Invitro expression analysis shows that the secretory form of gelsolin is the sole source of amyloid in gelsolin-related amyloidosis
In vitro expression analysis shows that the secretory form of gelsolin is the sole source of amyloid in gelsolin-related amyloidosisHannele Kangas*, Tiina Paunio, Nisse Kalkkinen1, Anu Jalanko and Leena Peltonen
Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finlandand 1Institute of Biotechnology, University of Helsinki, Helsinki,Finland
Received March 29, 1996;Revised and Accepted June 10, 1996
Amyloidoses are a group of diseases where abnormal fibrillar protein deposits accumulate in patients' tissues. In familial amyloidosis of the Finnish type (FAF), or gelsolin-related amyloidosis, the amyloid subunit protein consists of gelsolin peptides of amino acids 173-243 with the disease causing substitution at Asp187. Gelsolin is an actin-modulating protein and exists in both secretory and intracellular forms both encoded by a single gene in chromosome 9. We have previously shown that the FAF-associated forms of secretory gelsolin carrying the Asp187Asn or Asp187Tyr mutations are abnormally processed in cells, resulting in the secretion of an aberrant 68 kDa carboxyterminal fragment. Here we demonstrate by N-terminal sequencing that the amino terminus of this abnormal fragment is the amino acid 173 and thus represents the N-terminus of the FAF amyloid. We also provide evidence that the same truncated gelsolin can be found among the aberrant gelsolin fragments detected in patients' CSF. Finally, we also expressed the FAF-associated forms of intracellular gelsolin in COS-1 cells, and found no abnormality in their processing opposite to secretory form. Our results provide strong evidence that the secretory gelsolin is solely responsible for the amyloid formation in FAF.
Amyloidosis comprises a group of diseases where abnormal fibrillar protein deposits are found in the tissues of patients. These deposits have a [beta]-pleated structure showing typical green birefringence under Congo-red staining (1 ). Familial amyloidosis of the Finnish type (FAF), or gelsolin-related amyloidosis (AGel), is a familial amyloid polyneuropathy and is characterized by corneal lattice dystrophy and progressive cranial, especially facial, and peripheral neuropathy (2 ). There are 400-600 FAF patients in Finland (3 ), whereas only a few cases have been reported in other countries. The amyloid fibrils accumulating in the tissues of FAF patients are peptides consisting of amino acid residues 173-243 of gelsolin (4 ,5 ). Gelsolin is an actin-binding protein which exists both in intracellular and secretory (plasma) forms (6 ). A single gene in chromosome 9 encodes both these forms of gelsolin (7 ,8 ) but the transcription initiation sites and message splicing of the two forms are different (9 ). At the protein level they differ only by the presence of a 25 N-terminal amino acid extension (plasma extension) in secretory gelsolin (6 ). Both forms of gelsolin can sever actin filaments, nucleate actin filament assembly and block the fast-growing ends of actin under the control of calcium and polyphosphoinositides (10 ,11 ). Knock-out mice carrying a disrupted gelsolin gene have defects in platelet activation during hemostasis, inflammatory response, leukocyte motility and dermal fibroblast function in vivo (12 ).
FAF is caused by an amino acid substitution where Asp187 has changed either to Asn or to Tyr (13 -15 ). This replacement is due to a point mutation where the G654 nucleotide of gelsolin cDNA has changed either to A or to T (15 -17 ). FAF patients represent several aberrant, presumably carboxyterminal gelsolin fragments of size 60-70 kDa in their cerebrospinal fluid (CSF) and serum (18 -20 ). We have earlier shown that both disease-associated forms of secretorygelsolin (Asn187 and Tyr187) are abnormally processed in COS-1 cells, resulting in the secretion of a 68 kDa carboxyterminal gelsolin fragment into the culture medium. We have suggested that this aberrant cleavage would be the initial event which triggers the cascade of pathological events leading to FAF, and the 68 kDa secreted gelsolin fragment in all likelihood represents the precursor for FAF amyloid (21 ). However, the precise character of the abnormal cleavage as well as the interference of the FAF mutation in the processing of theintracellular form of gelsolin has so far remained unknown.
Here we ascertained the precise abnormal proteolytic cleavage site of the mutant secretory gelsolin. We also provide evidence that the aberrant fragments found in patients' CSF truly represent the carboxyterminal part of gelsolin and that the aberrant 68 kDa fragment secreted from the mutant COS cells is among them. We also monitored the cellular processing of the intracellular FAF gelsolin in the COS cell expression system, and our data suggest that mutant secretory gelsolin is the sole contributor to the formation of FAF amyloid.
COS-1 cells were transfected with the pCD-X expression construct coding for mutant (Asn187) secretory gelsolin as described earlier (21 ). Immunoblotting of the culture medium revealed both the 83 kDa full-size gelsolin and the aberrant 68 kDa fragment to be in accordance with our previous data (21 ). These fragments were partially purified and after SDS-PAGE and blotting were placed on a PVDF membrane for N-terminal sequencing. The 83 kDa band gave the sequence Ala-Thr-Ala-Ser-Arg-Gly-Ala-Ser-Gln-Ala, corresponding to the N-terminus of the wild type gelsolin. When the 68 kDa band was analyzed for 12 Edman degradation cycles, two amino acid residues could be detected in each cycle, indicating that the band consisted of a mixture of two proteins. One of the sequences from this combination, Ala-Thr-Glu-Val-Pro-Val-Ser-Trp-Glu-Ser-Phe Asn, revealed a 100% match to the gelsolin sequence starting from position 173 of the secretory gelsolin (Fig. 1 a). The calculated molecular weight for this fragment is 63876 Da, which is slightly less than the molecular weight assumed from its mobility on SDS-PAGE. The remaining part of the determined double sequence, Ile-Pro-Leu-Asp-Pro-Val-Ala-Gly-Tyr-Lys-Glu-Pro, corresponded to the N-terminal sequence of the [alpha]2-HS glycoprotein precursor (Fetuin) (GCG, SWISS PROT), a constituent of the cell growth medium.
The abnormal cleavage site determined by N-terminal sequencing was further confirmed by western blot analysis using the NH951 antibody, which recognizes the amino terminal part of gelsolin (amino acids 1-172). The expression constructs coding for wild type (Asp187) and mutant (Asn187) secretory gelsolin were used for transfection of COS-1 cells and the media was analyzed by immunoblotting. As expected, the NH951 antibody did not recognize the aberrant 68 kDa fragment of the mutant secretory gelsolin in western blot analysis (Figs 1 b, 2 ).
In order to monitor the cellular processing of the mutant secretory gelsolin, the transfected COS-1 cells were metabolically labeled with a mixture of 35S cysteine and 35S methionine for 2 h, chased for 0, 1, 4 and 16 h and the media were analyzed by immunoprecipitation with anti-gelsolin antibody followed by SDS-PAGE and fluorography. After 2 h pulse both the polypeptide band corresponding to the 83 kDa secretory gelsolin and the 68 kDa aberrant fragment were detected in the medium, indicating that mutant secretory gelsolin is secreted from the cells on a time scale close to that observed for the wild type (Fig. 4 ). After 1, 4 and 16 h chase the 68 kDa fragment could still be detected in the immunoprecipitate (data not shown). These results suggest that intracellular processing of the mutant secretory gelsolin closely resembles that of the wild type protein, the only aberration being the sensitivity of the mutant gelsolin to single proteolytic cleavage, which results in the secretion of the abnormal 68 kDa fragment.
To monitor the effect of FAF mutation on the metabolism of the intracellular form of gelsolin, constructs coding for the wild type (Asp187) and mutant (Asn187 and Tyr187) forms of intracellular gelsolin (Fig. 5 ) were expressed in COS-1 cells and both the media and cells were analyzed by immunoblotting using antibodies which recognize different epitopes of gelsolin (Fig. 2 ).
The processing of the intracellular wild type (Asp187) and mutant forms (Asn187 and Tyr187) of gelsolin were studied by pulse-chase experiments. The transfected COS-1 cells were metabolically labeled with 35S cysteine and 35S methionine for 2 h, chased for 0, 1, 4 and 16 h and analyzed by SDS-PAGE after immunoprecipitation with monoclonal anti-gelsolin antibody. After 2 h pulse time and the various chase times, no difference between the wild type and mutant forms of intracellular gelsolin could be detected. Both the wild type and mutant forms of intracellular gelsolin migrated with an apparent molecular weight of 80 kDa on western blots (Fig. 7 ).
Amyloidogenesis characteristically involves some specific amyloidogenic protein precursors, which are converted into amyloid fibrils by different potential mechanisms. Proteolysis, in particular, is likely to play a central role in various types of amyloidosis, as concluded by the observation that many amyloid peptides are fragments of larger precursor proteins. Our in vitro expression studies have earlier demonstrated that the FAF associated forms of secretory gelsolin (with Asn187 or Tyr187 mutation) are subjected to abnormal proteolysis in cells, resulting in the secretion of an aberrant 68 kDa carboxyterminal fragment (21 ). Here we determined the amino terminal sequence of this fragment with Asn187 mutation to be at amino acid position 173 of gelsolin. This provides strong evidence that the abnormal fragment truly represents the precursor protein for FAF amyloid, known to consist of amino-acids 173-244 of gelsolin (4 ,5 )
The possibility that mutant secretory gelsolin would be retained for exceptionally long times in the secretory pathway and become fragmented was ruled out by the pulse-chase experiments which showed that the aberrant gelsolin fragment is secreted within the same time scale as the normal full-length gelsolin. Since the proteolysis of mutant secretory gelsolin occurs in cells (21 ), this `first hit' seems to be a triggering prefibrillogenic event and thus crucial for the pathogenesis of FAF. Our demonstration of the novel proteolytic cleavage site at FAF gelsolin between amino acids 172-173 is in accordance with structural analyses of wild type gelsolin segment 1-actin complex, which have suggested that the FAF mutation at position 187 could cause local rearrangements in the gelsolin structure, leaving amino acid 172 (Arg) more accessible to proteases (22 ). Further, our data from western analysis strongly suggest that the mutant 68 kDa gelsolin fragment secreted from the cells can also be found among the abnormal carboxyterminal fragments in the CSF of the patients (18 ), where this polypeptide could represent the precursor for FAF amyloid, found extensively in the meninges of the patients (Fig. 9 , right) (23 ). To our knowledge, a similar pathological proteolytic processing of a larger precursor protein for amyloid has so far been demonstrated in cell cultures only in the case of Alzheimer's disease where the Swedish double mutation in APP altered the cleavage of the mutant APP within the secretory pathway (24 ).
The wild type (Asp187) and mutant (Asn187 and Tyr187) gelsolin cDNAs coding for secretory gelsolin (21 ) were digested with NarI (Pharmacia Biotech, USA) which cleaves gelsolin cDNA at nucleotide 129 and disrupts the initiation site of the secretory gelsolin. The intracellular gelsolin cDNA was then removed from the pCD-X vector (30 ) by EcoRI(Pharmacia Biotech, USA) digestion, blunt-ended and subcloned into the SmaI (New England BioLabs, USA) site of the pCD-X vector (Fig. 5 ). Clones with the insert in the correct orientation were identified by restriction enzyme mapping. The correct nucleotide sequence of the clones was confirmed by dideoxynucleotide sequencing (31 ).
Antibodies which recognize different epitopes of gelsolin were used in the assays (Fig. 2 ). K572 and AM904 antibodies are described in detail elsewhere (21 ). Monoclonal anti-gelsolin (Sigma Chemicals, St. Louis, USA) is specific for a 47 kDa peptide derived from a chymotryptic cleavage extending to the carboxyterminus of the protein (32 ). K572 rabbit antiserum was raised against a synthetic peptide of amino acid residues 231-242 located on the carboxyterminus of the amyloid part of gelsolin (21 ). AM904 rabbit antiserum was raised against gelsolin fragments extracted from amyloid in the tissues of a patient (21 ). To obtain the gelsolin polypeptides containing the amino terminal (amino acids 1-172, corresponding to the NH951 antibody) and amyloid-forming (amino acids 173-244, corresponding to the AM951 antibody) sequences of gelsolin, the gelsolin cDNAs coding for each part of the protein were amplified by PCR and cloned into the BamH1 site of the pGEX-2T fusion vector (Pharmacia Biotech, USA). The correct nucleotide sequence of each clone was confirmed by dideoxynucleotide sequencing (31 ). The polypeptides were expressed as a glutathione S-transferase (GST) fusion protein in E.coli strain BL21 and purified on 8 and 10% preparative SDS-PAGE. Rabbits were immunized by subcutaneous injection with 200-250 [mu]g of the fusion protein in Freund's complete adjuvant, which was repeated twice after 2 and 6 weeks. Blood was collected 1 week after the last immunization and the antibody titers and specificity were determined by western blotting (33 ).
Transfections were carried out as described earlier (21 ). Briefly, COS-1 cells (ATCC CLR 1650) (American Type Culture Collection) cultured in Dulbecco's modified Eagle's medium (DMEM) with antibiotics, glutamine and 10% fetal calf serum (FCS) were seeded at a confluence of 2-4 * 105 cells/3 cm dish 1 day prior to transfection. The cells were transfected either with 10 [mu]g of the pCD-X constructs using the DEAE dextran method (34 ) or with 3 [mu]g of the pCD-X constructs using the liposome transfection method (35 ). Serum-free medium was added 48 h after transfection, and after an overnight incubation, the medium was collected and the cells harvested by trypsinization. Protease inhibitors (EDTA, 1.2 mg/ml, Merck, Germany; PMSF, 1 mM, Sigma Chemicals, St. Louis, USA; E-64c, 0.1 mM, Boehringer Mannheim, Germany; Aprotinin 2 [mu]g/ml, Boehringer Mannheim, Germany) were used in collection and further experiments, unless stated otherwise. After harvesting, the cells were homogenized in DMEM, washed with ice-cold PBS and lysed by freeze-thawing in the phosphate buffer with 1% Triton X-100.
The protein concentrations of the samples were analyzed by Bio-Rad's protein detection assay (36 ). 10 [mu]g of cell samples in 1 * Laemmli buffer and 3 % [beta]-mercaptoethanol were run on 9 to 14% SDS polyacrylamide gel and electroblotted. The proteins were detected both by color detection using AP-conjugated secondary anti-mouse or anti-rabbit antibodies (DAKO, Denmark) or with enhanced chemiluminescence (ECL, Amersham, UK) using peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (DAKO, Denmark) as described earlier (21 ). For restaining, the primary and secondary antibodies were removed from the ECL filters by incubating in 62.5 mM Tris-HCl pH 6.7, 2% SDS, 100 mM [beta]-mercaptoethanol for 30 min at 50oC.
The COS-1 cells were transfected with the secretory form of mutant (Asn187) gelsolin as described earlier (21 ) and the cell growth medium (about 40 ml) was concentrated to 0.8 ml using a Novacell NC 10 Omega membrane concentrator (Filtron Technology Corporation, MA, USA). The concentrate was then subjected in 400 [mu]l portions to gel filtration in a Superdex HR 10/30 column (Pharmacia Biotech, Sweden) in 50 mM sodium phosphate pH 7.4, 150 mM NaCl at a flow rate of 0.5 ml/min. The collected fractions (0.5 ml) were analyzed by SDS-PAGE followed by immunoblotting. Gelsolin-containing fractions were further purified by reversed phase chromatography on a 0.21 * 4 cm TSK 250 TMS (C1, 5 [mu]m, 250 Å) column (Tosoh Corporation, Japan) using a linear gradient of acetonitrile (3-100% in 60 min) in 0.1% trifluoroacetic acid at a flow rate of 200 [mu]l/min. The collected fractions were analyzed by immunoblotting as above. The fractions containing the 83 kDa secretory gelsolin and the 68 kDa fragment of interest were dried in a vacuum centrifuge. Corresponding gelsolin fractions from six repeated reversed phase chromatography runs were combined and subjected to SDS-PAGE on 10% gel followed by blotting on a PVDF membrane (37 ). The protein bands on the membrane were visualized by staining with Coomassie Brilliant Blue. The 83 kDa bands and 68 kDa bands, also localized by parallel immunoblotting, were individually cut out and subjected to N-terminal sequence analysis on an Applied Biosystems Procise 494 A sequencer. The results were analyzed using an Applied Biosystems 610A Data Analysis System (Perkin Elmer Corporation, USA).
Transfected cells were starved in cysteine- and methionine-free medium for 30 min and thereafter labeled with a mixture of 100 [mu]Ci/ml of 35S cysteine and 100 [mu]Ci/ml of 35S methionine (Amersham, UK) for 2 h in cysteine- and methionine-free medium. The media and the cells were collected either after 2 h pulse time or subjected to 0 h, 1 h, 4 h and 16 h chase in DMEM without FCS. The cells and media were collected and the media concentrated by Centricon 3-30 (Amicon, Beverly, MA,USA). Immunoprecipitations were carried out with anti-gelsolin antibody at a 1:200 dilution as described earlier (21 ), with the following modifications: 0.1% SDS and 0.5% natriumdesoxycholate were added to the immunoprecipitation buffer and washing buffer I.
The cells were seeded on 12 mm coverslips and grown to 30-50% confluency overnight after transfections using the lipofectin method (35 ). The cells were fixed in freshly made 4% paraformaldehyde for 30 min at room temperature and after washing twice with PBS, permeabilized by incubating for 15 min in blocking buffer (PBS containing 0.5% BSA and 0.2% saponin). This was followed by incubation of anti-gelsolin antibody at a 1:500 dilution in the blocking buffer for 45 min. The cells were rinsed three times with blocking buffer and stained with a 1:50 dilution of rhodamine-conjugated rabbit anti-mouse IgG (DAKO, Denmark) in blocking buffer for 45 min at room temperature. After staining, the cells were rinsed three times with PBS and the coverslips were mounted in glycerol or in Mowiol and viewed with a Zeiss Axiophot immunofluorescence microscope using a 63* objective.
We thank Dr Jouni Vesa for helpful discussions, Dr Ilkka Julkunen and Tapani Ronni MSc for their advice on preparative SDS-PAGE and Dr Sari Kiuru and Dr Seppo Kaakkola for providing us the CSF patient and control samples. This study was supported by the Hjelt Foundation, the Paulo Foundation and the Maud Kuistila Foundation.
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