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• W2016997461 abstract "Patients homozygous for the Z mutant form of α1-proteinase inhibitor (α1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant α1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one α1-PI monomer between the central strands of the A β-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A β-sheet of α1-PI monomer and this insertion prevents α1-PI polymerization. Normal α1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal α1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for α1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A β-sheets may not occur during polymerization of this dimer. Patients homozygous for the Z mutant form of α1-proteinase inhibitor (α1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant α1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one α1-PI monomer between the central strands of the A β-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A β-sheet of α1-PI monomer and this insertion prevents α1-PI polymerization. Normal α1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal α1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for α1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A β-sheets may not occur during polymerization of this dimer. α1-Proteinase inhibitor (α1-PI) 1The abbreviations used are: α1-PI, α1-proteinase inhibitor (human); serpin, serine protease inhibitor; EM, electron microscopy; Mr, relative molecular weight; Mw, weight average molecular weight; GdnHCl, guanidine hydrochloride; SE-HPLC, size exclusion-high performance liquid chromatography; RCL, reactive center loop; SE-FPLC, size exclusion-chromatography performed with the ÄKTATM system of Amersham Biosciences; BSA, bovine albumin; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.1The abbreviations used are: α1-PI, α1-proteinase inhibitor (human); serpin, serine protease inhibitor; EM, electron microscopy; Mr, relative molecular weight; Mw, weight average molecular weight; GdnHCl, guanidine hydrochloride; SE-HPLC, size exclusion-high performance liquid chromatography; RCL, reactive center loop; SE-FPLC, size exclusion-chromatography performed with the ÄKTATM system of Amersham Biosciences; BSA, bovine albumin; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. protects tissue, lungs in particular, from damage primarily through its inhibition of neutrophil elastase (1Carrell R.W. Jeppsson J.-O. Laurell C.-B. Brennan S.O. Owen M.C. Vaughan L. Boswell D.R. Nature. 1982; 298: 329-334Crossref PubMed Scopus (498) Google Scholar). α1-PI is synthesized in hepatocytes and secreted into the circulation. Z mutant α1-PI (2Jeppsson J.-O. FEBS Lett. 1976; 65: 195-197Crossref PubMed Scopus (147) Google Scholar), although translated at normal levels, folds defectively and, thus, aggregates within hepatocytes and is secreted at greatly reduced levels (3Brantly M. Nukiwa T. Crystal R.G. Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Google Scholar, 4Kang H.A. Lee K.N. Yu M.H. J. Biol. Chem. 1997; 272: 510-516Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). As a result, individuals with such a mutation manifest extreme α1-PI deficiency (3Brantly M. Nukiwa T. Crystal R.G. Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Google Scholar, 5McElvaney N.G. Crystal R.G. Crystal R.G. West J.B. Barnes P.J. Weibel E.R. The Lung: Scientific Foundations. 2nd edition. Lippincott-Raven, Philadelphia, PA1997: 2537-2553Google Scholar, 6Memoranda Bull. World Health Org. 1997; 75: 397-415PubMed Google Scholar), suffer from progressive emphysema, and may experience severe liver disease (7Morrison E.D. Kowdley K.V. Postgrad. Med. 2000; 107: 147-159Crossref PubMed Scopus (17) Google Scholar, 8Perlmutter D.H. Clin. Liver Dis. 2000; 4: 387-408Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). α1-PI belongs to the serine protease inhibitor (serpin) super-family (9Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G.W. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar, 10Janciauskiene S. Biochim. Biophys. Acta. 2001; 1535: 221-235Crossref PubMed Scopus (159) Google Scholar), members of which share a highly conserved structure. Three β-sheets (A, B, and C) and nine α-helices comprise the basic tertiary structure of a serpin (11Stein P.E. Leslie A.G. Finch J.T. Carrell R.W. J. Mol. Biol. 1991; 221: 941-959Crossref PubMed Scopus (397) Google Scholar, 12Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (239) Google Scholar, 13Baumann U. Huber R. Bode W. Grosse D. Lesjak M. Laurell C.B. J. Mol. Biol. 1991; 218: 595-606Crossref PubMed Scopus (156) Google Scholar). The physiologically active form of a serpin has a metastable conformation, which is essential for its normal inhibitory function (14Carrell R.W. Lomas D.A. Sidhar S. Foreman R. Chest. 1996; 110: 243S-247SAbstract Full Text Full Text PDF PubMed Google Scholar, 15Lee C. Park S.-H. Lee M.-Y. Yu M.-H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7727-7731Crossref PubMed Scopus (96) Google Scholar). A critical structural element of a serpin is its mobile reactive center loop (RCL), exposed on the serpin surface, which normally targets the active site of the cognate serine protease. Upon binding of the serpin to the protease, a tight binary complex forms, the RCL is cleaved, and the segment of the cleaved RCL remaining in the N-terminal portion of the serpin inserts between central strands of the A β-sheet (16Schreuder H.A. de Boer B. Dijkema R. Mulders J. Theunissen H.J. Grootenhuis P.D. Hol W.G. Nat. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar, 17Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 18Stratikos E. Gettins P.G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 4: 453-458Crossref Scopus (137) Google Scholar). Under certain conditions, the intact RCL of a free serpin can also insert between central strands of its A β-sheet thereby giving rise to a latent form; this can occur both in vivo, e.g. with plasminogen activator inhibitor-1 (19Carrell R.W. Evans D.Ll. Stein P.E. Nature. 1991; 353: 576-578Crossref PubMed Scopus (269) Google Scholar, 20Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (520) Google Scholar) and antithrombin (19Carrell R.W. Evans D.Ll. Stein P.E. Nature. 1991; 353: 576-578Crossref PubMed Scopus (269) Google Scholar), and in vitro, e.g. with α1-PI (21Lomas D.A. Elliott P.R. Chang W.-S.W. Wardell M.R. Carrell R.W. J. Biol. Chem. 1995; 270: 5282-5288Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The metastability of a serpin that confers its inhibitory potential also renders it more susceptible to conformational changes, arising from a mutation, that promote its polymerization. Crucial positions implicated in α1-PI polymerization are found in the hinge region of the RCL in the common Z variant (E342K) (2Jeppsson J.-O. FEBS Lett. 1976; 65: 195-197Crossref PubMed Scopus (147) Google Scholar) and in the hydrophobic core of two rare variants, Mmalton (22Fraizer G.C. Harrold T.R. Hofker M.H. Cox D.W. Am. J. Hum. Genet. 1989; 44: 894-902PubMed Google Scholar) and Siiyama (23Seyama K. Nukiwa T. Takabe K. Takahashi H. Miyake K. Kira S. J. Biol. Chem. 1991; 266: 12627-12632Abstract Full Text PDF PubMed Google Scholar) (F52Δ and S53F, respectively). The detailed structure of α1-PI polymers formed in vivo is yet to be established. However, EM data demonstrate that these polymers are linear. Several models of polymerization have been proposed with each assuming an interaction between the RCL of one molecule and a β-sheet in an adjacent molecule. The crystal structures of antithrombin dimer (24Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and plasminogen activator inhibitor-1 (25Sharp. A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Structure Fold. Des. 1999; 7: 111-118Abstract Full Text Full Text PDF Scopus (161) Google Scholar) provide evidence that serpins are capable of forming RCL-β-sheet linkages, in which the RCL of one monomer binds to and forms the first strand in the C β-sheet (24Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) or the last strand in the A β-sheet (25Sharp. A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Structure Fold. Des. 1999; 7: 111-118Abstract Full Text Full Text PDF Scopus (161) Google Scholar) of an adjacent monomer, respectively. However, no direct structural evidence exists supporting the widely accepted model (26Schulze A.J. Baumann U. Knof S. Jaeger E. Huber R. Laurell C.-B. Eur. J. Biochem. 1990; 194: 51-56Crossref PubMed Scopus (174) Google Scholar, 27Lomas D.A. Evans D.Ll. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar) proposing an insertion of the intact RCL of one monomer between the central strands of the A β-sheet of an adjacent monomer. All models of α1-PI polymerization proposed thus far assume a head-to-tail arrangement of monomers forming a linear chain. Here, we show that α1-PI can assemble in a different manner to form linear polymers. We have studied polymerization of disulfide-linked (head-to-head) α1-PI dimer, which forms under mild denaturing conditions in the absence of reducing agent. This dimer, after folding, spontaneously polymerizes giving rise to linear polymers that appear to be multimers of dimer without any involvement of folded monomer, if present. None of the previously proposed models of polymerization appears able to accommodate the observed polymerization of this disulfide-linked dimer. Protein and Peptides—α1-PI, purified from pooled plasma, was a gift from the Aventis Behring Corp. This material, obtained frozen, was thawed and dialyzed into the standard buffer that consisted of 20 mm sodium phosphate and 130 mm NaCl, pH 7.4. The resulting ∼700 μm α1-PI stock solution was stored in small aliquots at –70 °C. α1-PI consisted of 99% monomer and 1% dimer, as determined by SE-HPLC analysis, and showed full inhibitory activity against bovine pancreatic trypsin. The inhibitory activity of α1-PI was determined by measuring the decrease in the steady state activity of the trypsin (with the substrate N-benzoyl-l-arginine p-nitroanalide hydrochloride, Ref. 28Erlanger B.F. Kokowsky N. Cohen W. Arch. Biochem. Biophys. 1961; 95: 271-278Crossref PubMed Scopus (2867) Google Scholar) caused by preincubation of the protease with α1-PI. The active concentration of the trypsin was determined with the active site titrant p-nitrophenyl-p′-guanidinobenzoate hydrochloride (29Chase Jr., T. Shaw E. Biochem. Biophys. Res. Commun. 1967; 29: 508-514Crossref PubMed Scopus (779) Google Scholar). The Mr of the α1-PI monomer was 50,600 and that of the dimer was 101,000, as determined by MALDI-TOF analysis. The extent of glycosylation was 12.5% based on a Mw of 44,300 for the unglycosylated protein, calculated from the frequency of occurrence and sequence of each of the four major variants. The A2800.1% for the α1-PI preparation was 0.431 and was determined by differential refractometry at 546 nm by using (dn/dc)546 values of 0.186 and 0.142 ml/g for the protein (30Doty P. Geiduschek E.P. Neurath H. Bailey K. The Proteins. 1. Academic Press, New York1953: 393-460Google Scholar) and glycosyl moieties, respectively. The (dn/dc)546 value used for the carbohydrate represents the average of values measured by us for d-mannose and N-acetyl-d-glucosamine, 0.137 and 0.147 ml/g, respectively, which are in the same range as values reported for other naturally occurring polysaccharides, e.g. 0.131–0.148 ml/g (31Heyer A.G. Schroeer B. Radosta S. Wolff D. Czapla S. Springer J. Carbohydr. Res. 1998; 313: 165-174Crossref PubMed Scopus (31) Google Scholar, 32Beleski-Carneiro E.B. Ganter J.L.M.S. Reicher F. Int. J. Biol. Macromol. 1999; 26: 219-224Crossref PubMed Scopus (5) Google Scholar, 33Villain-Simonnet A. Milas M. Rinaudo M. Int. J. Biol. Macromol. 2000; 27: 65-75Crossref PubMed Scopus (26) Google Scholar). The value of A2150.1% for the α1-PI preparation is 16.3 and was determined by comparing absorbance, corrected for dilution, measured at 215 nm with that measured at 280 nm. The RCL peptide of α1-PI (nP14), TEAAGAMFLEAIPM (i.e. Thr345–Met358), and a control peptide (cP14), EAPFTALEMGAMAI, designed by scrambling the sequence of the RCL peptide, both N-acetylated, were synthesized at the CBER Facility for Biotechnology Resources. Reagents and Solutions—GdnHCl (99+%), anhydrous dibasic sodium phosphate, bovine pancreatic trypsin, N-benzoyl-l-arginine p-nitroanalide hydrochloride, p-nitrophenyl p′-guanidinobenzoate hydrochloride, d-mannose, N-acetyl-d-glucosamine, β-mercaptoethanol, bovine serum albumin (molecular weight marker for non-denaturing PAGE), and iodoacetamide (SigmaUltra) were from Sigma Chemical Co. Monobasic sodium phosphate was from Fisher Biochemical, and sodium chloride was from J. T. Baker. All chemicals were ACS reagent grade. All GdnHCl solutions were prepared in the standard buffer. A concentrated stock solution of GdnHCl was prepared, and the concentration was determined by differential refractometry (34Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2402) Google Scholar) at 546 nm. Concentrations of all diluted solutions were determined on the basis of weight by utilizing appropriate measured densities. PAGE—PAGE was performed by using the Mini-PROTEAN® system of Bio-Rad with 7.5% Tris-HCl gels under both non-reducing and reducing, denaturing (SDS) conditions and under non-reducing, non-denaturing conditions. Simply BlueTM SafeStain (Invitrogen) was used to detect protein. SE-HPLC and SE-FPLC—Analytical SE-HPLC was performed on the System Gold® HPLC system of Beckmann Corp., controlled with 32 Karat Work station software and equipped with two TosoHaas TSK-3000SWXL columns (5 μm, 7.8 mm × 30 cm) connected in series and an SWXL guard column. The flow rate was 1 ml/min, and the absorbance was monitored at 280 and 215 nm; the latter was used for quantitative analysis. α1-PI dimer was purified by SE-FPLC with an Amersham Biosciences HiLoad® 16/60 Superdex 200 preparative grade column, by using a flow rate of 0.5 ml/min and monitoring the absorbance at 280 nm. Polymerization—Prior to polymerization, α1-PI dimer solutions were concentrated with microconcentrators (Amicon/Millipore) with a molecular weight cut-off of 30,000. All polymerization of disulfide-linked dimer was performed in buffer in the absence of reducing agent at protein concentrations and temperatures as indicated under “Results.” Polymerization of monomer was performed in 1.4 m GdnHCl at 25 °C or by heating in buffer in the presence and absence of 0.1% β-mercaptoethanol at 55 and 65 °C to generate polymer controls (see Figs. 4 and 8).Fig. 8Electron micrographs of α1-PI dimer, dimer polymerized in the absence and presence of RCL peptide, and polymerized α1-PI monomer. The scale given for the first electron micrograph is the same for all. The black arrows indicate free dimers, the white arrows straight, linear chains, and the white arrowheads curved, linear chains. Polymerized dimer was formed in buffer in the absence of reducing agent. A, 0.5 μm purified dimer. B, 50 μm dimer, after incubation at 37 °C for 80 h, diluted to 5 μm for EM. The inset is a portion of the field at the same magnification but at higher under focus. C,11 μm dimer with a 280-fold molar excess of nP14 after incubation at 37 °C for 50 h, diluted to 5 μm for EM. D,5 μm monomer after heating at 65 °C for 12 h in buffer in the presence of 0.1% β-mercaptoethanol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Electron Microscopy—Undiluted and diluted samples of α1-PI were stained and dried for EM examination. A 10-μl drop of each solution to be examined was placed onto a carbon-coated electron microscope grid for 2 min. The grid was blotted with filter paper, stained for 2 min with 2% (w/v) uranyl acetate solution, and air-dried. The prepared grid was examined with a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operated at 120 kV. Images were recorded at magnifications in the range of 3–60 k with a Gatan Multi-Scan 794 CCD camera by using DigitalMicrograph 3.4.4 software. Image processing was done with Adobe PhotoShop 5.5 software. Further Polymerization of Folded Intermediates—Unlike mutant forms of α1-PI, the normal form of the monomer, used in this study, although a metastable form, does not spontaneously polymerize at ≤37 °C (35Lomas D.A. Evans D.Ll. Stone S.R. Chang W.-S. W. Carrell R.W. Biochemistry. 1993; 32: 500-508Crossref PubMed Scopus (204) Google Scholar). Normal monomer must be partially unfolded by treatment with a low level of denaturant or by heating to induce polymerization. Previous unfolding studies of normal α1-PI monomer suggest the existence of an intermediate state at 1–2 m GdnHCl at 25 °C (36Bruch M. Weiss V. Engel J. J. Biol. Chem. 1988; 263: 16626-16630Abstract Full Text PDF PubMed Google Scholar, 37Hervé M. Ghélis C. Eur. J. Biochem. 1990; 191: 653-658Crossref PubMed Scopus (21) Google Scholar, 38Powell L.M. Pain R.H. J. Mol. Biol. 1992; 224: 241-252Crossref PubMed Scopus (76) Google Scholar, 39Tran S.T. Shrake A. Arch. Biochem. Biophys. 2001; 385: 322-331Crossref PubMed Scopus (5) Google Scholar) with the formation of a transiently stable distribution of small polymeric intermediate species after 1–2 h (39Tran S.T. Shrake A. Arch. Biochem. Biophys. 2001; 385: 322-331Crossref PubMed Scopus (5) Google Scholar) that further polymerize more slowly to form higher oligomers. The time course of polymerization of α1-PI in 1.5 m GdnHCl over 1 h analyzed by SE-HPLC in 1.5 m GdnHCl shows the progressive partial unfolding and decrease in the amount of monomer and increase in the amount of dimer and higher polymers (Ref. 39Tran S.T. Shrake A. Arch. Biochem. Biophys. 2001; 385: 322-331Crossref PubMed Scopus (5) Google Scholar and Fig. 4, A–E). However, the quantitation of partially unfolded intermediates by SE-HPLC in GdnHCl is difficult due to variable recovery of these species from the column. The species formed from 50 μm monomer after 3 h in 1.4 m GdnHCl at 25 °C in the absence of reducing agent were diluted 10-fold with buffer, allowed to fold for 16 h at 25 °C, and then analyzed by SE-HPLC in buffer; α1-PI monomer, dimer, and some larger polymers were detected (Fig. 1, lower tracing). Significantly, during further incubation (for 499 h) of these folded species in buffer at 25 °C (Fig. 1, upper tracing), the amount of monomer remained unchanged whereas the level of high molecular weight species increased primarily at the expense of dimer. Since dimer was the smallest species involved in further polymerization of the folded intermediates, we isolated folded dimer in order to study its polymerization. Throughout this article, the concentrations of all α1-PI species, e.g. dimer, are expressed in terms of monomer. Preparation and Properties of Dimer—To induce polymerization, 50 μm α1-PI monomer was incubated in 1.4 m GdnHCl at 25 °C for 3 h, and the resulting intermediate species were folded by 10-fold dilution with buffer and incubated overnight at 4 °C. Folded dimer was isolated by a two-step SE-FPLC purification procedure and characterized by SE-HPLC (Fig. 2A), SDS-PAGE (Fig. 2B), and native PAGE (Fig. 2C). The dimer stock solutions contained ≥96% dimer and trace amounts of tetramer (1.0–2.5%) and monomer (1.2–1.8%) (Fig. 2A). Non-reducing SDS-PAGE of purified dimer shows a dimer band with a trace of monomer (Fig. 2B, lanes 1 and 2, 0.2 and 0.5 μg of dimer, respectively) whereas reducing SDS-PAGE shows only a monomer band (Fig. 2B, lanes 4 and 5) thereby demonstrating that essentially all the purified dimer was disulfide linked. An α1-PI monomer control, containing a trace of dimer, shows the position of the monomer band under non-reducing and reducing conditions (Fig. 2B, lanes 3 and 6, respectively). To verify that the disulfide bond forms in GdnHCl, polymerization of monomer was carried out, as before, in 1.4 m GdnHCl at 25 °C for 3 h in the absence and presence of the sulfhydryl blocking agent iodoacetamide added at various times during polymerization at a 7-fold molar excess. After polymerization and refolding by dilution with buffer, the samples were analyzed by SDS-PAGE (Fig. 3). In Fig. 3, lane 1 is unpolymerized monomer (control), containing a trace of dimer, lane 2 is a sample polymerized in the absence of blocking agent, and lanes 3–5 are samples treated with blocking agent at 1, 2, and 3 h after the initiation of polymerization. Lane 6 is a sample of material obtained after polymerization of monomer that was blocked for1hat37 °C prior to the initiation of polymerization. This sample shows that the amount of dimer did not increase (relative to that in unpolymerized monomer, lane 1) during incubation of blocked monomer in GdnHCl in contrast to the substantial amount of dimer obtained in the absence of blocking agent (lane 2). Furthermore, for the samples treated with blocking agent during polymerization, increasing amounts of dimer were obtained with increasing incubation time in GdnHCl prior to the addition of blocking agent (lanes 3, 4, and 5, respectively). The sample in lane 5 was treated with blocking agent at the end of the 3-h polymerization, and the amount of dimer is comparable to that obtained in the absence of blocking agent (lane 2). All dimers observed in Fig. 3 were completely dissociated in SDS in the presence of reducing agent (data not shown) thereby showing that they were disulfide-linked. These results demonstrate that the disulfide bond of the dimer forms between partially unfolded monomers in 1.4 m GdnHCl. The disulfide-linked dimer did not show inhibitory activity against porcine elastase. 2X. Du, unpublished result. Stock solutions of dimer (0.5–0.7 μM) were stored at 4 °C; under such conditions, dimer polymerizes very slowly. During storage for 2 months at 4 °C, <10% of the dimer polymerized with the formation of tetramer. To assess the compactness of the α1-PI dimer, we have considered its hydrodynamic properties. The SE-HPLC elution volumes for the monomer and dimer of α1-PI are slightly greater than those for the monomer and dimer constituents of a BSA molecular weight marker for non-denaturing PAGE (data not shown). This observation is consistent with the lower molecular weights of the monomer and dimer of α1-PI (Mr 50,600 and Mr 101,000) relative to those of the BSA standard (Mr 66,000 and Mr 132,000). PAGE analysis of the α1-PI dimer and monomer and of the BSA standard under non-denaturing conditions (Fig. 2C) shows that the migration distance of α1-PI dimer is slightly less than that of BSA dimer. However, this small difference between dimers is a reflection of the slightly shorter migration distance of α1-PI monomer relative to that of the BSA monomer (Fig. 2C). Preliminary far UV CD and intrinsic protein fluorescence spectra (not shown) were used to estimate the extent of structural perturbation of the α1-PI monomer within the disulfide-linked dimer and indicate a substantial retention of structure. A deconvolution of the CD data with CDPro software (40CDPro Software Package, lamar.colostate.edu/~sreeram/CDPro (September 27, 2002)Google Scholar), which uses three algorithms (41Sreerama N. Venyaminov S.Yu. Woody R.W. Protein Sci. 1999; 8: 370-380Crossref PubMed Scopus (631) Google Scholar, 42Johnson W.C. Proteins: Struc. Func. Genet. 1999; 35: 307-312Crossref PubMed Scopus (617) Google Scholar, 43Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1857) Google Scholar), gives similar changes in secondary structure for the monomer within the dimer relative to free monomer for a given set of protein reference spectra. However, the use of three standard reference sets shows retention of ∼100% β-sheet and ∼86% α-helix whereas the use of two other sets shows a retention of ∼82% β-sheet and ∼84% α-helix. Fluorescence emission spectra with excitation at 280 or 295 nm show an increase of ≤1 nm in the emission maximum of dimer relative to that of monomer. With excitation at 295 nm, the shift of the maximum for the intermediate state in 1.5 m GdnHCl is ∼5 nm and that for the fully unfolded state in 7 m GdnHCl is ∼20 nm (39Tran S.T. Shrake A. Arch. Biochem. Biophys. 2001; 385: 322-331Crossref PubMed Scopus (5) Google Scholar). These results indicate minimal perturbation of the fluorophore environment in the dimer. Therefore, electrophoretic and chromatographic results demonstrate that the disulfide-linked dimer is a compact structure, and the CD and fluorescence measurements suggest a substantial retention of secondary and tertiary structure by the monomer within the dimer. Polymerization of Dimer—0.5 μm α1-PI dimer was concentrated to 9.2 μm and polymerized by incubation at 37 °C (to accelerate polymerization) for 24 h. As the control, 50 μm monomer was polymerized by heating at 55 °C for 2 h in the absence and presence of 0.1% β-mercaptoethanol. Native PAGE analysis of the products of polymerization of monomer in the absence and presence of reducing agent shows a ladder of species consisting of multiples of monomer (Fig. 4, lanes 4 and 5, respectively) with the latter showing sharper bands; the former shows a much weaker pentamer band, and this observation suggests that polymerization under non-reducing conditions may, in part, involve polymerization of initially formed dimer. The ladder of species obtained for the polymerized dimer in the absence of reducing agent (Fig. 4, lane 3) shows no trimer or pentamer bands and only a faint band of monomer, which is a trace impurity in the dimer preparation. Unpolymerized control samples of monomer and dimer each show the single, anticipated band (Fig. 4, lanes 1 and 2, respectively). Dimer was concentrated and then incubated at concentrations of 1, 11, 21, and 50 μm at 37 °C (Fig. 5A) and also at a concentration of 21 μm at 4, 25, 37, and 41 °C (Fig. 5B). Concentrating at 4 °C causes some polymerization of the dimer. Thus, samples analyzed immediately after concentration (at t = 0) had decreased levels of dimer relative to that of the initial stock solution (0.5 μm) in a concentration-dependent manner (Fig. 5A). During incubation at 37 °C, the amount of dimer decreased, the amount of tetramer increased, and substantial levels of higher oligomers were formed. For example, compare the SE-HPLC traces of 50 μm protein before and after incubation for 50 h at 37 °C (Fig. 5, C and D, respectively). The initial rate of dimer polymerization increased with increase in concentration and in temperature (Fig. 5, A and B, respectively). The polymers formed from the dimer were stable because they did not dissociate upon dilution even with further incubation and showed only very slight dissociation upon treatment with 1% β-mercaptoethanol (data not shown). Effect of RCL Peptide on Polymerization of α1-PI Dimer— Lack of polymerization of α1-PI monomer complexed with RCL peptide was an important observation leading to the proposition that α1-PI polymerizes by the insertion of the RCL of one molecule into the A β-sheet of another (26Schulze A.J. Baumann U. Knof S. Jaeger E. Huber R. Laurell C.-B. Eur. J. Biochem. 1990; 194: 51-56Crossref PubMed Scopus (174) Google Scholar, 27Lomas D.A. Evans D.Ll. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar). Thus, we investigated the effect of an RCL peptide on the polymerization of α1-PI dimer using a peptide, nP14, with the sequence of residues 345–358 of the RCL of α1-PI and a control peptide, cP14, with a s" @default.
• W2016997461 created "2016-06-24" @default.
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• W2016997461 creator A5054665013 @default.
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• W2016997461 date "2003-05-01" @default.
• W2016997461 modified "2023-09-30" @default.
• W2016997461 title "A Novel Mode of Polymerization of α1-Proteinase Inhibitor" @default.
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