Amyloidogenic and Associated Proteins in Systemic Amyloidosis Proteome of Adipose Tissue
2008; Elsevier BV; Volume: 7; Issue: 8 Linguagem: Inglês
10.1074/mcp.m700545-mcp200
ISSN1535-9484
AutoresFrancesca Lavatelli, David H. Perlman, Brian Spencer, Tatiana Prokaeva, Mark E. McComb, Roger Théberge, Lawreen H. Connors, Vittorio Bellotti, David C. Seldin, Giampaolo Merlini, Martha Skinner, Catherine E. Costello,
Tópico(s)Caveolin-1 and cellular processes
ResumoIn systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of αB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration. In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of αB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration. The amyloidoses constitute a heterogeneous group of diseases whose common pathological hallmark is the presence of extracellular or intracellular amyloid deposits that lead to cellular toxicity, disruption of anatomical architecture, and organ dysfunction (1Merlini G. Bellotti V. Molecular mechanisms of amyloidosis.N. Engl. J. Med. 2003; 349: 583-596Crossref PubMed Scopus (1467) Google Scholar). In the systemic forms, widespread extracellular amyloid deposition leads to severe dysfunction of vital organs such as the heart, kidney, and liver, resulting in poor prognosis for long term survival. Despite their lack of similarity in amino acid sequence, the amyloidogenic proteins share certain secondary structural similarities (e.g. the tendency to form β-pleated sheet structures) and generate morphologically similar fibrils that show apple green birefringence under polarized light when stained with Congo red. Diagnosis and type classification of amyloid diseases are based on detecting the insoluble amyloid aggregates and determining which protein is the primary constituent (2Westermark P. Benson M.D. Buxbaum J.N. Cohen A.S. Frangione B. Ikeda S.I. Masters C.L. Merlini G. Saraiva M.J. Sipe J.D. Amyloid: toward terminology clarification—report from the Nomenclature Committee of the International Society of Amyloidosis.Amyloid. 2005; 12: 1-4Crossref PubMed Scopus (285) Google Scholar, 3Obici L. Perfetti V. Palladini G. Moratti R. Merlini G. Clinical aspects of systemic amyloid diseases.Biochim. Biophys. Acta. 2005; 1753: 11-22Crossref PubMed Scopus (216) Google Scholar, 4Falk R.H. Comenzo R.L. Skinner M. Medical progress—the systemic amyloidoses.N. Engl. J. Med. 1997; 337: 898-909Crossref PubMed Scopus (1078) Google Scholar, 5Picken M.M. New insights into systemic amyloidosis: the importance of diagnosis of specific type.Curr. Opin. Nephrol. 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The most common systemic forms worldwide are light chain amyloidosis (AL) 1The abbreviations used are: AL, light chain amyloidosis; ATTR, transthyretin amyloidosis; 2D, two-dimensional; PMF, peptide mass fingerprinting; TTR, transthyretin; VL, variable region, light chain; FR, framework region; MOWSE, molecular weight search. and reactive amyloidosis in which the fibrils are derived, respectively, from monoclonal immunoglobulin light chains and from the acute phase reactant, serum amyloid A protein. However, hereditary amyloidoses, caused by a large and ever expanding list of mutations in genes that code for normally soluble plasma proteins such as transthyretin (TTR), lysozyme, and apolipoproteins A-I and A-II, are not uncommon and have a particularly high incidence in some geographic regions. Little is known of the mechanism underlying the loss of protein secondary and tertiary stability under physiological conditions and the resultant protein deposition as fibrils in target organs. Post-translational modifications of amyloidogenic and/or associated proteins or changes in the local tissue environment, particularly those related to aging and oxidative stress, have been hypothesized to be important factors in disease onset and progression and may also play a role in the variability of clinical presentation (6Saraiva M.J. Transthyretin amyloidosis: a tale of weak interactions.FEBS Lett. 2001; 498: 201-203Crossref PubMed Scopus (87) Google Scholar, 7Lim A. Prokaeva T. McComb M.E. O'Connor P.B. Theberge R. Connors L.H. Skinner M. Costello C.E. Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis.Anal. Chem. 2002; 74: 741-751Crossref PubMed Scopus (61) Google Scholar, 8McComb M.E. Lim A. Théberge R. Prokaeva T. Connors L.H. Skinner M. Costello C.E. 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Here we applied a 2D PAGE- and MS-based proteomics approach to characterize and identify proteins deposited in abdominal subcutaneous fat tissue aspirates from patients with different types of systemic amyloidoses. We found profound differences between the 2D adipose tissue proteomes of normal and amyloidosis patients that can be exploited as a diagnostic tool for amyloid typing as the approach utilizes tissue that is routinely collected as part of diagnosis. Furthermore we demonstrate the characterization of the deposited amyloidogenic proteins and their isoforms or fragments, the identification of co-deposited proteins that are known to be associated with amyloid fibrils in various tissues, and apparent variations in the regulation of proteins involved in chaperone activity, cellular energetics, and redox regulation in the adipocytes of diseased patients. Abdominal subcutaneous fat tissue samples used as controls were obtained, with institutional review board approval and informed consent, from seven non-amyloid-affected patients (four male and three female; median age, 72 years) undergoing abdominal surgery at Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Matteo, Pavia, Italy, during the course of the surgical procedure. Patients with neoplastic or other systemic diseases were excluded. Subcutaneous fat samples from patients with systemic amyloidosis were obtained by fine needle aspiration during the routine diagnostic procedure at the Amyloid Treatment and Research Program, Boston University School of Medicine. One sample (ATTR patient 05-152) was surgically obtained at Boston Medical Center with informed consent during inguinal herniectomy. All patients who had received specific treatments for disease were excluded from the study. Samples were obtained from a total of nine amyloidosis patients (six male and three female; seven patients were diagnosed with AL (six λ and one κ), and two were diagnosed with ATTR) with a median age of 65 years. The presence of amyloid fibrils was evaluated histologically with Congo red staining, and the amyloid load was graded with a score from 0 (negative) to 3 (intensely positive). All specimens were immediately frozen and stored at −80 °C until used. Matched blood samples were also obtained contemporaneously with fat tissue acquisition. Serum was separated by centrifugation and then kept frozen until used. Thawed adipose tissue samples were washed repeatedly with sterile isotonic saline and once with double distilled water. For large samples (in the range of mg to g of tissue), IEF buffer (7 m urea, 2 m thiourea, 4% CHAPS, 65 mm DTT) was added directly to samples at a ratio of 200 μl/100 mg of tissue. Samples were crushed with a disposable pestle and sonicated in ice-water four times (10 pulses of 5 s each time) at intervals of 15 min with gentle shaking at room temperature between intervals. Samples were then centrifuged at 80,000 × g for 1.5 h at 19 °C (23Corton M. Villuendas G. Botella J.I. San Millan J.L. Escobar-Morreale H.F. Peral B. Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide.Proteomics. 2004; 4: 438-441Crossref PubMed Scopus (54) Google Scholar). The central aqueous layer between the top lipid layer and the cell debris pellet was recovered, residual lipids were removed with a second centrifugation at 25,000 × g for 30 min at 4 °C, and the aliquots were stored at −80 °C. Small patient samples (in the range of 10–20 μg of tissue) were washed, resuspended in 100 μl of IEF buffer, sonicated as described above, and then centrifuged for 1 h at 25,000 × g at 4 °C. The central aqueous layer was recovered and then stored at −80 °C. Large and small sample preparation techniques produced identical results when compared with one another using the same samples. Total protein was quantitated relative to standards using the Bio-Rad Protein Assay. Protein extracts (amounts equivalent to 10–30 μg of protein) were diluted to a final volume of 300 μl with 100 μl of Destreak™ buffer (Amersham Biosciences), IEF buffer, and pI 3–10 ampholytes (Bio-Rad) at a final concentration 0.02%. For serum samples, an aliquot of 6.25 μl of serum was mixed with 10 μl of 10% SDS, 2.3% DTT; heated to 95 °C for 5 min; and diluted to 500 μl with IEF buffer (24Hughes G.J. Frutiger S. Paquet N. Ravier F. Pasquali C. Sanchez J.C. James R. Tissot J.D. Bjellqvist B. Hochstrasser D.F. Plasma protein map—an update by microsequencing.Electrophoresis. 1992; 13: 707-714Crossref PubMed Scopus (120) Google Scholar). Sixty-five microliters of this solution were then diluted to a final volume of 300 μl using IEF and Destreak buffers and ampholytes as described above for tissue samples. Seventeen-centimeter ReadyStrip™ IPG strips (Bio-Rad) with non-linear gradients of pH 3–10 were subjected to passive rehydration for 1 h and then to active rehydration at 50 V for 8 h. Isoelectric focusing was performed in a Protean™ IEF cell (Bio-Rad) as follows: 120 V for 1 h, 300 V for 30 min, a linear increase up to 3500 V over 3 h, 5000 V for 10 min, and 8000 V steady until a total of 67,000 V-h had elapsed. After IEF, the strips were subjected to standard disulfide reduction with DTT and cysteine alkylation with iodoacetamide followed by second dimension electrophoresis using 9–16% gradient ReadyGels™ (Bio-Rad). Gels were stained with fixative silver stain, PlusOne™ (Amersham Biosciences); the MS-compatible silver stain ProteoSilver™ Plus (Sigma-Aldrich); or GelCode™ colloidal Coomassie Blue (Pierce). All gels were imaged with an EDAS 290 (Eastman Kodak Co.) or a VersaDoc™ 3000 (Bio-Rad) imaging station, and the results were analyzed using PDQuest™ software (Bio-Rad). After electrophoresis, proteins were transferred to a Millipore™ Q PVDF membrane (Millipore, Billerica, MA) using a TransBlot™ semidry electrophoretic transfer cell (Bio-Rad) and probed with polyclonal rabbit anti-human immunoglobulin light chain or rabbit anti-human transthyretin antibodies (Dako, Glostrup, Denmark), both at a 1:1000 dilution, followed by an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Dako) used at a 1:5000 dilution. In-gel digestion was conducted as described previously (25Perlman D.H. Berg E.A. O'Connor P.B. Costello C.E. Hu J. Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9020-9025Crossref PubMed Scopus (99) Google Scholar). Briefly the protein spots of interest were punch-excised using wide orifice pipette tips. Gel pieces were destained with either Coomassie destain (100 mm NH4CO3, pH 9, 50% acetonitrile) or silver destain (the potassium ferrocyanide-based reagents of the ProteoSilver Plus kit). The gel pieces were washed three times with wash solution 1 (100 mm NH4HCO3, pH 9) and wash solution 2 (100 mm NH4HCO3, pH 9, 50% acetonitrile) followed by wash solution 3 (100% acetonitrile). Gel pieces were swelled in digestion solution (∼10 ng of Trypsin Gold (Promega, Madison, WI) in 50 mm NH4HCO3, pH 9, 5% acetonitrile) and incubated overnight at 37 °C. Peptides were extracted twice with extraction solution 1 (20 mm NH4HCO3, pH 9) and extraction solution 2 (1% trifluoroacetic acid, 50% acetonitrile) followed by extraction solution 3 (100% acetonitrile), each time for 20 min. The entire extraction process was performed in duplicate, and all extraction supernatants were pooled and dried. Peptides were desalted and prepared for MS using micro-reversed-phase chromatography (ZipTips™, Millipore). Mass spectra were obtained after purified peptides were co-crystallized with the matrix 2,5-dihydroxybenzoic acid onto AnchorChip™ (Bruker Daltonics, Billerica, MA) targets using the dried droplet technique (26Karas M. Bachmann D. Bahr U. Hillenkamp F. Matrix-assisted ultraviolet-laser desorption of nonvolatile compounds.Int. J. Mass Spectrom. 1987; 78: 53-68Crossref Scopus (1722) Google Scholar, 27Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4850) Google Scholar) using a Reflex IV™ (Bruker Daltonics) MALDI-TOF MS instrument operated in the positive ion, reflectron mode over the range m/z 400–8000. The intensity of the nitrogen laser irradiation (337 nm, 3-ns pulse duration) was adjusted to ∼3% above threshold value. Signals resulting from 100 to 200 laser shots were summed for each mass spectrum. External calibration of the spectra was achieved using Bruker peptide standards, and internal calibration within 50 ppm was achieved using the masses of known peptide ions as reference values. MALDI-TOF mass spectra were deisotoped, and peak lists were generated using the software MoverZ™ from Genomic Solutions (Ann Arbor, MI). Peak lists were submitted to the on-line database search engine Mascot™ at Matrix Science (London, UK) for peptide mass fingerprinting (PMF) analysis against the Swiss-Prot or National Center for Biotechnology Information (NCBI) non-redundant protein databases using the following restrictions: (a) Homo sapiens, (b) trypsin digestion with up to three missed cleavages, (c) ±100-ppm error, and (d) cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification. Identifications were assigned when the score values were returned with p < 0.05 for a false positive and were confirmed by individual inspection of the spectra. Comprehensive peak assignments were accomplished manually using theoretical digest values generated from Mascot-matched sequences (or in the case of immunoglobulin light chain samples, monoclonal light chain sequences established from genetic sequencing of the corresponding patient-derived bone marrow samples) with the on-line software tool MS-Digest (Protein Prospector at University of California, San Francisco, CA) (28Clauser K.R. Baker P. Burlingame A.L. Role of accurate mass measurement (±10 ppm) in protein identification strategies employing MS or MS/MS and database searching.Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (977) Google Scholar). Cells from bone marrow aspirates were treated with NH4Cl to lyse red blood cells, washed, pelleted, and frozen at −80 °C as described previously (29Comenzo R.L. Michelle D. LeBlanc M. Wally J. Zhang Y. Kica G. Karandish S. Arkin C.F. Wright D.G. Skinner M. McMannis J. Mobilized CD34+ cells selected as autografts in patients with primary light-chain amyloidosis: rationale and application.Transfusion. 1998; 38: 60-69Crossref PubMed Scopus (41) Google Scholar). Total RNA was extracted, and cDNA was synthesized, amplified by multiplex PCR with a set of 5′ primers specific for the framework (FR) 1 region of seven Vλ (VλI, VλII/V, VλIII, VλIVa, VλIVb, and VλVI) and four Vκ (VκI/IV, VκII, and VκIII) families and 3′ λ or κ constant region primers (Integrated DNA Technologies, Coralville, IA) (30Welschof M. Terness P. Kolbinger F. Zewe M. Dubel S. Dorsam H. Hain C. Finger M. Jung M. Moldenhauer G. Hayashi N. Little M. Opelz G. Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.J. Immunol. Methods. 1995; 179: 203-214Crossref PubMed Scopus (71) Google Scholar), cloned, and sequenced as described previously (31Weichman K. Dember L.M. Prokaeva T. Wright D.G. Quillen K. Rosenzweig M. Skinner M. Seldin D.C. Sanchorawala V. Clinical and molecular characteristics of patients with non-amyloid light chain deposition disorders, and outcome following treatment with high-dose melphalan and autologous stem cell transplantation.Bone Marrow Transplant. 2006; 38: 339-343Crossref PubMed Scopus (61) Google Scholar). The clonal sequence was determined on the basis of identity of at least 50% of multiple independently cloned and sequenced products. Sequences were compared using the Jellyfish™ gene analysis software package (Field Scientific, LLC). Once the light chain was identified, additional PCR amplification was performed to correct for minor nucleotide sequence errors introduced by the FR1 primers, and resequencing was carried out. For these experiments, 5′ primers specific for the appropriate VL leader regions and 3′ primers for the appropriate constant region were used. VL genes with the corrected FR1 sequences were evaluated for their homology to the germ line donor sequences using a database of rearranged immunoglobulin genes, V BASE, and the International Immunogenetics Information System, IMGT/V-QUEST (32Giudicelli V. Chaume D. Lefranc M.P. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis.Nucleic Acids Res. 2004; 32: W435-W440Crossref PubMed Scopus (248) Google Scholar). The assignation of germ line gene counterpart was done based on maximum homology of the nucleotide sequences. Homology to germ line sequence was determined for complete VL genes with the exception of the codons associated with the variable-joining junction and FR4. All sequences were functional without stop codons, frameshifts, or pseudogenes. Genomic DNA was extracted from the peripheral blood leukocytes by standard phenol/chloroform procedure. Exon 4 of the TTR gene was amplified by PCR as described elsewhere (33Lim A. Prokaeva T. Connors L.H. Falk R.H. Skinner M. Costello C.E. Identification of a novel transthyretin Thr59Lys/Arg104His. A case of compound heterozygosity in a Chinese patient diagnosed with familial transthyretin amyloidosis.Amyloid. 2002; 9: 134-140Crossref PubMed Scopus (20) Google Scholar) The PCR product was subjected to electrophoresis in a 1.5% agarose gel, stained with ethidium bromide, visualized with ultraviolet light, purified by QIAquick purification kit (Qiagen, Valencia, CA), and sequenced in both directions. As a basis for comparison with samples from patients with amyloidosis, it was essential to establish a proteomic reference map of non-amyloid adipose tissue. The Congo red-negative abdominal subcutaneous fat samples from individuals (n ≥ 4) without evidence of amyloid disease were analyzed by 2D PAGE followed by Coomassie or silver staining. A representative control 2D map is shown in Fig. 1. Selected landmark spots (Fig. 1, circled spots, spot numbers 1–14), which were relatively abundant and present in roughly equal amounts across all the samples, were identified by in-gel protease digestion, MALDI-TOF MS, and PMF analyses as listed in Table I. Notably although low levels of some blood proteins were detected, the thorough washing of the specimens had minimized contamination by red cell or plasma proteins, and the vast majority of the observed spots were derived from adipose tissue-resident proteins.Table IAssignments of selected protein spots from 2D PAGE, MALDI-TOF MS, and PMF analysis of control fat tissueSpot no. (Fig. 1)Spot IDNCBInr accession no.MOWSE scoreNumber of matching peptides (sequence coverage)1Albumingi_5566991034251 (76%)2Actin βgi_1425040110623 (54%)3Vimentingi_6241428935056 (74%)4GP96gi_616566079125 (29%)5Glycerol-3-P dehydrogenasegi_170802611416 (48%)6Annexin A2gi_1864516742461 (94%)7Aldo-keto reductase Igi_450328510016 (53%)8Peroxiredoxin 6gi_475863810616 (68%)9Mn-SOD, mitochondrialgi_3850333915219 (77%)10αB-Crystallin isoformgi_450305712016 (75%)11αB-Crystallin isoformgi_45030579314 (64%)12Hemoglobin βgi_44285012117 (100%)13Adipocyte fatty acid-binding proteingi_52695841927 (51%)1414-3-3gi_8375446712116 (40%) Open table in a new tab Abdominal subcutaneous fat samples were obtained from nine amyloidosis patients, and their amyloid infiltration was graded according to the extent of their Congo red-positive staining. As noted above, all the control samples were negative for amyloid deposition. The results for the patient samples are shown in Table II. For all patients, the type of amyloidosis had been characterized by the demonstration of
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