©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Antitrypsin Mmalton (Phe-deleted) Forms Loop-Sheet Polymers in Vivo
EVIDENCE FOR THE C SHEET MECHANISM OF POLYMERIZATION (*)

David A. Lomas (1)(§), Peter R. Elliott (1), Sanjiv K. Sidhar (3), Richard C. Foreman (3), John T. Finch (2), Diane W. Cox (4), James C. Whisstock (1), Robin W. Carrell (1)

From the (1)Department of Haematology, University of Cambridge and the (2)Medical Research Council Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom, the (3)Department of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton S09 3TU, United Kingdom, and the (4)Departments of Paediatrics and Genetics, The Hospital for Sick Children, Toronto M5G 1X8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Z (Glu Lys) and Siiyama (Ser Phe) deficiency variants of -antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a -pleated sheet of a second. We show here that antitrypsin Mmalton (Phe-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A -sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation.

Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R.(1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.


INTRODUCTION

-Antitrypsin (-proteinase inhibitor) is a 52-kDa 394-amino acid glycoprotein and the archetypal member of the serpin()superfamily(1) . Members of this family each have a unique inhibitory specificity, but share a similar molecular structure based on a dominant five-strand A -sheet(2, 3, 4, 5, 6, 7, 8, 9, 10) . The intact inhibitor has an exposed reactive center loop with the P-P` residues()acting as ``peptide bait'' for the cognate proteinase(11, 12) . The precise conformation of the reactive center loop in the active inhibitor when it docks with an enzyme is unknown, but is likely to lie between the three-turn helical conformation of ovalbumin (3, 10) and the canonical structure of the small inhibitors(13, 14) . For serpins to adopt a canonical conformation, the helical reactive center loop must unfold with its N-terminal stalk inserted into the gap between strands 3 and 5 of the A sheet(15, 16) . After docking with the enzyme, the loop is presumed to insert further into the A sheet to form an extended canonical structure that fits the substrate-binding site of the cognate proteinase, the locking conformation(17) . Dissociation of the enzyme-inhibitor complex may result in the release of the intact serpin (18) or cleavage of the loop at the P-P` residues(19) . Reactive center loop cleavage results in a profound conformational rearrangement in which the loop is fully inserted into the A sheet of the molecule (2, 20, 21).

It is clear that the sequence of events involved in proteinase inhibition requires the loop to be mobile and to adopt a variety of conformations. A consequence of this is the formation of the so-called ``latent'' conformation (by analogy with the corresponding reactivable form of plasminogen activator inhibitor-1(6) ), in which the intact loop is fully incorporated into and stabilizes the A sheet (6, 9, 22). A second consequence is the formation of loop-sheet polymers in which the reactive center loop of one molecule is inserted into a -pleated sheet of a second. This interaction accounts for the biologically important retention of Z (23, 24) and Siiyama (25) antitrypsin in the endoplasmic reticulum of the liver and hence the accompanying plasma deficiency. Polymerization of Z antitrypsin is blocked by annealing homologue reactive loop peptides to the A sheet (24), and on this basis, it was reasonably deduced that loop-sheet polymerization occurs due to the insertion of the loop of one molecule into the A sheet of the next. The phenomenon of loop-sheet polymerization is not restricted to antitrypsin and has now been reported in plasminogen activator inhibitor-2 (26) and in association with point mutations in C1-inhibitor(27, 28) , -antichymotrypsin(29) , and antithrombin(30) . Indeed, recent structural findings with antithrombin (9, 30, 31) suggest an alternative mechanism involving interaction between the loop of one molecule and the C sheet of the next. The occurrence of C sheet-linked polymers of antithrombin raised the likelihood that this is a general mechanism and that antitrypsin also forms polymers by a C, rather than an A, sheet interaction.

Here we examine a third antitrypsin deficiency variant, Mmalton (Phe-deleted(32, 33, 34) ), which gives the same liver inclusions (33, 35) and plasma aggregates as Z (23) and Siiyama (25) antitrypsin. The aggregates from plasma have been characterized and shown to have the properties of loop-sheet polymers. The physical properties of these polymers and the associated monomeric antitrypsin Mmalton, together with data from the Z and Siiyama variants described previously(24, 25) , fit convincingly with the C sheet, rather than A sheet, mechanism of loop-sheet polymerization.


MATERIALS AND METHODS

Study Patient

The donor was a 45-year-old male who is a lifelong nonsmoker and has had no evidence of liver or lung disease over a follow-up period of 18 years. He is the father of the propositus, who had previously been identified as PI-type MmaltonZ (36). DNA sequence analysis and isoelectric focusing confirmed him to be an Mmalton/QO (null) bolton heterozygote(32, 37) ; and therefore, all the circulating antitrypsin must be derived from the Mmalton allele.

Purification of M, Z, and Mmalton Antitrypsin

M, Z, and Mmalton antitrypsin were purified from 200, 250, and 130 ml of plasma, respectively, by 50 and 75% ammonium sulfate fractionation followed by thiol exchange and Q-Sepharose chromatography as detailed previously (25, 38). The protein was stored in 50 mM Tris, 50 mM KCl, pH 7.4, with 0.1% (v/v) -mercaptoethanol, and total protein was determined from the absorbance at 280 nm (39) and amino acid analysis. Purity was assessed by 7.5-15% (w/v) SDS-PAGE with the antitrypsin band being detected by Western blot analysis (40) and amino-terminal sequencing following transfer onto Problot membrane(41) . Mmalton polymers were further purified by antitrypsin antibody affinity chromatography. Bound antitrypsin was eluted with 0.2 M glycine, pH 2.5, and collected onto 1 g of solid Tris to restore physiological pH. The Tris was removed by concentration/dialysis into 50 mM Tris, 50 mM KCl, pH 7.4, in an Amicon concentrating cell.

Assessment of Antitrypsin Conformations

SDS, nondenaturing, and transverse urea gradient polyacrylamide gel electrophoresis; electron microscopy; heat stability assays; the assessment of the rate of insertion of a synthetic reactive center loop peptide (Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile) into the A sheet of antitrypsin; and limited proteolysis of the reactive center loop of M and Mmalton antitrypsin with Staphylococcus aureus V8 proteinase were performed as detailed previously(22) . Limited proteolysis of the reactive center loop of antitrypsin with papaya proteinase IV (Calbiochem-Novobiochem Ltd., Nottingham, United Kingdom) was achieved by incubation in 30 mM potassium phosphate, 30 mM NaCl, pH 7.0, for 2 h at 37 °C. C-terminal peptides were separated by reverse-phase high performance liquid chromatography (HPLC) on a PLRP-s column as described by Pemberton et al.(42) .

Determination of the Association Kinetics of Antitrypsin Mmalton with Bovine -Chymotrypsin and Human Neutrophil Elastase

The active-site values of M, Z, and Mmalton antitrypsin were determined against bovine -chymotrypsin as described previously(38) . Kinetic parameters for the interaction of antitrypsin Mmalton with bovine -chymotrypsin and human neutrophil elastase were determined at 37 °C in the presence of substrate by analyzing the progress curves for the formation of p-nitroanilide(38) .

Preparation of Latent M and Mmalton Antitrypsin

Latent antitrypsin was prepared by incubating isolated antitrypsin (final concentration of 0.2 mg/ml) at 67 °C for 12 h in 20 mM Tris, pH 7.4, and 0.7 M sodium citrate as described previously(22) . The citrate was removed by dialysis against 20 mM Tris, pH 8.6, and the antitrypsin was concentrated to 1.0 mg/ml. This material was then heated at 60 °C for 3 h to convert any residual active antitrypsin to polymer(38) . The presence of residual thermostable latent antitrypsin was detected by nondenaturing PAGE and a concomitant loss of inhibitory activity against bovine -chymotrypsin(22) .

Assessment of Antitrypsin Secretion from the Xenopus Oocyte

The construction of Z and Mmalton mutants, in vitro transcription, and assessment of secretion from Xenopus oocytes were performed as described by Sidhar et al.(43) .


RESULTS

Antitrypsin Mmalton was isolated from 130 ml of plasma and eluted from the Q-Sepharose anion-exchange column as two distinct peaks: one at a sodium chloride concentration similar to that of M and Z antitrypsin (38) (denoted fraction 1) and the other at a sodium chloride concentration similar to that of antitrypsin Siiyama (25) (denoted fraction 2). The protein in each of these fractions was characterized separately. The antitrypsin in fraction 1 migrated as a 52-kDa band on SDS-PAGE (Fig. 1), but protein estimation from the extinction coefficient resulted in underloading when compared with M and Z antitrypsin controls. Amino acid analysis confirmed the extinction coefficient () of antitrypsin Mmalton to be 11.1 rather than 5.3 (39) for M antitrypsin. Amino-terminal sequencing following transfer onto Problot membrane showed that antitrypsin Mmalton in the early eluting peak of fraction 1 was cleaved at the amino terminus (DAAQK-), whereas that of the main peak was intact (EDPQG-) as described previously for Z antitrypsin(38) . Fraction 2 migrated as multiple bands on SDS-PAGE, and Western blot analysis revealed the antitrypsin to be present as 52- and 48-kDa species (Fig. 1); both were intact at the N terminus (EDPQG-), and the 48-kDa species had the same electrophoretic mobility as reactive center loop-cleaved M antitrypsin. The cleaved protein was detected in all four purifications of antitrypsin Mmalton, in Western blot analysis of the starting material, and following antitrypsin antibody immunopurification of fraction 2 (Fig. 2). The C-terminal peptide liberated following reactive loop cleavage was not detected by SDS-PAGE of fraction 2 (Fig. 2) or by reverse-phase HPLC. The specific activities of fractions 1 and 2 were 66 and <1%, respectively, when the total protein in fraction 1 was determined by amino acid analysis. The value for fraction 1 compares with 77% for M antitrypsin and 50% for Z antitrypsin purified using the same protocol; the reduced activity of Z compared with M antitrypsin reflects contaminating loop-sheet dimers.


Figure 1: Left panel, 7.5-15% (w/v) SDS-PAGE of Mmalton/QO (null) bolton fractions eluted from the Q-Sepharose anion-exchange column. Lane1, molecular mass markers (in kilodaltons); lane2, M antitrypsin (5 µg); lane3, antitrypsin Mmalton fraction 1 (5 µg); lane4, antitrypsin Mmalton fraction 2 (20 µg); lane5, M antitrypsin cleaved at the reactive center loop with S. aureus V8 proteinase (5 µg); lane6, M antitrypsin (5 µg). Middle panel, Western blot for antitrypsin of 7.5-15% (w/v) SDS-PAGE of Mmalton/QO (null) bolton fractions eluted from the Q-Sepharose anion-exchange column. Lane1, M antitrypsin (5 µg); lane2, antitrypsin Mmalton fraction 1 (5 µg); lane3, antitrypsin Mmalton fraction 2 (20 µg); lane4, M antitrypsin cleaved at the reactive center loop with S. aureus V8 proteinase (5 µg); lane5, M antitrypsin (5 µg). Right panel, Western blot for antitrypsin of 7.5-15% (w/v) nondenaturing PAGE of Mmalton/QO (null) bolton fractions eluted from the Q-Sepharose anion-exchange column. Lane1, M antitrypsin (5 µg); lane2, antitrypsin Mmalton fraction 1 (5 µg); lane3, antitrypsin Mmalton fraction 2 (20 µg); lane4, M antitrypsin (5 µg).




Figure 2: A, 7.5-15% (w/v) SDS-PAGE of Mmalton polymers and M antitrypsin following immunopurification. Lane 1, molecular mass markers (in kilodaltons); lane2, M antitrypsin (5 µg); lane3, M antitrypsin control eluted from the antitrypsin affinity column; lane4, antitrypsin Mmalton polymers (fraction 2) eluted from the antitrypsin affinity column; lane5, M antitrypsin cleaved at the reactive loop with S. aureus V8 proteinase (5 µg). Arrows represent native (N) and reactive center loop-cleaved (C) antitrypsin. Both bands were shown to be antitrypsin by Western blot analysis (data not shown). B, overloaded 7.5-15% (w/v) SDS-PAGE to assess the C-terminal peptide from antitrypsin Mmalton polymers (fraction 2) formed in vivo. Lane1, molecular mass markers (in kilodaltons); lane2, M antitrypsin (5 µg); lanes3 and 4, M antitrypsin cleaved at the reactive loop with S. aureus V8 proteinase (5 and 10 µg, respectively); lanes5-7, antitrypsin Mmalton polymers (20, 40, and 60 µg, respectively). peptide shows the electrophoretic mobility of the 4-kDa C-terminal peptide from reactive loop-cleaved antitrypsin.



Western blot imaging of antitrypsin on nondenaturing PAGE (Fig. 1) showed fraction 1 to be monomeric, while fraction 2 contained high molecular mass polymers that were present in Western blot analysis of starting plasma. As fraction 2 contains no monomeric antitrypsin, the reactive center loop-cleaved antitrypsin must be an integral component of the polymers. The conformation and stability of antitrypsin in these two fractions were assessed by unfolding on transverse urea gradient gel electrophoresis. Monomeric antitrypsin Mmalton (fraction 1) unfolded with a profile similar to that of native M and Z antitrypsin (Fig. 3). M and Mmalton antitrypsin cleaved at the reactive center loop with S. aureus V8 proteinase failed to unfold in 8 M urea, indicating that the A -sheet had been stabilized by insertion of the reactive loop peptide. Similarly, M and Mmalton antitrypsin polymers formed by heating at 60 °C and antitrypsin Mmalton polymers isolated from the plasma were resistant to unfolding in 8 M urea. The antitrypsin Mmalton polymers isolated from the plasma migrated farther into the gel than M and Mmalton antitrypsin polymers formed by heating at 60 °C. This reflects the nondenaturing PAGE and electron micrograph observations (Fig. 4) that circulating antitrypsin Mmalton polymers are shorter and hence of lower molecular mass than M and Mmalton antitrypsin polymers formed in vitro.


Figure 3: 7.5% (w/v) transverse urea gradient gel electrophoresis of M, Z, and Mmalton antitrypsin monomers and polymers. All lanes contained 50 µg of protein; the left of each gel represents 0 M urea, and the right represents 8 M urea. Toprowleft, native M antitrypsin; toprowright, native Z antitrypsin; secondrowleft, native antitrypsin Mmalton monomer; secondrowright, M antitrypsin cleaved at the reactive center loop with S. aureus V8 proteinase; thirdrowleft, antitrypsin Mmalton cleaved at the reactive center loop with S. aureus V8 proteinase; thirdrowright, M antitrypsin polymers formed by heating M antitrypsin (0.2 mg/ml) at 60 °C for 3 h; bottomrowleft, antitrypsin Mmalton polymers formed by heating antitrypsin Mmalton (0.2 mg/ml) at 60 °C for 3 h; bottomrowright, Western blot for antitrypsin of Mmalton polymers isolated from plasma.




Figure 4: Electron micrographs of antitrypsin Mmalton monomers (upper panel) and polymers (middle panel) isolated from plasma. Shown in the lower panel, for comparative purposes, are M antitrypsin polymers formed by heating M antitrypsin (0.25 mg/ml) at 65 °C for 12 h. All preparations were stained with 1% (w/v) uranyl acetate. As antitrypsin Mmalton polymers isolated from plasma were only partially pure, the polymers were shown to be antitrypsin by the attachment of polyclonal antitrypsin antibodies, which were then visualized by negative staining and electron microscopy (data not shown).



Isolated monomeric antitrypsin Mmalton polymerized less readily than Z antitrypsin, but more readily than M antitrypsin, after heating at 0.6 mg/ml and 40 °C (Fig. 5). The resulting polymers showed similar electrophoretic mobility to Z antitrypsin polymers formed under identical conditions. Monomeric antitrypsin Mmalton accepted an 11-mer antithrombin reactive center loop peptide at a significantly slower rate than M or Z antitrypsin (t for M antitrypsin = 30-60 min, for Z antitrypsin = 2 h, and for Mmalton = 24 h) (Fig. 6). This implies that the Phe deletion closes or distorts the gap between strands 3 and 5 of the A sheet or that the reactive center loop adopts a conformation that hinders exogenous peptide insertion.


Figure 5: 7.5-15% (w/v) nondenaturing PAGE showing the polymerization of M (upper panel), Mmalton (middle panel), and Z (lower panel) antitrypsin (0.6 mg/ml) in 50 mM Tris, 50 mM KCl, pH 7.4, at 40 °C. All lanes contained 10 µg of protein. Lane1, 0 h; lane2, 12 h; lane3, 24 h; lane4, 48 h; lane5, 72 h; lane6, 6 days; lane7, 12 days.




Figure 6: 7.5-15% (w/v) nondenaturing PAGE showing the rate of insertion of an 11-mer antithrombin reactive center loop peptide into M, Z, and Mmalton antitrypsin. Antitrypsin (0.6 mg/ml in storage buffer) was incubated with an equal volume of a 100-fold molar excess of peptide at 37 °C. Shown are M (upperpanel), Z (middle panel), and Mmalton (lower panel) antitrypsin at the following time points: lane1, 0 h; lane2, 0.5 h; lane3, 1 h; lane4, 2 h; lane5, 4 h; lane6, 12 h; lane7, 24 h; lane8, 48 h; lane9, 72 h. Each lane contained 5 µg of protein. N, native M, Z, or Mmalton antitrypsin; BC, protein containing the synthetic reactive center loop peptide.



The inhibitory conformation requires the partial insertion of the loop into the A sheet(14, 15, 16) , and it is plausible that distortion of this domain may perturb the loop and so affect the inhibitory kinetics with serine proteinases. Antitrypsin Mmalton had an essentially normal association rate constant and K value with human neutrophil elastase (2.9 ± 0.03 10M s and <5 pM (Fig. 7A), respectively, compared with 5.3 ± 0.06 10M s and <5 pM for M antitrypsin(38) ) and with bovine -chymotrypsin (3.0 ± 0.02 10M s and <5 pM, respectively, compared with 2.6 ± 0.2 10M s and 9.2 ± 2.7 pM for M antitrypsin(38) ) and was able to form SDS-stable complexes (Fig. 7B). Limited proteolysis of the reactive loop with S. aureus V8 proteinase (which cleaves at P-P) and papaya proteinase IV (which cleaves at P-P) showed no differences between Mmalton and native M antitrypsin (data not shown).


Figure 7: A, progress curve showing antitrypsin Mmalton inhibition of human neutrophil elastase. The reaction was started with 155 pM elastase, and the curves represent 0 (), 66 (), 132 (), 197 (▾), 263 (), and 329 () pM antitrypsin Mmalton. B, M and Mmalton antitrypsin complex formation with bovine -chymotrypsin. The enzyme and inhibitor were incubated together in 50% (v/v) reaction buffer at room temperature for 15 min before heating at 95 °C for 2 min and separating by 7.5-15% SDS-PAGE. All lanes contained 5 µg of protein, and ratios are based on activity. Molecular mass markers (in kilodaltons) are shown on the left. Lane1, M antitrypsin; lanes2-4, M antitrypsin:chymotrypsin ratios of 1:0.5, 1:1 and 1:2, respectively; lane5, Mmalton antitrypsin monomer; lanes 6-8, antitrypsin Mmalton monomer:chymotrypsin ratios of 1:0.5, 1:1, and 1:2, respectively.



Distortion of the A sheet did not prevent the insertion of the reactive center loop peptide after cleavage as antitrypsin Mmalton monomer was able to undergo the S to R (active to cleaved) transition (Fig. 8). More specifically, antitrypsin Mmalton was induced to polymerize upon heating at temperatures in excess of 60 °C, and this was prevented by the insertion of the reactive loop peptide into the A sheet. Similarly, heating both M and Mmalton antitrypsin at 67 °C for 12 h in 0.7 M sodium citrate produced a thermostable monomeric species (data not shown) that we have recently characterized as the latent conformation(22) .


Figure 8: Heat stability of native () and reactive center loop-cleaved () antitrypsin Mmalton (0.2 mg/ml). The xaxis represents increasing temperature, and the yaxis represents the percentage of antigen remaining in the solution after filtration measured against the value at 30 °C. The assay was performed in 20 mM Tris, pH 7.4, and all points are the average of duplicate values.



In vitro studies demonstrated that purified Z antitrypsin was able to polymerize more readily than antitrypsin Mmalton, which in turn polymerized more readily than M antitrypsin. To confirm this observation in vivo and to show that antitrypsin Mmalton is retained at the same point of processing as Z antitrypsin, these mutants, along with an M antitrypsin control, were expressed in Xenopus oocytes. M antitrypsin secreted from the oocytes was 65 ± 7.6% (S.E.) of the total synthesized antitrypsin. Both Z and Mmalton antitrypsin had reduced secretion of 11.1 ± 3.3 and 19.8 ± 3.1%, respectively. The experiments were performed on at least four occasions for each variant, and on each occasion, the secretion was the sum of 20 injected oocytes. The retained Z and Mmalton antitrypsin were sensitive to digestion with endoglycosidase H (Fig. 9), localizing them to a pre-Golgi compartment, most likely the endoplasmic reticulum.


Figure 9: 12.5% (w/v) SDS-PAGE showing the digestion of intracellular and secreted forms of antitrypsin with endoglycosidase H. Samples 1-3 are oocyte extracts from M, Z, and Mmalton antitrypsin, respectively, with (+) and without (-) endoglycosidase H (Endo H) digestion. Samples 4-6 are the corresponding secreted forms of the inhibitor with and without enzyme.




DISCUSSION

We have previously shown that Z antitrypsin forms intracellular inclusions in the liver of Z homozygotes by a unique protein-protein interaction, loop-sheet polymerization(24) . Similar loop-sheet polymers have been isolated from the plasma of an individual who is a homozygote for the Siiyama mutation(25) . This mutation, Ser Phe, is located in the B helix and similarly results in the formation of hepatic inclusions(44) . The only other identified mutation that predisposes to the formation of inclusions is Phe deletion, which has been variously named Mmalton (33, 34, 37), Mnichinan(45) , and Mcagliari(46) . We demonstrate here that as predicted(24) , this point mutation also favors the formation of loop-sheet polymers. Furthermore, several associated findings provide strong support for the proposal (9, 28, 30, 31) that the polymerization occurs through the linking of the reactive loop of one molecule to the C, rather than the A, sheet of the next. Specifically, the findings are compatible with the occurrence of a spontaneous and inappropriate change in conformation of the variant antitrypsin toward that normally adopted by the inhibitor in the stable complex with the proteinase(47) . The structure of this ``locking'' conformation has not been determined, but there is evidence to support the conclusion (47) that it involves a partial reincorporation of the loop (48) together with release of strand 1C of the molecule(28) .

Antitrypsin Mmalton was isolated from the plasma of an Mmalton/QO (null) bolton heterozygote as two distinct peaks that represented monomer and polymer. Antitrypsin Mmalton polymers were inactive as an inhibitor of bovine -chymotrypsin and were resistant to unfolding on transverse 0-8 M urea gradient gels. These data support our hypothesis that polymers result from the linkage of the reactive loop of one molecule with the -pleated sheet of a second. Such an interaction obscures the scissile P-P` bond of the reactive center loop, rendering the protein inactive, but also stabilizes the A sheet of the receptor molecule. Antitrypsin Mmalton formed long chain polymers in vitro (Fig. 5), but the polymers isolated from plasma were predominantly only 2-5 molecules in length (Fig. 1). This differed from the plasma findings in the Siiyama homozygote, in which the polymers were formed by 15-20 molecules of antitrypsin Siiyama (25). The findings do, however, resemble those recently described for a variant of antithrombin, Rouen VI Asn Asp(30) , which forms short chain polymers. These polymers were truncated at 2-3 units by the complete conversion of the terminal molecule to the latent form. We initially thought that antitrypsin Mmalton might similarly undergo a spontaneous transition to the latent form and hence result in short polymers. Although it was shown that the variant antitrypsin could be induced into the latent form, this conformation was not detected in either the monomeric or polymeric fraction from plasma.

The clue to the process limiting the length of Mmalton polymers in vivo was provided by the finding that a proportion of the molecules in the polymers isolated from plasma had been cleaved at the reactive center (Fig. 1). This finding is in keeping with a mechanism in which polymerization of antitrypsin Mmalton occurs as a result of a preceding transition to the locking conformation that is normally adopted to stabilize the proteinase-inhibitor complex(47) . The combination of a partially exposed reactive center loop together with a released strand from the C sheet will allow the formation of sequential loop-sheet linkages to give polymerization. In vitro, and presumably in the protected environment of the endoplasmic pathway, this sequential linkage will allow the formation of long chain polymers as shown in Fig. 5. However, within the plasma, the presence of extraneous proteinases explains the truncation of polymerization by cleavage of the exposed loop of the terminal molecule. The apparent absence of the C-terminal peptide from the reactive loop-cleaved component of the plasma polymers is both unexpected and intriguing (Fig. 2). This peptide is usually tightly bound to the body of the molecule of serpins cleaved in vitro and can only be dissociated by strong denaturing conditions. The absence of the peptide from the cleaved component of the Mmalton polymers implies that dissociation may follow cleavage of the locking conformation in which strand 1C release has occurred.

As compared with the Z and Siiyama variants, antitrypsin Mmalton is relatively stable. The majority of antitrypsin Mmalton present in plasma is in the monomeric form, has essentially normal inhibitory kinetics, and forms SDS-stable complexes with bovine -chymotrypsin. Moreover, Mmalton has the same limited proteolysis profile as antitrypsin following digestion with bovine -chymotrypsin (P-P`), S. aureus V8 proteinase (P-P), and papaya proteinase IV (P-P). This compares with Z antitrypsin, which has modified inhibitory kinetics (38, 49, 50) and is not susceptible to cleavage at P-P(38) . Monomeric antitrypsin Mmalton forms long chain polymers on incubation in vitro at a faster rate than M antitrypsin, but at a slower rate than Z antitrypsin (Fig. 5). All of these findings fit with a common molecular pathology that explains the accumulation of these three variants of antitrypsin within the endoplasmic reticulum of the liver. We believe the shared abnormality is a tendency for overinsertion of the reactive loop in the inhibitor molecule to give a spontaneous conformational change with release of strand 1C. The consequent formation of loop-C sheet-linked polymers will vary in rate depending on the nature of the underlying mutation. Polymerization is more marked in the Siiyama variant, in which all the plasma antitrypsin is polymerized(25) , and less so in the Z variant, which is predominantly present in plasma as the monomer, although the inaccessibility of its P-P bond to proteolysis infers partial insertion of its loop(38) . Mmalton circulates in a virtually normal functional form, but its decreased rate of formation of binary complexes (Fig. 6) indicates that its own loop is likely to be overinserted and to inhibit homologue peptide insertion. The difference in severity of the molecular lesions of the two antitrypsin variants is confirmed by the reduced secretion of Z as compared with Mmalton in the Xenopus oocyte expression system. Z and Mmalton, but not M, antitrypsin were retained within the oocyte at a pre-Golgi stage most likely to be in the endoplasmic reticulum. Interestingly, the secretion of antitrypsin Mmalton was almost twice that of Z antitrypsin, reflecting the reduced rate of forming loop-sheet polymers. These and other results(9, 30, 31) , taken together with the observations of Eldering et al.(28) , favor the conclusion that mutations that produce hepatic inclusions lead to overinsertion of the reactive loop and a likely C sheet mechanism of polymerization, although they do not exclude the alternative possibility of a loop-A sheet interaction.


FOOTNOTES

*
This work was supported by the Medical Research Council (United Kingdom) and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Medical Research Council Clinician Scientist Fellow. To whom correspondence and reprint requests should be addressed. Fax: 44-1223-336827.

The abbreviations used are: serpin, serine proteinase inhibitor; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

The structure of the loop is defined by the nomenclature of Schechter and Berger (51), with residues 358 and 359 denoted as P-P`.


ACKNOWLEDGEMENTS

We thank Dr. L. Packman (Department of Biochemistry, University of Cambridge) for peptide synthesis, amino acid analysis, and N-terminal sequence analysis. We are indebted to the donor, T. P., who has consistently supported our research.


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