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Characterization of amyloid fibril protein from a case of cerebral amyloid angiopathy showing immunohistochemical reactivity for both β protein and cystatin C
38
Neuroscience Letters, 144 (1992) 38 42 ,~: 1992 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00
NSL 08913
Characterization of amyloid fibril protein from a case of cerebral amyloid angiopathy showing immunohistochemical reactivity for both fl protein and cystatin C K e i k o M a r u y a m a ", F u y u k i K a m e t a n i b, Shu-ichi Ikeda", T o k u h i r o I s h i h a r a ~ a n d N o b u o Y a n a g i s a w a ~' "Department o[ Medicine [Neurology], Shinshu University School of Medicine, Matsumoto (Japan). t'Department of Molecular Biology, 7bkyo Institute (if'Psychiatry, Tokyo /Japan) and "The First Department of Pathology, Yamaguchi University School of Medicine, Ube (Japan)
(Received 11 May 1992; Revised version received9 June 1992;Accepted9 June 1992) Key wor&." Cerebral amyloidangiopathy: Amyloid;fl-Protein;Cystatin C: Amino acid sequence
We isolated and carried out a chemicalanalysis of the amyloidfibril protein from the leptomeningealvesselsof a case with non-hereditarycerebral amyloid angiopathy (CAA) showing dual immunohistochemicalreactivitywith antibodies to both fl-protein and cystatin C. A crude amyloid fibril fraction reacted only with anti-fl-proteinantibody, and cystatin C immunoreactivitywas observedin the first PBS supernatant. Completeamino acid sequence of this cystatin C-immunoreactiveprotein showed a homologousstructure to that of normal cystatin C. It is concludedthat cystatin C is not an intrinsic component of the amyloidfibril in this type of CAA.
Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within the walls of leptomeningeal and cortical blood vessels [17]. This pathological condition is observed in several brain degenerative disorders [4], and is also recognized as a cause of intractable and recurrent cerebral hemorrhages [17, 20]. Previous biochemical and immunohistochemical studies have revealed four different amyloid fibril proteins in cerebrovascular amyloids: (1)fl-protein in Alzheimer's disease, aged Down's syndrome, Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) and non-demented elderly persons [5, 8, 9, 14], (2) cystatin C in Icelandic-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-I) [7, 13], (3) prion protein (PrP 27-30) in Creutzfeldt-Jakob disease and sheep scrapie [2, 16], (4) transthyretin in type I familial amyloid polyneuropathy [12]. Recently, we have revealed that CAA seen in some sporadic aged individuals can show a dual immunohistochemical reactivity towards antibodies to both fl protein and cystatin C [14]. However, it has not been determined whether cystatin C is actually an integral component of cerebrovascular amyloid fibrils in these cases. In the present study we biochemically characterized the amyCorrespondence." S.-i.Ikeda, DepartmentofMedicine(Neurology),Shinshu UniversitySchool of Medicine, Matsumoto 390, Japan.
loid fibrils from one of the previously reported CAA cases with apparent coexistence offl-protein and cystatin C [14]. The patient was a 74-year-old man with two episodes of subcortical cerebral hemorrhages. He had not been suffering from either hypertension or dementia, and there was no family history of strokes. Routine histopathological examination revealed cerebrovascular amyloid deposits without significant numbers of senile plaques or neurofibrillary tangles (Fig. 1, inset), and immunohistochemical stainings showed that CAA in this case was specifically labeled by both antibodies to fl-protein and cystatin C (Fig. 1). Amyloid fibrils were isolated from the meninges of the patient according to our modification of the method used by Glenner and Wong [8, 12]. Briefly, the meninges (ca. 3.9 g) were homogenized in phosphate-buffered saline (PBS). After centrifugation, the brownish top layer of the resultant pellet which was enriched in amyloid fibrils was digested with collagenase and centrifuged. The resultant pellet (crude amyloid fraction, wet weight, ca. 60 mg) was dissolved in 140/11 of 99% formic acid and incubated overnight at room temperature. After centrifugation, the supernatant fraction was applied to a column of Superose 12 equilibrated with 70% formic acid. The fractions containing the amyloid fibril protein were purified by reverse phase HPLC using an Aquapore RP-300 col-
39
Fig. 1. Histopathological findings of CAA. Polarized view of the section after Congo red staining shows extensive deposition of amyloid in both leptomeningeal and cortical blood vessels. Arrowheads indicate an identical vessel (A inset). All amyloid-laden vessels were invariably immunostained with anti-fl-protein antibody (A), and some of these vessels also reacted with the anti-cystatin C antiserum (B). Note that the cystatin C immunoreactive areas were mainly seen in the outer media to adventitia in the involved vessels (B). The immunohistochemical staining methods employed in this study have been described previously [15]. Bars= 100/.tm.
umn.The purified amyloid fibril protein was subjected to automatic amino acid sequence analysis (Applied Biosystems, 477A and 120A gas-phase sequencer).
1
2
3
4
5
Conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out for all of the protein fractions obtained. The primary
6 1
2
3
4
5
6
KDa 106.0 80.0 49.5 32.5 27.5 18.5
A
KDa 106.0 m 80.0 49.5 B -
32.5 27.5 18.5
-
I
II
B
Fig. 2. SDS-PAGE and immunobloning of the crude amyloid fraction and purified proteins. A: lanes 1 3, crude amyloid fraction; lanes 4-6, purified amyloid fibril protein. Lanes 1 and 4, CBB staining; lanes 2 and 5, immunoblotting with anti-fl-protein antibody; lanes 3 and 6, immunoblotting with anti-cystatin C antibody. Both the crude amyloid fraction and the purified amyloid fibril protein reacted specifically with the anti-fl-protein antibody. B: lanes 1-3, first PBS extraction; lanes 4-6, purified cystatin C-immunoreactive protein. Lanes 1 and 4, CBB staining; lanes 2 and 5, immunoblotting with anti-cystatin C antibody; lanes 3 and 6, immunoblotting with anti-fl-protein antibody.
40
-protein Purified amyloid fibril protein main 75% minor 25%
i0 20 30 40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI IGLMVGGVVIAT
1.28
DAEFRHDSGYEVHhQKLvFFAEDVg AEFRHDSGYEVh
Fig. 3. N-terminal amino acid sequence of purified amyloid fibril protein. The sequence from the amyloid fibril protein displayed N-terminal length heterogeneity with 25% of the molecules starting at position 2. Tentative identifications are denoted with the lower case single-letter amino acid code. This sequence is partially coincident with that offlprotein [8, 9].
antibodies employed in immunochemical assay were an antiserum raised against a synthetic peptide consisting of residues 1 24 offl-protein (R-17) [11] and an antiserum to human cystatin C (AG8206) [13]. The specimens were immunostained with the 1:500 1:1000 diluted antisera. For the purification of cystatin C-immunoreactive protein, the supernatant fraction obtained from first PBS extraction was dialyzed against 10 m M Tris-HC1, pH 8.8, and applied onto a DEAE-Sepharose CL-6B column equilibrated with the same buffer. The unbound materials were loaded onto a Bakerbond WP butyl reverse phase HPLC. The purified cystatin C-immunoreactive protein was digested with lysyl endopeptidase (EC 3.4.21.50, E/S = 1/25) in 0.1 M a m m o n i u m bicarbonate, pH 8.3 at 37°C for 4 h. The digests were separated and purified by reverse phase H P L C using a column of Aquapore RP-300, and then the collected peptides were sequenced as described above. The crude amyloid fibril fraction was separated on a column of Superose 12 and was further purified by reverse phase H P L C (data not shown). The amyloid fibril protein obtained from CAA in this case had a molecular weight of 5 kDa. The protein fractions including the crude amyloid one reacted only with the anti-fl-protein antibody, and cystatin C immunoreactivity was not seen in either the crude amyloid fibril fraction or the purified amyloid fibril protein (Fig. 2A). The N-terminal amino acid sequence of the purified amyloid fibril protein was identical with that offl-protein as shown in Fig. 3. The anti-cystatin C antibody showed a specific immunoreactivity with the first supernatant fraction from the PBS extraction (Fig. 2B). The protein purified from this fraction (Fig. 4) had a molecular weight of 14 kDa, and 5 peptides were obtained by reverse phase H P L C after the lysyl endopeptidase digestion. The complete amino acid sequence of these peptides were determined (Table I). The alignment of these peptides was deduced by homology with the corresponding region of cystatin C, and it was clear that the cystatin C-immunoreactive protein isolated from the cerebrovascular walls of the patient was normal cystatin C as shown in Fig. 5. CAA is seen in a variety of brain degenerative diseases [4], and especially in the brains of elderly individuals this
0.64
4
~;
4 Time (rain)
Fig. 4. Elution profile of cystatin C-immunoreactive fractions. Crosshatched peak indicates cystatin C-immunoreactive fractions. Separation was achieved on a Bakerbond WP butyl column (4.6 x 300 mm) eluted with a linear gradient of 10 50% acetonitrile in 0.05% trifluoroacetic acid at 0.5 ml/min flow rate.
vasculopathy is not a rare finding [17]. However, the incidence of cerebral hemorrhages caused by CAA is reported to range from 2% to 10% in these persons [10] and the reason for this low occurrence of CAA-related intracerebral bleeding remains unclear. We previously reported that CAA seen in some non-hereditary aged persons (the vast majority of these persons had a history of subcortical hemorrhages, possibly attributable to CAA) revealed a peculiar immunohistochemical staining pattern: all amyloid-laden vessels strongly reacted with antifl-protein antibody, and moreover cystatin C imi0
Cystatin C Purified
protein
: :
20
30
40
SSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASND SSPGKPPRLVGGPMDASVEEEGVRRALDFAvGEYNkASND LI I}---
50 60 *70 80 MYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNL MYHSRALQVVRARKQIVAGVNYFLDVELGRTTxTKTQPNL --L2t} L3 t F-90 100 110 120 DNCPFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA DNxPFHDQPHLKRKAFxSFQIYAVPWQGTMTLSKSTxQDA --k4 IH} L6 { I--L7-~ L5 Fig. 5. Complete amino acid sequence of cystatin C-immunoreactive protein. Upper line: the established sequence of cystatin-C [3]. Lower line: amino acid sequence of the purified cystatin C-immunoreactive protein. The primary structure was determined by direct sequencing of the purified peptides after digestion with lysyl endopeptidase. The alignment of these peptides was deduced by homology with the corresponding region of cystatin C. L3-4 consisted of peptides at positions 55 75 and 76 92 of cystatin C linked by the disulfide bond, and L(~ 7 consisted of the peptides at positions 95- 114 and 115 120 also linked by the disulfide bond. In HCHWA-I amyloid protein, an amino acid substitution of glutamine (Q) for leucine (L) is seen at position 68, as indicated by an asterisk. Cysteineresidues (shown as x) are not detectable in this systemand tentative identificationsare indicated by the lower case single-letter amino acid code.
41
munoreactivity was also seen on some of the vessels involved, mainly in the outer media to the adventitia of larger vessels [14]. These observations were confirmed by other investigators [6, 18, 19, 21]. In the present study we have shown that the cystatin C immunoreactive protein isolated from the leptomeningeal vessels of a patient with this type of CAA is not a component of cerebrovascular amyloid fibrils, and also the amino acid sequence of this cystatin C shows no structural abnormality. Therefore, the pathogenesis of CAA with cystatin deposition in this patient seems to be different from that of Icelandic-type hereditary CAA caused by a mutation in the cystatin C gene [15].
Cystatin C is a low-molecular-weight cysteine proteinase inhibitor, especially for cathepsin B [1, 3]. A relatively high amount of this protein has been demonstrated to be present in cerebrospinal fluid, seminal plasma and milk [1, 3], but the physiological properties or metabolism of this cysteine proteinase inhibitor in the central nervous system is still incompletely understood. In patients with CAA showing coexistence of fl-protein and cystatin C, an abnormal (presumably secondary) accumulation of cystatin C seems to be induced by the flprotein amyloid deposits on the diseased vessels. On the other hand, it is becoming clear that extensive deposition of amyloid in the cerebral vessels is a primary and impor-
TABLE I A M I N O A C I D SEQUENCES OF THE PEPTIDES OBTAINED F R O M LYSYL E N D O P E P T I D A S E D I G E S T I O N
Yields of PTH-amino acid (pmol) Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
L1 S S P G K P P R L V G G P M D A S V E E E G V R R A L D F A v G E Y N k
10.1 10.7 10.0 21.7 10.2 15.1 15.2 12.5 5.7 2.9 12.0 15.1 8.7 0.8 6.5 3.7 0.8 0.4 1.9 4.3 2.0 1.8 0.7 1.3 1.8 0.5 0.9 1.7 1.0 0.9 0.1 1.4 0.9 0.6 0.8 0.1
L2 A 26.2 S 10.1 N 16.0 D 19.9 M 3.1 Y4.7 H 4.7 S 10.1 R 12.7 A 4.6 L 5.1 Q 9.5 v 1.8 V 2.3 R 2.5 A 4.6 R 3.4 K 1.6
L3~ Q I V A G V N Y F L D V E L G R T T x T K
17.1 14.9 9.7 14.8 17.8 9.3 12.6 11.6 9.3 9.4 12.9 3.3 6.2 8.0 6.4 1.6 4.2 7.1 3.5 2.0
L5 T 46.2 Q 19.3 P 20.4 N 15.7 L 13.6 P 17.3 N 12.6 x P 9.7 F 5.5 H 3.1 D 10.2 Q 4.6 P 5.6 H 2.5 L 5.5 K 2.2
R 28.0 K 18.6 -
L6-7 A F x S F Q I Y A V P W Q G T M T
29.9 15.6 12.8 10.4 16.0 12.8 5.8 7.2 2.9 6.9 3.9 4.6 6.9 6.2 1.5 4.2
L 4.7 S 1.1 K 2.0
S T x Q D A
31.0 25.9 15.8 13.1 7.4
42
rant factor in the pathogenesis of CAA-related brain hemorrhage [17, 20], and the exact role ofcystatin C and related proteinase activity in this pathological condition has not been determined. However, on the basis of previous clinicopathological studies [14, 18, 19, 21], it is suggested that cystatin C, a non-amyloid component in the vascular wall, may play an additional significant role in the development of cerebral bleeding in CAA-affected patients. We thank Dr. D. Allsop for critical reading of the manuscript, and Miss K. Tanaka for her technical assistance. Antiserum to cystatin C (AG8206) was kindly provided by Dr. A.O. Grubb and this study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture and a grant t¥om the Primary Amyloidosis Research Committee of the Ministry of Health and Welfare, Japan. I Abrahamson, M., Barrett, A.J., Salvensen, G. and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids, J. Biol. Chem., 24 (19861 11282 11289. 2 Allsop, D., Ikeda, S., Bruce, M. and Glenner G.G., Cerebrovascnlar amyloid in scrapie-affected sheep reacts with antibodies to priori protein, Neurosci. Lett., 92 (1988) 234 239. 3 Barret, A.J., Davies, M.E. and Grubb, A., The place of human y-trace (cystatin C) amongst the cysteine proteinase inhibitors Biochem. Biophys. Res. Commun., 12(I (19841 631 636. 4 Castafio, E.M. and Frangione, B., Human amyloidosis, Alzheimer's disease and related disorders, Lab. Invest., 58 (19881 122 132. 5 Van Duinen, S.G., Castafio, E.M., Prelli, F., Bots, G.T.A.M., Luyendijk, W. and Frangione, B., Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer's disease, Proc. Natl. Acad. Sci. USA, 84 (1987) 5991 5994. 6 Fujihara, S., Shimode, K., Nakamura, M., Kobayashi, S. and Tsunematsu, T., Cerebral amyloid angiopathy with the deposition of cystatin C (gamma-trace) and ,6-protein. In K. Iqbal, H.M. Wisniewski and B. Winblad (Eds.), Alzheimer's Disease and Related Disorders, Alan R. Liss, New York, 1989, pp. 939 944. 7 Ghiso, J., Jensson, O. and Frangione, B., Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of T-trace basic protein (cystatin C), Proc. Natl. Acad. Sci. USA, 83 (19861 2974 2978. 8 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular
9
10
1l
12
13
14
15
16 17 18
19
20
21
amyloid protein, Biochem. Biophys. Res. Commun., 120 (19841 885- 891. Joachim, C.L., Duff'y, L.K., Morris, J.H. and Selkoe, D.J., Protein, chemical and immunohistochemical studies of meningovascular flamyloid protein in Alzheimer's disease and normal aging, Brain Res.. 474 (19881 100 111. Joachim, C.L., Morris, J.H. and Selkoe, D.J., Clinically diagnosed Alzheimer's disease: autopsy results in 150 cases, Ann. Neurol., 24 (19881 50 56. Ishii, T., Kametani, F., Haga, S. and Sato, M., The immunohislochemical demonstration of subsequence of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease, Neuropathol. Appl. Neurobiol., 15 (1989) 135-t47. Kametani, F., Ikeda, S., Yanagisawa, N., lshii, T. and Hanyu, N., Characterization of a transthyretin-related amyloid fibril protein from cerebral amyloid angiopathy in type I familial amyloid polyneuropathy, J. Neurol. Sci., 108 (19921 178 183. L6fberg, H., Grubb, A.O., Nilsson, E.K., Jensson, O., Gudmundsson, G., Blondal, H., Arnason. A. and Thorsteinsson, L., Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis, Stroke, 18 (1987)431 44(I. Maruyama, K., lkeda, S., lshihara, T., Alssop, D. and Yanagisawa. N., lmmunohistochemical characterization of cerebrovascular amyloid in 46 autopsied cases using antibodies to fl protein and cystatin C, Stroke, 21 (199(t) 397 4113. Palsdottir, A., Abrahamson, M., Thorsteinsson, L., Arnason, A., Olafsson, 1. and Grubb, A., Mutation in cystatin C gene causes hereditary brain haemorrhage, Lancet, ii (1988) 603 604. Prusiner, S.B. and DeArmond, S.J., Prions causing nervous system degeneration, Lab. Invest., 56 (1987} 349 363. Tomonaga, M., Cerebral amyloid angiopathy in the elderly, J. Am. Geriatr. Soc., 29 (19811151 157. Vinters, H.V., Nishimura, G.S., Secor, D.L. and Pardridge, W.M.. hnmunoreactive A4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer's disease, Am. J. Pathol., 137 (1990) 233 240. Vinters. H.VI, Secor, D.L., Pardridge, W.M. and Gray, F., lmmunohistochemical study of cerebral amyloid angiopathy. Ill. Widespread Alzheimer A4 peptide in cerebral microvessel walls colocalizes with gamma trace in patients with leukoencephalopathy, Ann. Neurol., 28 (1990) 34 42. Vonsattel, J.P.G., Myers, R.H., Hedley-Whytc, E.T., Ropper, A.H.. Bird. E.D. and Richardson, E.P., Cerebral anayloid angiopaIhy without and with cerebral hemorrhages: a comparative histological study. Ann. Neurol., 30 ( 1991 ) 637 649. Yong, W.H., Robert, M.E., Secor, D.L., Kleikamp, T.J. and Vinters, H.V.. Cerebral hemorrhage with biopsy-proved amyloid angiopathy, Arch. Neurol.. 49 (19921 51 58.
Neuroscience Letters, 144 (1992) 38 42 ,~: 1992 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00
NSL 08913
Characterization of amyloid fibril protein from a case of cerebral amyloid angiopathy showing immunohistochemical reactivity for both fl protein and cystatin C K e i k o M a r u y a m a ", F u y u k i K a m e t a n i b, Shu-ichi Ikeda", T o k u h i r o I s h i h a r a ~ a n d N o b u o Y a n a g i s a w a ~' "Department o[ Medicine [Neurology], Shinshu University School of Medicine, Matsumoto (Japan). t'Department of Molecular Biology, 7bkyo Institute (if'Psychiatry, Tokyo /Japan) and "The First Department of Pathology, Yamaguchi University School of Medicine, Ube (Japan)
(Received 11 May 1992; Revised version received9 June 1992;Accepted9 June 1992) Key wor&." Cerebral amyloidangiopathy: Amyloid;fl-Protein;Cystatin C: Amino acid sequence
We isolated and carried out a chemicalanalysis of the amyloidfibril protein from the leptomeningealvesselsof a case with non-hereditarycerebral amyloid angiopathy (CAA) showing dual immunohistochemicalreactivitywith antibodies to both fl-protein and cystatin C. A crude amyloid fibril fraction reacted only with anti-fl-proteinantibody, and cystatin C immunoreactivitywas observedin the first PBS supernatant. Completeamino acid sequence of this cystatin C-immunoreactiveprotein showed a homologousstructure to that of normal cystatin C. It is concludedthat cystatin C is not an intrinsic component of the amyloidfibril in this type of CAA.
Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within the walls of leptomeningeal and cortical blood vessels [17]. This pathological condition is observed in several brain degenerative disorders [4], and is also recognized as a cause of intractable and recurrent cerebral hemorrhages [17, 20]. Previous biochemical and immunohistochemical studies have revealed four different amyloid fibril proteins in cerebrovascular amyloids: (1)fl-protein in Alzheimer's disease, aged Down's syndrome, Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) and non-demented elderly persons [5, 8, 9, 14], (2) cystatin C in Icelandic-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-I) [7, 13], (3) prion protein (PrP 27-30) in Creutzfeldt-Jakob disease and sheep scrapie [2, 16], (4) transthyretin in type I familial amyloid polyneuropathy [12]. Recently, we have revealed that CAA seen in some sporadic aged individuals can show a dual immunohistochemical reactivity towards antibodies to both fl protein and cystatin C [14]. However, it has not been determined whether cystatin C is actually an integral component of cerebrovascular amyloid fibrils in these cases. In the present study we biochemically characterized the amyCorrespondence." S.-i.Ikeda, DepartmentofMedicine(Neurology),Shinshu UniversitySchool of Medicine, Matsumoto 390, Japan.
loid fibrils from one of the previously reported CAA cases with apparent coexistence offl-protein and cystatin C [14]. The patient was a 74-year-old man with two episodes of subcortical cerebral hemorrhages. He had not been suffering from either hypertension or dementia, and there was no family history of strokes. Routine histopathological examination revealed cerebrovascular amyloid deposits without significant numbers of senile plaques or neurofibrillary tangles (Fig. 1, inset), and immunohistochemical stainings showed that CAA in this case was specifically labeled by both antibodies to fl-protein and cystatin C (Fig. 1). Amyloid fibrils were isolated from the meninges of the patient according to our modification of the method used by Glenner and Wong [8, 12]. Briefly, the meninges (ca. 3.9 g) were homogenized in phosphate-buffered saline (PBS). After centrifugation, the brownish top layer of the resultant pellet which was enriched in amyloid fibrils was digested with collagenase and centrifuged. The resultant pellet (crude amyloid fraction, wet weight, ca. 60 mg) was dissolved in 140/11 of 99% formic acid and incubated overnight at room temperature. After centrifugation, the supernatant fraction was applied to a column of Superose 12 equilibrated with 70% formic acid. The fractions containing the amyloid fibril protein were purified by reverse phase HPLC using an Aquapore RP-300 col-
39
Fig. 1. Histopathological findings of CAA. Polarized view of the section after Congo red staining shows extensive deposition of amyloid in both leptomeningeal and cortical blood vessels. Arrowheads indicate an identical vessel (A inset). All amyloid-laden vessels were invariably immunostained with anti-fl-protein antibody (A), and some of these vessels also reacted with the anti-cystatin C antiserum (B). Note that the cystatin C immunoreactive areas were mainly seen in the outer media to adventitia in the involved vessels (B). The immunohistochemical staining methods employed in this study have been described previously [15]. Bars= 100/.tm.
umn.The purified amyloid fibril protein was subjected to automatic amino acid sequence analysis (Applied Biosystems, 477A and 120A gas-phase sequencer).
1
2
3
4
5
Conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out for all of the protein fractions obtained. The primary
6 1
2
3
4
5
6
KDa 106.0 80.0 49.5 32.5 27.5 18.5
A
KDa 106.0 m 80.0 49.5 B -
32.5 27.5 18.5
-
I
II
B
Fig. 2. SDS-PAGE and immunobloning of the crude amyloid fraction and purified proteins. A: lanes 1 3, crude amyloid fraction; lanes 4-6, purified amyloid fibril protein. Lanes 1 and 4, CBB staining; lanes 2 and 5, immunoblotting with anti-fl-protein antibody; lanes 3 and 6, immunoblotting with anti-cystatin C antibody. Both the crude amyloid fraction and the purified amyloid fibril protein reacted specifically with the anti-fl-protein antibody. B: lanes 1-3, first PBS extraction; lanes 4-6, purified cystatin C-immunoreactive protein. Lanes 1 and 4, CBB staining; lanes 2 and 5, immunoblotting with anti-cystatin C antibody; lanes 3 and 6, immunoblotting with anti-fl-protein antibody.
40
-protein Purified amyloid fibril protein main 75% minor 25%
i0 20 30 40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI IGLMVGGVVIAT
1.28
DAEFRHDSGYEVHhQKLvFFAEDVg AEFRHDSGYEVh
Fig. 3. N-terminal amino acid sequence of purified amyloid fibril protein. The sequence from the amyloid fibril protein displayed N-terminal length heterogeneity with 25% of the molecules starting at position 2. Tentative identifications are denoted with the lower case single-letter amino acid code. This sequence is partially coincident with that offlprotein [8, 9].
antibodies employed in immunochemical assay were an antiserum raised against a synthetic peptide consisting of residues 1 24 offl-protein (R-17) [11] and an antiserum to human cystatin C (AG8206) [13]. The specimens were immunostained with the 1:500 1:1000 diluted antisera. For the purification of cystatin C-immunoreactive protein, the supernatant fraction obtained from first PBS extraction was dialyzed against 10 m M Tris-HC1, pH 8.8, and applied onto a DEAE-Sepharose CL-6B column equilibrated with the same buffer. The unbound materials were loaded onto a Bakerbond WP butyl reverse phase HPLC. The purified cystatin C-immunoreactive protein was digested with lysyl endopeptidase (EC 3.4.21.50, E/S = 1/25) in 0.1 M a m m o n i u m bicarbonate, pH 8.3 at 37°C for 4 h. The digests were separated and purified by reverse phase H P L C using a column of Aquapore RP-300, and then the collected peptides were sequenced as described above. The crude amyloid fibril fraction was separated on a column of Superose 12 and was further purified by reverse phase H P L C (data not shown). The amyloid fibril protein obtained from CAA in this case had a molecular weight of 5 kDa. The protein fractions including the crude amyloid one reacted only with the anti-fl-protein antibody, and cystatin C immunoreactivity was not seen in either the crude amyloid fibril fraction or the purified amyloid fibril protein (Fig. 2A). The N-terminal amino acid sequence of the purified amyloid fibril protein was identical with that offl-protein as shown in Fig. 3. The anti-cystatin C antibody showed a specific immunoreactivity with the first supernatant fraction from the PBS extraction (Fig. 2B). The protein purified from this fraction (Fig. 4) had a molecular weight of 14 kDa, and 5 peptides were obtained by reverse phase H P L C after the lysyl endopeptidase digestion. The complete amino acid sequence of these peptides were determined (Table I). The alignment of these peptides was deduced by homology with the corresponding region of cystatin C, and it was clear that the cystatin C-immunoreactive protein isolated from the cerebrovascular walls of the patient was normal cystatin C as shown in Fig. 5. CAA is seen in a variety of brain degenerative diseases [4], and especially in the brains of elderly individuals this
0.64
4
~;
4 Time (rain)
Fig. 4. Elution profile of cystatin C-immunoreactive fractions. Crosshatched peak indicates cystatin C-immunoreactive fractions. Separation was achieved on a Bakerbond WP butyl column (4.6 x 300 mm) eluted with a linear gradient of 10 50% acetonitrile in 0.05% trifluoroacetic acid at 0.5 ml/min flow rate.
vasculopathy is not a rare finding [17]. However, the incidence of cerebral hemorrhages caused by CAA is reported to range from 2% to 10% in these persons [10] and the reason for this low occurrence of CAA-related intracerebral bleeding remains unclear. We previously reported that CAA seen in some non-hereditary aged persons (the vast majority of these persons had a history of subcortical hemorrhages, possibly attributable to CAA) revealed a peculiar immunohistochemical staining pattern: all amyloid-laden vessels strongly reacted with antifl-protein antibody, and moreover cystatin C imi0
Cystatin C Purified
protein
: :
20
30
40
SSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASND SSPGKPPRLVGGPMDASVEEEGVRRALDFAvGEYNkASND LI I}---
50 60 *70 80 MYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNL MYHSRALQVVRARKQIVAGVNYFLDVELGRTTxTKTQPNL --L2t} L3 t F-90 100 110 120 DNCPFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA DNxPFHDQPHLKRKAFxSFQIYAVPWQGTMTLSKSTxQDA --k4 IH} L6 { I--L7-~ L5 Fig. 5. Complete amino acid sequence of cystatin C-immunoreactive protein. Upper line: the established sequence of cystatin-C [3]. Lower line: amino acid sequence of the purified cystatin C-immunoreactive protein. The primary structure was determined by direct sequencing of the purified peptides after digestion with lysyl endopeptidase. The alignment of these peptides was deduced by homology with the corresponding region of cystatin C. L3-4 consisted of peptides at positions 55 75 and 76 92 of cystatin C linked by the disulfide bond, and L(~ 7 consisted of the peptides at positions 95- 114 and 115 120 also linked by the disulfide bond. In HCHWA-I amyloid protein, an amino acid substitution of glutamine (Q) for leucine (L) is seen at position 68, as indicated by an asterisk. Cysteineresidues (shown as x) are not detectable in this systemand tentative identificationsare indicated by the lower case single-letter amino acid code.
41
munoreactivity was also seen on some of the vessels involved, mainly in the outer media to the adventitia of larger vessels [14]. These observations were confirmed by other investigators [6, 18, 19, 21]. In the present study we have shown that the cystatin C immunoreactive protein isolated from the leptomeningeal vessels of a patient with this type of CAA is not a component of cerebrovascular amyloid fibrils, and also the amino acid sequence of this cystatin C shows no structural abnormality. Therefore, the pathogenesis of CAA with cystatin deposition in this patient seems to be different from that of Icelandic-type hereditary CAA caused by a mutation in the cystatin C gene [15].
Cystatin C is a low-molecular-weight cysteine proteinase inhibitor, especially for cathepsin B [1, 3]. A relatively high amount of this protein has been demonstrated to be present in cerebrospinal fluid, seminal plasma and milk [1, 3], but the physiological properties or metabolism of this cysteine proteinase inhibitor in the central nervous system is still incompletely understood. In patients with CAA showing coexistence of fl-protein and cystatin C, an abnormal (presumably secondary) accumulation of cystatin C seems to be induced by the flprotein amyloid deposits on the diseased vessels. On the other hand, it is becoming clear that extensive deposition of amyloid in the cerebral vessels is a primary and impor-
TABLE I A M I N O A C I D SEQUENCES OF THE PEPTIDES OBTAINED F R O M LYSYL E N D O P E P T I D A S E D I G E S T I O N
Yields of PTH-amino acid (pmol) Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
L1 S S P G K P P R L V G G P M D A S V E E E G V R R A L D F A v G E Y N k
10.1 10.7 10.0 21.7 10.2 15.1 15.2 12.5 5.7 2.9 12.0 15.1 8.7 0.8 6.5 3.7 0.8 0.4 1.9 4.3 2.0 1.8 0.7 1.3 1.8 0.5 0.9 1.7 1.0 0.9 0.1 1.4 0.9 0.6 0.8 0.1
L2 A 26.2 S 10.1 N 16.0 D 19.9 M 3.1 Y4.7 H 4.7 S 10.1 R 12.7 A 4.6 L 5.1 Q 9.5 v 1.8 V 2.3 R 2.5 A 4.6 R 3.4 K 1.6
L3~ Q I V A G V N Y F L D V E L G R T T x T K
17.1 14.9 9.7 14.8 17.8 9.3 12.6 11.6 9.3 9.4 12.9 3.3 6.2 8.0 6.4 1.6 4.2 7.1 3.5 2.0
L5 T 46.2 Q 19.3 P 20.4 N 15.7 L 13.6 P 17.3 N 12.6 x P 9.7 F 5.5 H 3.1 D 10.2 Q 4.6 P 5.6 H 2.5 L 5.5 K 2.2
R 28.0 K 18.6 -
L6-7 A F x S F Q I Y A V P W Q G T M T
29.9 15.6 12.8 10.4 16.0 12.8 5.8 7.2 2.9 6.9 3.9 4.6 6.9 6.2 1.5 4.2
L 4.7 S 1.1 K 2.0
S T x Q D A
31.0 25.9 15.8 13.1 7.4
42
rant factor in the pathogenesis of CAA-related brain hemorrhage [17, 20], and the exact role ofcystatin C and related proteinase activity in this pathological condition has not been determined. However, on the basis of previous clinicopathological studies [14, 18, 19, 21], it is suggested that cystatin C, a non-amyloid component in the vascular wall, may play an additional significant role in the development of cerebral bleeding in CAA-affected patients. We thank Dr. D. Allsop for critical reading of the manuscript, and Miss K. Tanaka for her technical assistance. Antiserum to cystatin C (AG8206) was kindly provided by Dr. A.O. Grubb and this study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture and a grant t¥om the Primary Amyloidosis Research Committee of the Ministry of Health and Welfare, Japan. I Abrahamson, M., Barrett, A.J., Salvensen, G. and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids, J. Biol. Chem., 24 (19861 11282 11289. 2 Allsop, D., Ikeda, S., Bruce, M. and Glenner G.G., Cerebrovascnlar amyloid in scrapie-affected sheep reacts with antibodies to priori protein, Neurosci. Lett., 92 (1988) 234 239. 3 Barret, A.J., Davies, M.E. and Grubb, A., The place of human y-trace (cystatin C) amongst the cysteine proteinase inhibitors Biochem. Biophys. Res. Commun., 12(I (19841 631 636. 4 Castafio, E.M. and Frangione, B., Human amyloidosis, Alzheimer's disease and related disorders, Lab. Invest., 58 (19881 122 132. 5 Van Duinen, S.G., Castafio, E.M., Prelli, F., Bots, G.T.A.M., Luyendijk, W. and Frangione, B., Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer's disease, Proc. Natl. Acad. Sci. USA, 84 (1987) 5991 5994. 6 Fujihara, S., Shimode, K., Nakamura, M., Kobayashi, S. and Tsunematsu, T., Cerebral amyloid angiopathy with the deposition of cystatin C (gamma-trace) and ,6-protein. In K. Iqbal, H.M. Wisniewski and B. Winblad (Eds.), Alzheimer's Disease and Related Disorders, Alan R. Liss, New York, 1989, pp. 939 944. 7 Ghiso, J., Jensson, O. and Frangione, B., Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of T-trace basic protein (cystatin C), Proc. Natl. Acad. Sci. USA, 83 (19861 2974 2978. 8 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular
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