A Novel Mode of Polymerization of {alpha}1-Proteinase Inhibitor*

Ewa Marszal {ddagger}, Dganit Danino §  and Andrew Shrake {ddagger} ||

From the {ddagger}Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 and the §Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 19, 2002 , and in revised form, March 19, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients homozygous for the Z mutant form of {alpha}1-proteinase inhibitor ({alpha}1-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 {alpha}1-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 {alpha}1-PI monomer between the central strands of the A {beta}-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A {beta}-sheet of {alpha}1-PI monomer and this insertion prevents {alpha}1-PI polymerization. Normal {alpha}1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal {alpha}1-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 {alpha}1-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 {beta}-sheets may not occur during polymerization of this dimer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}1-Proteinase inhibitor ({alpha}1-PI)1 protects tissue, lungs in particular, from damage primarily through its inhibition of neutrophil elastase (1). {alpha}1-PI is synthesized in hepatocytes and secreted into the circulation. Z mutant {alpha}1-PI (2), although translated at normal levels, folds defectively and, thus, aggregates within hepatocytes and is secreted at greatly reduced levels (3, 4). As a result, individuals with such a mutation manifest extreme {alpha}1-PI deficiency (3, 5, 6), suffer from progressive emphysema, and may experience severe liver disease (7, 8).

{alpha}1-PI belongs to the serine protease inhibitor (serpin) super-family (9, 10), members of which share a highly conserved structure. Three {beta}-sheets (A, B, and C) and nine {alpha}-helices comprise the basic tertiary structure of a serpin (11, 12, 13). The physiologically active form of a serpin has a metastable conformation, which is essential for its normal inhibitory function (14, 15). 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 {beta}-sheet (16, 17, 18). Under certain conditions, the intact RCL of a free serpin can also insert between central strands of its A {beta}-sheet thereby giving rise to a latent form; this can occur both in vivo, e.g. with plasminogen activator inhibitor-1 (19, 20) and antithrombin (19), and in vitro, e.g. with {alpha}1-PI (21). 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 {alpha}1-PI polymerization are found in the hinge region of the RCL in the common Z variant (E342K) (2) and in the hydrophobic core of two rare variants, Mmalton (22) and Siiyama (23) (F52{Delta} and S53F, respectively).

The detailed structure of {alpha}1-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 {beta}-sheet in an adjacent molecule. The crystal structures of antithrombin dimer (24) and plasminogen activator inhibitor-1 (25) provide evidence that serpins are capable of forming RCL-{beta}-sheet linkages, in which the RCL of one monomer binds to and forms the first strand in the C {beta}-sheet (24) or the last strand in the A {beta}-sheet (25) of an adjacent monomer, respectively. However, no direct structural evidence exists supporting the widely accepted model (26, 27) proposing an insertion of the intact RCL of one monomer between the central strands of the A {beta}-sheet of an adjacent monomer.

All models of {alpha}1-PI polymerization proposed thus far assume a head-to-tail arrangement of monomers forming a linear chain. Here, we show that {alpha}1-PI can assemble in a different manner to form linear polymers. We have studied polymerization of disulfide-linked (head-to-head) {alpha}1-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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein and Peptides—{alpha}1-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 {alpha}1-PI stock solution was stored in small aliquots at –70 °C. {alpha}1-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 {alpha}1-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. 28) caused by preincubation of the protease with {alpha}1-PI. The active concentration of the trypsin was determined with the active site titrant p-nitrophenyl-p'-guanidinobenzoate hydrochloride (29).

The Mr of the {alpha}1-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 for the {alpha}1-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 (30) 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 (31, 32, 33). The value of for the {alpha}1-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 {alpha}1-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, {beta}-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 (34) 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 x 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. {alpha}1-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, {alpha}1-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% {beta}-mercaptoethanol at 55 and 65 °C to generate polymer controls (see Figs. 4 and 8).



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FIG. 4.
Polymerized samples of purified dimer and of monomer analyzed by native PAGE. 9.2 µM purified {alpha}1-PI dimer was polymerized by incubation for 24 h at 37 °C in buffer in the absence of reducing agent. 50 µM {alpha}1-PI monomer was polymerized by heating for 2 h at 55 °C in the absence and presence of 0.1% {beta}-mercaptoethanol. The monomer, dimer, trimer, tetramer, and pentamer bands are designated by M, D, Tri, Tet, and Pent, respectively. Lanes 1 and 2, unpolymerized samples (controls) of 2.6 µg of monomer and 0.3 µg of dimer, respectively; lane 3, 2.4 µg of dimer polymerized in the absence of reducing agent; lanes 4 and 5, 7.8 µg of monomer polymerized in the absence and presence of reducing agent, respectively.

 


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FIG. 8.
Electron micrographs of {alpha}1-PI dimer, dimer polymerized in the absence and presence of RCL peptide, and polymerized {alpha}1-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% {beta}-mercaptoethanol.

 

Electron Microscopy—Undiluted and diluted samples of {alpha}1-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Further Polymerization of Folded Intermediates—Unlike mutant forms of {alpha}1-PI, the normal form of the monomer, used in this study, although a metastable form, does not spontaneously polymerize at ≤37 °C (35). 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 {alpha}1-PI monomer suggest the existence of an intermediate state at 1–2 M GdnHCl at 25 °C (36, 37, 38, 39) with the formation of a transiently stable distribution of small polymeric intermediate species after 1–2 h (39) that further polymerize more slowly to form higher oligomers. The time course of polymerization of {alpha}1-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. 39 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; {alpha}1-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 {alpha}1-PI species, e.g. dimer, are expressed in terms of monomer.



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FIG. 1.
SE-HPLC analysis of {alpha}1-PI monomer after polymerizing in 1.4 M GdnHCl, folding in buffer, then further incubating in buffer. 50 µM {alpha}1-PI monomer was incubated for 3 h in 1.4 M GdnHCl in the absence of reducing agent at 25 °C, diluted 10-fold with buffer, and then incubated in buffer at 25 °C. 150-µl samples were analyzed by SE-HPLC. The monomer and dimer peaks are designated by M and D, respectively. Lower tracing, chromatogram after 16 h of incubation in buffer in order to allow folding of the sample. Upper tracing, chromatogram after incubating the sample for an additional 499 h in buffer.

 

Preparation and Properties of Dimer—To induce polymerization, 50 µM {alpha}1-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).



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FIG. 2.
Purified {alpha}1-PI dimer analyzed by SE-HPLC, SDS-PAGE, and native PAGE. Monomer, dimer, and tetramer peaks/bands are designated by M, D, and Tet, respectively. A, SE-HPLC profile of a 150-µl sample of 0.73 µM purified {alpha}1-PI dimer. B, SDS-PAGE of non-reduced (lanes 1–3) and reduced (lanes 4–6) {alpha}1-PI samples, which were prepared by boiling for 5 min in the presence of 1.3% SDS and in the absence and presence of 3.3% {beta}-mercaptoethanol, respectively. Lanes 1 and 4, 0.2 µg of purified dimer; lanes 2 and 5, 0.5 µg of purified dimer; lanes 3 and 6, 1.3 µg of monomer (control). C, native PAGE. Lanes 1 and 4, 5 µg of BSA standard; lane 2, 5 µg of {alpha}1-PI monomer; lane 3, 5 µg of {alpha}1-PI dimer.

 

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 {alpha}1-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.



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FIG. 3.
Monomer polymerized in 1.4 M GdnHCl in the absence and presence of iodoacetamide, folded, and analyzed by non-reducing SDS-PAGE. 500 µM {alpha}1-PI monomer was incubated 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. Samples were analyzed after dilution with buffer and incubation in buffer for 2.5 h; the protein load was 12.5 µg in each lane. Lane 1, unpolymerized monomer (control); lane 2, monomer polymerized in the absence of iodoacetamide; lanes 3–5, monomer polymerized and treated with iodoacetamide 1, 2, and 3 h after the initiation of polymerization, respectively; lane 6, monomer polymerized after treatment with iodoacetamide at 37 °C for 1 h.

 

The disulfide-linked dimer did not show inhibitory activity against porcine elastase.2 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 {alpha}1-PI dimer, we have considered its hydrodynamic properties. The SE-HPLC elution volumes for the monomer and dimer of {alpha}1-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 {alpha}1-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 {alpha}1-PI dimer and monomer and of the BSA standard under non-denaturing conditions (Fig. 2C) shows that the migration distance of {alpha}1-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 {alpha}1-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 {alpha}1-PI monomer within the disulfide-linked dimer and indicate a substantial retention of structure. A deconvolution of the CD data with CDPro software (40), which uses three algorithms (41, 42, 43), 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% {beta}-sheet and ~86% {alpha}-helix whereas the use of two other sets shows a retention of ~82% {beta}-sheet and ~84% {alpha}-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 (39). 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 {alpha}1-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% {beta}-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% {beta}-mercaptoethanol (data not shown).



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FIG. 5.
Time course of polymerization of purified {alpha}1-PI dimer as a function of concentration and of temperature. Samples were taken at various time points during the polymerization of purified dimer for SE-HPLC analysis. Elution profiles were monitored at 280 and 215 nm, and data at 215 nm were used to determine the relative dimer concentrations, which were used to monitor the extent of polymerization. A, percent dimer content as a function of time with incubation at 37 °C for total protein concentrations of 1 ({diamondsuit}), 11 ({blacksquare}), 21 ({blacktriangleup}), and 50 (•) µM. B, percent dimer content as a function of time for a total protein concentration of 21 µM with incubations at 4 ({diamondsuit}), 25 ({blacksquare}), 37 ({blacktriangleup}), and 41 °C (•). C and D, SE-HPLC profiles of a 2-µl sample of 50 µM total protein after 0 and 50 h at 37 °C, respectively. Monomer, dimer, and tetramer peaks in the chromatograms are designated by M, D, and Tet, respectively.

 

Effect of RCL Peptide on Polymerization of {alpha}1-PI Dimer— Lack of polymerization of {alpha}1-PI monomer complexed with RCL peptide was an important observation leading to the proposition that {alpha}1-PI polymerizes by the insertion of the RCL of one molecule into the A {beta}-sheet of another (26, 27). Thus, we investigated the effect of an RCL peptide on the polymerization of {alpha}1-PI dimer using a peptide, nP14, with the sequence of residues 345–358 of the RCL of {alpha}1-PI and a control peptide, cP14, with a sequence obtained by scrambling that of nP14 ("Experimental Procedures").

The nP14 peptide differs from the peptides used previously; the 14-mer peptide of Schulze et al. (26) had valine substituted for proline at residue 357 of {alpha}1-PI, and the sequence of the 13-mer peptide of Lomas et al. (27) was from the sequence of the RCL of antithrombin. Therefore, we tested the ability of nP14 to form a complex with {alpha}1-PI monomer. As anticipated, nP14 formed a binary complex with {alpha}1-PI monomer at a 100-fold molar excess of peptide (10 µM total protein) during incubation at 37 °C for 24 h as demonstrated by native PAGE (Fig. 6). The progressive appearance of a second band (Fig. 6, lanes 3–6, BC band) having greater mobility than that of the free monomer (Fig. 6, lanes 1 and 2, M band) demonstrates formation of the binary complex (26). Furthermore, this monomer-peptide complex did not polymerize when subsequently incubated in 1.4 M GdnHCl in the presence of a 100-fold molar excess of nP14 at 25 °C (data not shown) in agreement with previous reports for other RCL peptides (26, 27). In contrast, the control peptide cP14 did not form a complex with {alpha}1-PI monomer under the same conditions (Fig. 6, lanes 7–10), and monomer preincubated in the presence of a 100-fold molar excess of cP14 did polymerize in 1.4 M GdnHCl at 25 °C as does {alpha}1-PI monomer without peptide (data not shown).



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FIG. 6.
Binary complex formation between {alpha}1-PI monomer and RCL peptide monitored by native PAGE analysis. 10 µM {alpha}1-PI monomer was incubated at 37 °C for 24 h alone, in the presence of 1 mM nP14 (RCL peptide), and in the presence of 1 mM cP14 (control peptide). Samples taken at various time points (0, 2, 5, and 24 h) were stored at 4 °C prior to native PAGE analysis of a 2.6 µg sample of each. The free monomer band and the binary complex band are designated by M and BC, respectively. Lanes 1 and 2, no peptide added; lanes 3–6, nP14 added; lanes 7–10, cP14 added.

 

A 100-fold molar excess of nP14 did not decrease the rate or extent of polymerization of the dimer (10 µM total protein) in buffer at 37 °C and the same effect was observed with cP14 (Fig. 7A). Moreover, incubation of dimer (11 µM total protein) at 37 °C in the presence of a 280-fold molar excess of nP14 actually slightly promoted polymerization (Fig. 7B); this effect was similar for both peptides and, thus, seems to be nonspecific. In addition, nP14 added at a 200-fold molar excess of peptide to polymer, previously formed from the dimer in the absence of peptide, did not cause polymer dissociation (data not shown).



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FIG. 7.
Effect of the presence of peptide on the time course of polymerization of purified {alpha}1-PI dimer. Relative concentrations of {alpha}1-PI dimer during its polymerization at 37 °C were determined by SE-HPLC analysis as performed in Fig. 5 to monitor extent of polymerization. A, percent dimer as a function of time with a total protein concentration of 10 µM in the absence of peptide ({diamondsuit}) and in the presence of nP14 ({blacksquare}) and of cP14 ({blacktriangleup}), both at a 100-fold molar excess of peptide. B, percent dimer as a function of time with a total protein concentration of 11 µM in the absence of peptide ({diamondsuit}) and in the presence of nP14 ({blacksquare}) and of cP14 ({blacktriangleup}), both at a 280-fold molar excess of peptide. C, native PAGE analysis of 5.8 µg samples from panel B after 50 h of polymerization. Lane 1, no peptide added; lane 2, nP14 added; lane 3, cP14 added. Binary complex, monomer, dimer, and tetramer bands are designated by BC, M, D, and Tet, respectively.

 

Native PAGE analysis of dimers incubated at 37 °C for 50 h alone and in the presence of nP14 and of cP14, with a 280-fold molar excess of each peptide, (Fig. 7C, lanes 1, 2, and 3, respectively) shows that monomer present as a trace contaminant in the dimer preparation served as an internal control by demonstrating incorporation of nP14 (Fig. 7C, lane 2, BC band) and of a small amount of cP14 (Fig. 7C, lane 3, BC band). Binding of cP14 to monomer under these conditions probably results from the high concentration of peptide used (a 280-fold molar excess) since no binding was observed with a 100-fold molar excess (Fig. 6, lanes 7–10). In contrast, no differences in the mobility of the dimer or higher oligomers were observed in the absence and presence of the peptides. However, the resolution of this assay may not be high enough to determine whether a dimer-peptide complex forms given that sieving and charge-to-mass ratio effects operate in opposition. Preliminary intrinsic protein fluorescence emission spectra at 280 nm (not shown) show a slight red-shift in the emission maximum for dimer incubated in the presence of cP14 and a greater red-shift for dimer incubated in the presence of nP14 thereby suggesting a stronger interaction of dimer with nP14 and a weaker interaction with cP14.

Electron Microscopy of Dimer, Polymerized Dimer, and Polymerized Monomer—Electron micrographs compare purified dimer before and after 80 h of incubation at 37 °C (Fig. 8, A and B, respectively). Fig. 8A shows the almost exclusive presence of small molecular species (black arrows), which are free dimers. In Fig. 8B, these species are also present but with the additional presence of linear species (white arrows), which appear to be polymers formed from the dimers, with some of the polymers showing noticeable curvature (white arrowhead). The inset in Fig. 8B presents a portion of an EM field at higher contrast showing polymer and free dimer structures. Heating dimer in the presence of a 280-fold molar excess of nP14 for 50 h at 37 °C (Fig. 8C) also produces linear polymers.

Polymerization of monomer in the presence of 0.1% {beta}-mercaptoethanol under conditions used by Lomas et al. (44), heating at 65 °C for 12 h, resulted in the generation of linear polymers (Fig. 8D), as anticipated. Furthermore, heating {alpha}1-PI monomer under the same conditions but in the absence of reducing agent also produced linear polymers (data not shown). All EM results demonstrate that the polymers arising from the polymerization of the dimer are linear chains as are those formed by polymerization of the monomer.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The loop-sheet models of polymers of {alpha}1-PI have two features in common: all {alpha}1-PI monomers within the polymer are oriented head-to-tail and the RCL of each monomer inserts into a {beta}-sheet of a neighboring monomer. In contrast, the formation of linear polymers of {alpha}1-PI reported here involves association of disulfide-linked (head-to-head) dimers. As a result, the arrangement of monomers within these polymers must be substantially different from that for loop-sheet (head-to-tail) models.

The conformation of the monomer within the dimer must differ somewhat from that of the free monomer since the disulfide-linked dimer is prone to polymerization whereas the normal {alpha}1-PI monomer does not spontaneously polymerize. However, preliminary studies of the monomer and dimer, involving CD, intrinsic protein fluorescence, and SE-HPLC and native PAGE analysis, suggest a substantial retention of structure by the monomer within a compact dimer. Fig. 9A shows the crystallographic structure of free monomer in which helices are in black and {beta}-sheets are represented by gray ribbons. Fig. 9B is a schematic representation of the monomer within the dimer that distinguishes two general kinds of surfaces; the black surface contains the single free sulfhydryl, C232, and the white surface contains the A {beta}-sheet. Parts of these surfaces appear to be involved in the formation of the disulfide-linked dimer (black surface) and the polymerization of dimer (white surface). Fig. 9C represents two possible, extreme (cis- and trans-) conformations of this dimer. The disulfide-linked dimer may have either of the conformations shown in Fig. 9C or intermediate conformation(s) generated by rotation of the constitutive monomers about the disulfide bond.



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FIG. 9.
Extremes of possible structures for disulfide linked dimer of {alpha}1-PI. A, ribbon diagram of the {alpha}1-PI monomer from the Protein Data Bank (PDB code 1qlp [PDB] ). The helices are in black and {beta}-sheets are represented by gray ribbons. B, schematic representation of {alpha}1-PI monomer within the dimer. C, two extreme conformations for the disulfide-linked dimer of {alpha}1-PI, cis- and trans-conformations. Disulfide-linked dimer is represented by two arrows joined by an S–S bond (line) connecting the sulfhydryl containing surfaces (black surfaces).

 

The positions of the oligosaccharide moieties in {alpha}1-PI suggest that the association of disulfide-linked dimers to form linear polymers may occur through an interaction of the dimer surfaces containing the A {beta}-sheets (Fig. 9C, white surfaces). The oligosaccharide at N83 is located on the side opposite to the A {beta}-sheet (Fig. 9A). Oligosaccharides at N46, not shown, and N247 are located on either side of the monomer with the latter facing the reader and the former facing the opposite direction. Fig. 10A is a schematic representation of possible structures of polymers formed from disulfide-linked dimer and illustrates the interaction between monomers comprising the disulfide-linked dimer (black surface/black surface) and the interaction between dimers within the linear polymers (white surface/white surface). Fig. 10B shows possible species resulting from polymerization of monomer in the absence of reducing agent and involving the interactions described in Fig. 10A. The cis-conformation of the disulfide-linked dimer is used here exclusively to simplify the schematic representations; however, the population of disulfide-linked dimer containing species may actually involve all possible dimer conformations.



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FIG. 10.
Schematic representations of {alpha}1-PI polymers involving disulfide-bond formation and of {alpha}1-PI loop-sheet polymers. Each black/white arrow designates an {alpha}1-PI monomer as in Fig. 9B. See "Discussion" for the nature of the interactions. A, polymers resulting from polymerization of disulfide-linked dimer. B, polymers resulting from polymerization of monomer in the absence of reducing agent. C, loop-sheet polymers. Polymers that result from the proposed insertion of the RCL of a monomer into a {beta}-sheet of an adjacent monomer are represented by the head-to-tail, white arrows.

 

The role disulfide bonding plays in the formation of {alpha}1-PI polymers in the endoplasmic reticulum (ER) of hepatocytes is uncertain. The thiol-disulfide redox potential within the ER lumen promotes the formation of intraprotein disulfide bonds in secretory proteins (45). {alpha}1-PI, which contains a single cysteine, can form mixed disulfides in vivo (46, 47). However, normal {alpha}1-PI appears not to form the disulfide-linked homodimer in vivo (46) or in vitro at ≤37 °C in buffer. Nevertheless, mutants less stable than normal {alpha}1-PI may fold more slowly in vivo and, thus, may experience prolonged exposure of the free sulfhydryl during folding thereby facilitating formation of disulfide-linked dimer, which then polymerizes further.

Polymerization of {alpha}1-PI (35) and other serpins, e.g. neuroserpin (48), does not require the formation of disulfide bonds. However, attempts to dissociate {alpha}1-PI inclusion bodies in the absence of reducing agent have yielded mixed results (49, 50). Thus, although {alpha}1-PI can polymerize without the formation of disulfide bonds, data suggesting a lack of disulfide bond formation during polymerization in hepatocytes are not definitive.

Nevertheless, polymers formed from the disulfide-linked dimer, as seen by EM (Fig. 8, B and C), appear to be linear chains as do those formed in vivo by the Z (27), S (51), and Mmalton (44) mutants and by neuroserpin (48) as well as those formed in vitro by proteolytically cleaved {alpha}1-PI (52), by the I (53) and Z (27) mutants, and by normal {alpha}1-PI monomer incubated at elevated temperature in the presence or absence of reducing agent (see "Results" and Ref. 54). All these polymers are bead-like, linear chains that are occasionally curved. Thus, polymers formed with or without the participation of disulfide bonds are of the same general linear form.

The structure of {alpha}1-PI polymers formed without disulfide bonds may be similar to that of polymers containing disulfide bonds and, thus, involve both types of monomer-monomer interactions (Fig. 10B). Furthermore, such polymeric structures, with and without disulfide bonds, may be able to better accommodate curved structures observed by EM than the loop-A sheet model, which according to Carrell and co-workers (55) may not have sufficient flexibility.

Current {alpha}1-PI polymerization models propose that the RCL of one molecule inserts into a {beta}-sheet of an adjacent molecule to form linear polymers through a head-to-tail interaction as represented schematically in Fig. 10C. The first and most widely accepted loop-sheet polymerization model (i.e. loop-A sheet model) represents the polymer as a chain of monomers linked by insertion of the RCL of one monomer between central strands of the A {beta}-sheet (s3A and s5A) of the next monomer in the chain (26, 27). The loop-C sheet model (24) is based on the crystal structure of an antithrombin dimer consisting of a molecule with its RCL partially inserted as an edge strand (s1C) into the C {beta}-sheet of a second molecule in the latent form. The most recent loop-sheet model (s7A model) derives from the crystal structure of plasminogen activator inhibitor-1 (25); in this structure, linear chains are formed by partial insertion of the RCL of one monomer into the A {beta}-sheet of an adjacent monomer as an edge strand (s7A). However, the last two modes of insertion, involving formation of an edge strand, are reported to be unstable since they dissociate upon dilution (24, 25, 56) whereas polymers of {alpha}1-PI are stable.

In contrast to the loop-C sheet and strand 7A models, the loop-A sheet model has never been directly supported with detailed structural data. Two crystal structures of {alpha}1-PI polymers composed of monomers each with its RCL cleaved demonstrate insertion of a portion of the cleaved RCL into the A {beta}-sheet of the adjacent monomer (55, 57). However, the relevance of these structures to the loop-A sheet model is not clear. The crystal structure of {alpha}1-PI monomer with the RCL cleaved at the scissile bond reveals an ~7 nm separation between the C and N termini generated by cleavage of the RCL (58) and, thus, differs substantially from that of the intact Z monomer, which forms polymers in vivo.

The loop-A sheet model was initially based on the observation that insertion of the RCL peptide prevents polymerization of {alpha}1-PI monomer (26, 27). This model was further supported by the observation that addition of an RCL peptide to {alpha}1-PI polymer formed previously from the Z mutant in the presence of reducing agent results in at least partial dissociation of the polymer to monomers (35), presumably with bound peptide. Both observations show that {alpha}1-PI-peptide complexes are more stable than the corresponding polymers. However, such data demonstrate only energetic differences between the monomer-peptide complex and the polymer and provide no information concerning the structure of the polymers.

In contrast to unstable loop-C sheet and strand 7A associations, disulfide-linked dimer formed stable polymers in buffer as shown by the lack of dissociation upon dilution and when treated with nP14. In addition, polymerization of this dimer was not inhibited in the presence of the RCL peptide, as reported here, even though preliminary data suggest an interaction of nP14 with dimer. Therefore, RCL insertion may not occur during polymerization of disulfide-linked dimer. However, even if the dimer does not bind RCL peptide, RCL insertion during polymerization may not occur.

Results presented here suggest that under mild denaturing conditions in the absence of reducing agent, monomeric intermediates with free sulfhydryl groups tend to form disulfide linked dimeric species and that this process is most likely not easily reversible whereas reversibility may be feasible in the absence of disulfide bond formation. Previously, a transiently stable distribution of intermediate species was identified in 1.5 M GdnHCl after 1–2 h and was associated with an apparent equilibrium among monomeric and polymeric intermediates (39). However, the level of free sulfhydryl in the preparation used previously was not determined.

In summary, our studies of the polymerization of disulfide-linked dimer demonstrate that monomers in {alpha}1-PI polymers can be arranged differently than previously proposed. We suggest that polymers of {alpha}1-PI and perhaps of other serpins formed in vivo, even without involvement of disulfide bonds, may have structures similar to those formed from disulfide-linked dimer. Our model provides an alternative to earlier polymer models. However, the physiological relevance of all models remains to be demonstrated. The mechanism of serpin polymerization may depend upon the nature of the variant and the reaction conditions.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Present address: Dept. of Food Engineering and Biotechnology, Technion, IIT, Haifa 32000, Israel. Back

|| To whom correspondence should be addressed. Tel.: 301-402-4635; Fax: 301-402-2780; E-mail: shrake{at}cber.fda.gov.

1 The abbreviations used are: {alpha}1-PI, {alpha}1-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. Back

2 X. Du, unpublished result. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Jacqueline Muller (Division of Viral Products, Office of Vaccines Research and Review, CBER, Food and Drug Administration) for preliminary EM data demonstrating the formation of linear polymers from purified {alpha}1-PI dimer. We would like to express our appreciation to Dr. Jenny Hinshaw (Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health) for supporting the collaboration of DD. We also thank Robert Boykins (Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccines Research and Review, CBER, Food and Drug Administration) for performing the MALDI-TOF measurements and Dr. Joan May (Division of Manufacturing and Product Quality, Office of Compliance and Biologics Quality, CBER, Food and Drug Administration) for determining moisture levels in D-mannose and N-acetylglucosamine.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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