©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutations Which Impede Loop/Sheet Polymerization Enhance the Secretion of Human -Antitrypsin Deficiency Variants (*)

Sanjiv K. Sidhar (1), David A. Lomas (2)(§), Robin W. Carrell (2), Richard C. Foreman (1)(¶)

From the (1) Department of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton SO16 7PX and the (2) Department of Hematology, University of Cambridge, Medical Research Council Center, Hills Road, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

-Antitrypsin plasma deficiency variants which form hepatic inclusion bodies within the endoplasmic pathway include the common Z variant (Glu Lys) and the rarer -antitrypsin S(Ser Phe). It has been proposed that retention of both abnormal proteins is accompanied by a common mechanism of loop-sheet polymerization with the insertion of the reactive center loop of one molecule into a -pleated sheet of another. We have compared the biosynthesis, glycosylation, and secretion of normal, Z and Svariants of -antitrypsin using Xenopus oocytes. Sand Z -antitrypsin both duplicated the secretory defect seen in hepatocytes that results in decreased plasma -antitrypsin levels. Digestion with endoglycosidase H localized both variants to a pre-Golgi compartment. The mutation Phe Leu abolished completely the intracellular blockage of S-antitrypsin and reduced significantly the retention of Z -antitrypsin. The secretory properties of M and Z -antitrypsin variants containing amino acid substitutions designed to decrease loop mobility and sheet insertion were investigated. A reduction in intracellular levels of Z -antitrypsin was achieved with the replacement of Palanines by valines. Thus a decrease in Z and S-antitrypsin retention was observed with mutations which either closed the A sheet or decreased loop mobility at the loop hinge region.


INTRODUCTION

-Antitrypsin variants Z (1, 2) and S(3) are associated with a deficiency of the inhibitor in the blood and its partial retention within the endoplasmic reticulum of synthesizing hepatocytes. Recent evidence indicates that this retention is accompanied by polymerization of the abnormal protein by the process of loop-sheet linkage in which the reactive center loop of one molecule inserts into a -pleated sheet of another (4, 5) . The mutation in Z -antitrypsin, Glu Lys, occurs at the junction of strand 5 of the six-membered sheet A of the molecule (6) with the base of the mobile loop. This alteration in the hinge region is thought to interfere with normal refolding of the reactive center into the A sheet, thus favoring intermolecular loop insertion with the sequential formation of loop-sheet polymers (5) . The A sheet polymerization model is also compatible with structural studies on -antitrypsin Sin which a Ser Phe mutation is predicted to cause an opening of the A sheet between strands s3A and s5A (7, 8) .

Recent structural studies (9, 10) suggest a related but more complex process may be involved in the loop-sheet polymerization of the serpins. The structure of a dimer of antithrombin shows one molecule in the latent form in which the reactive loop is totally incorporated into the A sheet of the molecule. This releases a strand from the C sheet and it is this strand that is replaced by the reactive loop of the second molecule in the dimer. This alternative mechanism of C sheet polymerization is not incompatible with the A sheet model, since the common first step in either polymerization process would be increased rate of refolding of the loop into the A sheet.

Here we examine the comparative secretion of M, Z, and S-antitrypsins from Xenopus oocytes to further explore the link between loop-sheet interactions and defects in secretion. Yu and colleagues (11) have identified a mutation, Phe Leu, which stabilizes -antitrypsin against polymerization, predictably by locking its A sheet in the closed position. In particular we examine the secretion of this mutant -antitrypsin and of its chimer with Z -antitrypsin (Phe Leu/Glu Lys) and S-antitrypsin (Phe Leu/Ser Phe) to establish whether, as predicted, stabilization of the A sheet will prevent the polymerization that otherwise occurs in these variants with a consequent failure in protein export.

Although the structural and other evidence strongly indicates that the underlying defect in Sis due to an opening of the A sheet allowing the ready formation of polymers, the structural deductions with respect to the Z mutant are more ambivalent. The Z mutation is at residue P, at the base of the hinge region on which the reactive center loop pivots, and may result in either an opening of the A sheet or an impedance of loop insertion into the sheet. An inhibition of loop folding would be expected to favor polymerization, but if this is so, the mechanism would be that of A sheet rather than C sheet polymerization. We test this possibility here using constructs of M and Z antitrypsin containing replacements in the hinge region designed to constrain loop mobility but which should not effect the mechanism of A sheet opening. The secretory properties of these M and Z chimeric mutants provide evidence as to the mechanism underlying the intracellular polymerization of Z antitrypsin.


MATERIALS AND METHODS

Chemicals and Reagents

DNA and RNA modifying enzymes were from Promega Corp. or Boehringer Mannheim. -S-dATP (specific activity > 1000 Cimmol) and L-[S]methionine (specific activity > 1000 Cimmol) were supplied by Amersham International plc. Oligonucleotides were synthesized by Oswel DNA Service, Edinburgh, United Kingdom. Endoglycosidase H (cloned from Streptomyces plicatus) was from Boehringer Mannheim. All other reagents were analytical grade or better and provided by Sigma.

Construction of -Antitrypsin Mutants

Mutants were generated from full-length -antitrypsin cDNA (12) using the polymerase chain reaction-based procedure for site-directed mutagenesis described by Landt et al. (13) . PstI-compatible ends were added to the 5`- and 3`-flanking primers to facilitate cloning of the mutated CDNA into the PstI site of SP64T-RCF, a modified SP64T transcription vector (14) . Clones were sequenced using Sequenase reagents (Amersham International plc.) to ensure that the desired mutation was in place. cDNAs were transcribed in vitro as described previously (14) using the Promega Ribomax transcription system.

Secretion from Xenopus Oocytes

The preparation and micro-injection of Xenopus laevis oocytes were as described by Colman (15) . Healthy oocytes were incubated for 7 h in Barth's saline supplemented with 0.2 mCiml L-[S]methionine. At the end of this period radiolabeling medium was replaced with Barth's saline containing 10 m M methionine and the incubation continued overnight. Oocytes were homogenized and the oocyte extracts and incubation media immunoprecipitated using anti-human -antitrypsin (Dako) as described previously (15, 16) . The products were analyzed on 12.5% (w/v) SDS-polyacrylamide gels (SDS-PAGE)() followed by fluorography using ``Amplify'' (Amersham International plc). Quantitation of radio-labeled proteins was performed by scintillation counting of excised gel fragments.

Endoglycosidase H Digestion

immunoadsorbed pellets were suspended in 60 µl of 50 m M Tris-HCl, pH 5.5, 1% (w/v) SDS, 20% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol and incubated for 5 min at 95 °C. After cooling, 10 µl of 1 milliunit/µl endoglycosidase H containing 1 m M phenylmethylsulfonyl fluoride was added, and samples were digested for 16 h at 37 °C (17) and then analyzed by SDS-PAGE as described above.


RESULTS

Xenopus Oocyte Processing of the Siiyama Variant

Expression of PiM-, PiZ-, and S-encoding RNAs in Xenopus oocytes produced a 54-kDa partially glycosylated intracellular form of the inhibitor (Fig. 1) and a 56-kDa secreted protein. However, it was evident from the fluorograph that secretion of normal, M -antitrypsin was far greater than that of both Z and Svariants. The estimated molecular mass of secreted -antitrypsin as reported by various authors, ranges over 52-58 kDa, depending on the electrophoretic conditions and molecular mass standards used. Immunoprecipitated protein was treated with endoglycosidase H (18) prior to analysis by SDS-PAGE to determine the intracellular location of the 54-kDa -antitrypsin seen in oocytes. As shown in Fig. 1 , the intracellular 54-kDa species was converted to a single band of molecular mass 46 kDa on endoglycosidase H digestion. Intracellular PiZ and Sproteins were sensitive to digestion, indicating that both mutant proteins are retained in a pre-Golgi compartment, probably the ER. Digestion with the enzyme had no effect on secreted M, Z, and Sproteins which were terminally glycosylated.


Figure 1: Synthesis of M, Z, and S antitrypsins in Xenopus oocytes and the endoglycosidase H sensitivity of secreted and retained forms. Twenty oocytes were injected with messenger RNA for a given antitrypsin variant and radiolabeled with L-[S]methionine. Radiolabeled proteins were immunoprecipitated from cell extracts and incubation media then either separated by SDS-PAGE or digested with endoglycosidase H prior to electrophoresis. O represents oocyte extract and S represents material secreted into the medium. Control oocytes were injected with an equivalent volume of distilled water. Molecular mass was determined by the co-migration of standard protein markers.



To quantify differences in the extent of secretion of -antitrypsin PiM, PiZ, and Sbands were excised and counted and the experiment repeated with different batches of oocytes to eliminate, as far as possible, oocyte variation. Fig. 2( open bars) shows the amount of inhibitor secreted expressed as a percentage of the total immunoprecipitable material. S(13.7% secreted ± 1.2) and Z (10.0% secreted ± 1.0) variants are similarly retained in oocytes whereas M -antitrypsin (63.3% secreted ± 3.9) is more readily secreted from these cells during the incubation.


Figure 2: Quantitation of the relative amounts of Phe Leu M, Z, and S antitrypsins secreted from microinjected oocytes. Bands were excised from gels and radioactivity determined by liquid scintillation counting. Amounts of secreted antitrypsin are expressed as a percentage of the total immunoprecipitable protein. Each column represents the mean of at least seven different experiments using five different animals. Each experiment involved the labeling of at least 20 oocytes. Values shown are expressed as the arithmetic mean ± the standard error of the mean. Shaded columns denote constructs bearing the Phe Leu change.



Effect of the Phe Leu Mutation on -Antitrypsin Secretion

The Phe Leu mutant was constructed using polymerase chain reaction mutagenesis of M and Z cDNAs. The double mutant Phe Leu, Ser Phe was constructing using M cDNA as template. The effect of the Phe Leu mutation on M, Z, and Santitrypsin secretion is shown in Fig. 2 ( hatched bars). A 3-fold enhancement of Z antitrypsin secretion was recorded in the chimer Phe Leu, Glu Lys (28.9% secreted ± 4.2). Moreover, the SLeuchimer was secreted (68.9% ±3.1) as efficiently as normal M antitrypsin. Thus the Phe Leu mutation fully restores secretion of the Svariant to that of the normal phenotype and reduces retention of Z antitrypsin.

Effect of the Loop Hinge Mutants on -Antitrypsin Secretion

The 20-residue reactive center loop of -antitrypsin extends 15 residues (P P) amino-terminal and 5 residues (P` P`) carboxyl-terminal to the reactive center. The influence of residues Pand P(near the base of the loop) on the secretory properties of M and Z antitrypsins was examined by replacing the normal residues with those present in the noninhibitory serpin ovalbumin; namely PThr Arg and PAla Val. Fig. 3displays the extent of secretion of antitrypsin as a percentage of the total immunoprecipitable protein. M PArg shows a significant reduction in secretion (41.7% ± 2.3) compared with normal M antitrypsin (62.4% ± 3.1). No significant difference was observed between Z antitrypsin (12.4% ± 1.3) and the Z PArg double mutant (13.7% ± 1.3). The secretion defect observed with Z antitrypsin is partially corrected if the Palanines are substituted for the larger valine residues as indicated by the increased secretion of Z PVal (25.2% ± 2.7) compared with Z antitrypsin.


Figure 3: Quantitation of the relative amounts of PVal and PArg loop hinge M, Z, and S antitrypsins secreted from microinjected oocytes. Bands were excised from gels and radioactivity determined by liquid scintillation counting. Amounts of secreted antitrypsin are expressed as described in the legend to Fig. 2. Each column represents the mean of at least four different experiments using oocytes from five animals.




DISCUSSION

Secretion-defective -Antitrypsin Variants

It is now realized that the abnormalities of serpins associated with deficiency have a common molecular pathology in that they can spontaneously undergo a conformational transition which results in partial retention within the endoplasmic pathway and, particularly with -antitrypsin, is accompanied by polymer formation. The liver disease in Z homozygotes is coincident with the intracellular retention of polymerized inhibitor in inclusion bodies within the endoplasmic reticulum of the hepatocytes (2, 19) . Recently another variant, -antitrypsin S, was shown to have the same association with plasma deficiency and the identical histological finding of hepatic inclusions of mutant inhibitor (3, 7) . Both the Z and Smutants have amino acid substitutions, that although well separated from each other, will theoretically have the same effect, that is to open the A sheet between the third and fifth strands and thus promote the process of loop sheet polymerization (4) .

Z and S-antitrypsin will form long chain polymers with equal facility (7) , and Fig. 1demonstrates that they are secreted from oocytes with comparable efficiency, Z 10.0% and S13.7% of immunoprecipitable material. In mammalian cells only a small proportion of nonsecreted Z protein accumulates in the ER; the majority is rapidly degraded (20) . We attempted to measure the degradation of nonsecreted material in oocytes by quantifying -antitrypsin before and after the chase incubation. We were unable to detect significant proteolysis, mainly because the slow rate of equilibration of the large intracellular amino acid pool means that labeling continues well into the chase incubation. Other experiments following the fate of retained Z protein in oocytes suggest that little degradation occurs over the time scale of our experiments (21) . The synthesis of Z antitrypsin does stimulate the activity of a number of lysosomal enzymes in injected oocytes (22) , but this response may not facilitate the removal of retained protein since, in mammalian cells, degradation has been shown to occur in a post ER nonlysosomal compartment (23) . Both Z and Svariants are retained in a similar, if not identical, pre-Golgi compartment within the oocyte as indicated by the pattern of endoglycosidase H digestion. These findings emphasize the link between loop sheet insertion and the secretory block and suggest that, as with Z, the plasma deficiency of the Svariant can be explained solely in terms of a failure in protein export. We have investigated the phenomenon of loop sheet polymerization by two approaches involving the prevention of entry of the reactive center loop into the A sheet. First, mutations which close the gap between strands s3A and s5A and then mutations in the loop-hinge region which restrict entry into the sheet due to steric hindrance.

Secretion and Sheet Accessibility

The Smutation (Ser Phe) is located in the B helix which underlies the A sheet and provides a surface on which strand s3A slides in order for the sheet to open (8) . Substitution with a large, aromatic side chain at this position is thought to lock the sheet in the open conformation and thereby promote loop sheet polymerization (7, 8) . Recently, Yu and colleagues (11) have reported that substitution of the Phe residue at position 51 by a small, nonpolar residue such as leucine enhances the thermal stability and decreases heat-induced polymerization of wild type M -antitrypsin. Here we show that the stabilizing properties of this change at position 51 have an ameliorating influence on the Z and Ssecretion-defective mutations (Fig. 2). This effect, however, is not equivalent for both dysfunctional proteins; secretion of the Z/Phe Leu double mutant is increased nearly 3-fold, whereas secretion of S/Phe Leu is equal to M -antitrypsin (a greater than 5-fold increase). The increased effect on Smay simply be a matter of proximity in that removal of Phemay correct an aberrant conformation of the B helix, induced by the introduction of a third contiguous phenylalanine at position 53, and thus allow closure of the A sheet. The location of the Z mutation, at the hinge of the reactive center loop, will influence both A sheet opening and loop mobility. Changes at position 51 are liable to reverse the former but not the latter and would be predicted to allow only a partial correction of the Z secretory defect.

Secretion and Loop Mobility

A profound structural transformation from a stressed S conformation to a more ordered, heat-stable, and relaxed R state is observed upon reactive center cleavage of inhibitory serpins. This S to R transition is dependent upon the insertion of the mobile reactive center loop into sheet A after cleavage of the P-P` peptide bond. Inappropriate insertion of the intact loop into sheet A is a feature of the polymerization of aberrant serpins, but partial insertion of the uncleaved loop into the sheet is thought to facilitate formation of the canonical form of the active inhibitor (24, 25, 26) . The reactive center loops of all inhibitory serpins are characterized by the conservation of small hydrophobic amino acids, particularly at positions P, P, and Pat the base of the loop (27) . These residues are orientated with their side chains facing the hydrophobic interior of the molecule (6) , and as a consequence, there is a constraint on their size and polarity if loop insertion is to occur. The absence of inhibitory activity and the S to R transition in ovalbumin and angiotensinogen can be explained by the appearance of larger and/or more polar residues in these critical positions (27) . Similarly, several natural mutants of antithrombin III, C1-inhibitor, and other serpins have been identified with point mutations at positions Pand P, and in most cases these are proteinase substrates, not inhibitors (26, 28) . Schulze et al. (29) have shown that substitution of PThr by Arg converts a reactive center mutant of -antitrypsin from an inhibitor to a substrate that fails to undergo a detectable conformational change. However, a contradictory case has been made by Hood et al. (30) , who constructed a PThr Arg -antitrypsin which retained the ability to complex with several cognate proteinases and underwent the S to R transition.

Although the effect of such mutations on the thermal stability and inhibitory activity of normal serpins are well established, their influence on polymerization and secretion have yet to be explored. The secretion of Z -antitrypsin from oocytes was not improved by the replacement of PThr by arginine. This may mean that aggregation is unhampered by partial exclusion of strand 4A from the sheet, alternatively the gap between strands 3A and 5A may be wider as a result of the lysine residue at position 342 and thus able to accommodate the larger and more polar arginine side chain. The assertion that an arginine residue at Pdoes not prevent polymerization of the Z variant, and presumably does not materially affect loop insertion, is in general agreement with the observation that such a change in the normal inhibitor is compatible with the S R transition and maintenance of inhibitory function (30) . Nevertheless, the PArg mutation may have other structural consequences in addition to its effect on loop mobility, since the secretion of M type -antitrypsin PThr Arg was significantly reduced, but not to the level observed for Z -antitrypsin.

Alanine to valine substitutions at Pand Pwere more effective in overcoming the block in secretion imposed by the Z mutation, causing a 2-fold increase in export of this mutant -antitrypsin (Fig. 3). Recent results (28) indicate that insertion up to Pis required for the release of strand 1 from the C sheet. The presence of valine at P, and perhaps P, will inhibit this insertion and hence the availability of the S1C position for C sheet polymerization. Similarly, a naturally occurring mutation of the human C1-inhibitor with PAla Glu was not an effective inhibitor and did not undergo the S to R conformational change and also showed no tendency to polymerize (26) . Alanine to valine substitutions at both Pand Pwill consequently serve to hinder this more extensive incorporation of the loop, although single replacement with threonine at Pdoes not prevent M type antitrypsin from undergoing the S to R transition (31) . Thus an increase in secretion of Z antitrypsin with valine residues in both the Pand Ppositions is most readily explained by a C sheet mechanism of polymerization.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council, the Wessex Medical Trust, 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.

To whom correspondence and reprint requests should be addressed. Fax: 44-703-594319.

§
Medical Research Council Clinician Scientist.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.