop et al. 1986), an affinity-purified rabbit antiserum (AG8206) to human cystatin C (L/Sfberg et al. 1987) and an affinity-purified antibody to human TTR (Dako Japan Co., Ltd.). The specimens were immunostained with the 1:500 to 1 : 1000 diluted antisera or ascites fluid described above.
Sequence analysis of amyloid fibril protein Purified protein was digested with TPCK-trypsin (EC 3.4.21.4, Worthington, E / S = 1/20) in 50 mM Tris-HCl, pH 8.0 at 37°C for 16 h. The digests were separated and purified by reverse-phase HPLC as described above. The purified peptides were sequenced with an automatic sequencer (Applied Biosystems 477A and 120A).
Results
The autopsy case aged 51 originated from a Japanese FAP family, and a detailed clinical picture of this family has been reported as atypical FAP showing cerebellar ataxia and pyramidal tract signs in addition to a sensorimotor and autonomic peripheral neuropathy (lkeda et ai. 1987). Using the radioimmunoassay method described by Nakazato et al. (1984b), a variant TTR with a methionine for valine substitution at position 30 was detected in his serum (serum concentration: 9.47 mg/dl), and the mutation of the TI'R gene responsible for this amino acid substitution was also demonstrated (Fig. 1). Histopatbological examinations revealed severe deposits of TTR-immunoreactive amyloid in systemic organs including peripheral nerves, and in the brain this amyloid deposition was mainly seen on the leptomeningeal vessels and pia-arachnoid membranes (Fig. 2). The detailed neuropathological find-
180
M1
2 3 o
0.32¢::
o
8 .~ 5(
Fig. 1. TTR gene analysis. Lane M: DNA molecular weight pHY marker; 1: non-digested DNA; 2: DNA after digestion with Bail; 3: DNA after digestion with NsiI. Two abnormal bands (consisting of 505 and 347 bp fragments after digestion with Bali and of 498 and 347 bp fragments after digestion with NsiI) are seen in addition to the normal 852 band. This DNA analysis pattern is specific for the heterozygousgene abnormality of type I FAP (Sakaki et al, 1989).
ings of this case and their relation to the central nervous system dysfunctions will be reported elsewhere (Ikeda et al., in preparation). The crude amyloid preparation was separated by Superose 12 chromatography into 4 fractions as shown in Fig. 3. Each fraction was analyzed by SDS-PAGE and immunoblotting. All 4 fractions contained antiTI'R immunoreactive materials, but the immunoreactivities to fraction IV were weaker than the other fractions as shown in Fig. 4. The reason of this phe-
I
o
-'--T
T-
20
80 Time (min)
Fig. 3. Elution profile of crude amyloid fraction on a Superose 12 column. Each of the fractions indicated by horizontal bars was pooled and examined further.
nomenon is not clear. Fraction III (MW 35 kDa) and IV (MW 15 kDa) which represented major protein components seemed to be dimeric and monomeric forms of TTR, respectively. Antibodies t o / ~ / A 4 protein and cystatin C did not react with these materials (data not shown). Fraction IV (MW 15 kDa) was
k
Fig. 2. Immunohistochemicalfindings of cerebral amyloidangiopathy. TTR-immunoreactive amyloiddeposits are seen on many subarachnoidal vesselsand leptomeninges.Bar ffi I00/~m.
181 1
2
3
4
5
6
7
8
9
10 GP
I0 20 T G T G E S K C P L M V K V L D A V R G S ............................>
30 NVAMHV
PAl
(v)
I. . . . . . . . . . . I . . . . . . . . . . . t. . . . . . . . . . . . . . . . . . . . .
M
T2
(k
T3
T4
40 50 60 F R K A A D D T W E P F A S G K T S E S G E L H G L T T E -->I
...........................
i
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E EF
I ...............................
T5-6
T?
70 80 90 I YKVEI D T K S Y W K A L G I S P F H E H A E V V F T .......... • I .......... > I ...... > I ............................... T8 T9 TI0
VEG
100 A N D S G P R R Y T .............
110 IAALL
S PYSYS
I .....................
T11-12a
120 TTAVVTNPK
I .........................
E I
T12b-13
Fig. 6. Complete amino acid sequence of purified amyloid fibril protein. The primary structure was established by direct sequencing of the purified amyloid protein and its individual tryptic peptides. Since the amino terminal sequence of the protein is heterogeneous, the data obtained for the major sequence starting at position 4 are
shown. Fig. 4. SDS-PAGE and immunoblotting of the separated amyloid fractions. Lanes 1-5: CBB staining; lanes 6-10: immunoblotting with anti-Tl'R antibody. Lanes 1 and 6, crude amyloid fraction; lanes 2 and 7, fraction I; lanes 3 and 8, fraction lI; lanes 4 and 9, fraction Ill; lanes 5 and 10, fraction IV.
E co
8
°
2
i
0.32 -
further purified by reverse phase HPLC and sequenced. Purified 15-kDa amyloid fibril protein displayed ragged N-terminal amino acid sequences. However, the main sequence (37%) was GTGESKxPLMVKVLDand was identical with the sequence of TCR starting at position 4. As shown in Figs. 5 and 6, tryptic peptides obtained from the purified 15-kDa amyloid protein were analyzed by automated Edman degradation and placed by homology to normal TTR (Kanda et al. 1974). Amino acid sequences of all of the tryptic peptides except for the T4 peptide corresponded to subsequences of normal TTR as shown in Fig. 6. The T4 peptide was GSPAINVAMHVFR and had a substitution of methionine for valine at position 9 of this peptide as shown in Fig. 6. Thus, this amyloid fibril protein had an identical sequence to normal TTR except for a methionine for valine substitution at position 30.
Discussion
0
60
Time (rain) Fig. 5. Elution profile of tryptic digests of purified amyloid fibril protein. Separation was achieved on an Aquarpare RP-300 column (2.1 x 100 ram) with a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid at 0.2 ml/min flow rate. Peak 1, T8; peak 2, 3"9; peak 3, T2 and T3; peak 4, T12b-13; peak 5, T5-6; peak 6, T4; peak 7, TI0; peak 8, T7; peak 9, Tll-12a.
Type I FAP is a systemic amyloidosis presumably caused by a variant TYR due to a gene mutation product responsible for a valine to methionine ~ubstitution at position 30 (Sasaki et al. 1984; Sakaki et al. 1989). In this disease, severe amyloid deposits composed of this variant TI'R are seen in the peripheral n e r v e s and various visceral organs (Andrade 1952; Ikeda et al. 1987; Hanyu et al. 1989). Recently, one of us (S.I.) has found that CAA is a common pathological finding in the brain of patients with type I FAP. The amyloid fibril protein comprising this form of CAA was demonstrated to be antigenically related to T r R , and any immunohistochemicai reactions for anti-/3/A4 protein antibody or anti-cystatin C antiserum were not seen, eve.,., a~cr formic acid pretreatment (Ushiyama et aL 1991). In the present study, to confirm this observa-
182 tion, we characterized the amyloid fibril protein obtained from the meninges in a type I FAP patient for the first time. It was found that crude amyloid fibril proteins reacted with only anti-TrR antibody and did not react with antibodies to well-known cerebral amyloid fibril proteins, /3/A4 protein and cystatin C. The major protein component had a molecular weight of 15 kDa, the same as that of normal TI'R. Complete sequence analysis revealed that this protein was a variant TTR with a substitution of methionine for valine at position of 30. Additionally, we also found minor components with molecular weights of 5-8 kDa and their N-terminal sequences were LTI'EEEFVE and IYKVEIDTK corresponded to positions at 58 and 68 of TTR, respectively (data not shown). It is well known that a variant TI'R with a methionine for valine substitution at position 30 is present in the serum of patients with type I FAP (Nakazato et al. 1984a; Suzuki et al. 1987). Therefore, these results suggest that the amyloid fibril protein in CAA of type I FAP is derived from a serum variant TI'R. CAA is observed in a variety of brain degenerative diseases (Castafio and Frangione 1988) and this type of amyloid vascular change selectively involves the leptomeningea! and cortical vessels of the brain (Tomonaga 1981; Vinters and Gilbert 1983; Vinters 1987). In the central nervous system of type 1 FAP patients, leptomeningeal vessels and pia-arachnoid membranes are the principal site of TTR-related amyloid deposition and this amyloid does not significantly affect the brain parenchyma (Ushiyama et al. 1991). Amyloid fibril protein isolated from meninges of a patient with type I FAP was identified with the variant TTR as described above. Similar pattern of amyloid deposits but more heavy involvement is seen in familial oculoleptomeningeal amyloidosis (Hamburg 1971; Okayama et al. 1978; Goren et aL 1980; Uitti et al. 1988), and amyloid fibril protein in the disease was immunohistochemically shown to be TTR (Uitti et al. 1988). However, this unusual form of hereditary amyloidosis mainly involves the central nervous system, and the patients with this disorder usually lack distinct clinical manifestations ascribable to amyloidotic polyneuropathy, autonomic disorder or visceral lesions. Accordingly, the clinical pictures of familial oculoleptomeningeal amyloidosis differ from those of type l FAP. The pathological mechanism of amyloid deposition on the cerebral vessels is incompletely understood, and the origin of cerebrovascular amyloid (/~/A4 protein, cystatin C and PrP protein) still remains controversial. In considering the morphological similarity of CAA seen in diverse disorders (consisting of biochemically different amyloid fibril proteins), TI'R-type CAA observed in patients with type I FAP is likely to be a
valuable model for the study of cerebrovascular amyloid deposits derived from an abnormal serum protein, the causative gene abnormality of which has been clearly demonstrated. Further studies are required to clarify the exact pathogenesis of CAA. Acknowledgements We thank Dr. M. Nakazato for the testing of serum variant TTR, Dr. D. AIIsop for the 4D12/2/6 antibody and critical reading of the manuscript, Dr. A.O. Grubb for the AG8206 antibody, Miss. K. Tanaka for her excellent technical support, Mr. K. Kato and Mr. A. Kagiwada for preparing the photographs. This research was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture and grants from Kanae Foundation of New Medicine and from the Primary Amyloidosis Research Committee of the Ministry of Health and Welfare, Japan.
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