α1-Antitrypsin Mmalton (Phe52-deleted) Forms Loop-Sheet Polymers in Vivo.

Abstract

The Z (Glu342 → Lys) and Siiyama (Ser53 → Phe) deficiency variants of α1-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 β-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-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 β-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. The Z (Glu342 → Lys) and Siiyama (Ser53 → Phe) deficiency variants of α1-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 β-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-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 β-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. α1-Antitrypsin (α1-proteinase inhibitor) is a 52-kDa 394-amino acid glycoprotein and the archetypal member of the serpin1 1The abbreviations used are: serpinserine proteinase inhibitorPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatography. 1The abbreviations used are: serpinserine proteinase inhibitorPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatography. superfamily(1Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1479) Google Scholar). Members of this family each have a unique inhibitory specificity, but share a similar molecular structure based on a dominant five-strand A β-sheet(2Loebermann H. Tokuoka R. Deisenhofer J. Huber R. J. Mol. Biol. 1984; 177: 531-556Crossref PubMed Scopus (610) Google Scholar, 3Stein P.E. Leslie A.G.W. Finch J.T. 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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 (3Stein P.E. Leslie A.G.W. Finch J.T. Turnell W.G. McLaughlin P.J. Carrell R.W. Nature. 1990; 347: 99-102Crossref PubMed Scopus (346) Google Scholar, 10Wei A. Rubin H. Cooperman B.S. Christianson D.W. Nature Struct. Biol. 1994; 1: 251-258Crossref PubMed Scopus (167) Google Scholar) and the canonical structure of the small inhibitors(13Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1936) Google Scholar, 14Bode W. Huber R. Curr. Opin. Struct. Biol. 1991; 1: 45-52Crossref Scopus (85) Google Scholar). 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(15Stein P.E. Leslie A.G.W. Finch J.T. Carrell R.W. J. Mol. Biol. 1991; 221: 941-959Crossref PubMed Scopus (403) Google Scholar, 16Mast A.E. Enghild J.J. Salvesen G. Biochemistry. 1992; 31: 2720-2728Crossref PubMed Scopus (187) Google Scholar). 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(17Skriver K. Wikoff W.R. Patston P.A. Tausk F. Schapira M. Kaplan A.P. Bock S.C. J. Biol. Chem. 1991; 266: 9216-9221Abstract Full Text PDF PubMed Google Scholar). Dissociation of the enzyme-inhibitor complex may result in the release of the intact serpin (18Longstaff C. Gaffney P.J. Biochemistry. 1991; 30: 979-986Crossref PubMed Scopus (64) Google Scholar) or cleavage of the loop at the P1-P1′ residues(19Matheson N.R. van Halbeek H. Travis J. J. Biol. Chem. 1991; 266: 13489-13491Abstract Full Text PDF PubMed Google Scholar). 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). serine proteinase inhibitor polyacrylamide gel electrophoresis high performance liquid chromatography. serine proteinase inhibitor polyacrylamide gel electrophoresis high performance liquid chromatography. 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(6Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (526) Google Scholar)), 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 β-pleated sheet of a second. This interaction accounts for the biologically important retention of Z (23Cox D.W. Billingsley G.D. Callahan J.W. FEBS Lett. 1986; 205: 255-260Crossref PubMed Scopus (36) Google Scholar, 24Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar) and Siiyama (25Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar) 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 (26Mikus P. Urano T. Liljeström P. Ny T. Eur. J. Biochem. 1993; 218: 1071-1082Crossref PubMed Scopus (75) Google Scholar) and in association with point mutations in C1-inhibitor(27Aulak K.S. Eldering E. Hack C.E. Lubbers Y.P.T. Harrison R.A. Mast A. Cicardi M. Davis III, A.E. J. Biol. Chem. 1993; 268: 18088-18094Abstract Full Text PDF PubMed Google Scholar, 28Eldering E. Verpy E. Roem D. Meo T. Tosi M. J. Biol. Chem. 1995; 270: 2579-2587Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), α1-antichymotrypsin(29Faber J.-P. Poller W. Olek K. Baumann U. Carlson J. Lindmark B. Eriksson J. 1993; Full Text PDF PubMed Scopus Google Scholar), and D. D. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; PubMed Scopus Google Scholar). findings with antithrombin R.W. Stein P.E. Fermi G. Wardell M.R. 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Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar) antitrypsin. The from plasma have been and to have the of loop-sheet The of polymers and the associated monomeric antitrypsin with from the Z and Siiyama variants D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar, D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar), with the than A mechanism of loop-sheet polymerization. The was a is a and has had of liver or a of is the of the had been as sequence and to be an Mmalton/QO (null) bolton M. D.W. PubMed Scopus Google Scholar, D.W. J. Google and the antitrypsin must be from the Mmalton M, Z, and Mmalton antitrypsin were from and of by and by and as The protein was in with and protein was from the at J. D. Scopus Google Scholar) and acid was by with the antitrypsin by D.A. Evans D.L. C. G. Carrell R.W. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and H. T. J. U. A. PubMed Scopus Google Scholar). Mmalton polymers were further by antitrypsin chromatography. antitrypsin was with M and of to The was by into in an and transverse urea gradient polyacrylamide gel the of the of insertion of a reactive center loop peptide into the A sheet of and of the reactive center loop of M and Mmalton antitrypsin with proteinase were as D.A. Wardell M.R. Carrell R.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of the reactive center loop of antitrypsin with proteinase was by in for at peptides were by high performance liquid on a as by P.A. Stein P.E. Carrell R.W. Nature. PubMed Scopus Google Scholar). The of M, Z, and Mmalton antitrypsin were as D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). for the interaction of antitrypsin Mmalton with and were at in the of by the for the formation of D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). antitrypsin was by isolated antitrypsin of at for in and M as D.A. Wardell M.R. Carrell R.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The was by and the antitrypsin was to This was at for 3 to active antitrypsin to D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). The of latent antitrypsin was by and a of inhibitory D.A. Wardell M.R. Carrell R.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The of Z and Mmalton in vitro and of from Xenopus were as by D.A. Carrell R.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Mmalton was isolated from of plasma and from the as one at a similar to that of M and Z antitrypsin D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar) and the other at a similar to that of antitrypsin Siiyama (25Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar) The protein in each of was The antitrypsin in as a 52-kDa on but protein from the in when with M and Z antitrypsin acid the of antitrypsin Mmalton to be than J. D. Scopus Google Scholar) for M antitrypsin. that antitrypsin Mmalton in the of was at the that of the was intact as for Z D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). as on and the antitrypsin to be as and were intact at the and the had the same as reactive center loop-cleaved M antitrypsin. The protein was in of antitrypsin in of the and antitrypsin of The peptide reactive loop cleavage was not by of or by The of and were and when the protein in was by acid The for with for M antitrypsin and for Z antitrypsin the same the of Z with M antitrypsin loop-sheet of Mmalton polymers and M antitrypsin molecular M antitrypsin M antitrypsin from the antitrypsin antitrypsin Mmalton polymers from the antitrypsin M antitrypsin at the reactive loop with proteinase native and reactive center loop-cleaved antitrypsin. were to be antitrypsin by not to the peptide from antitrypsin Mmalton polymers formed in vivo. molecular M antitrypsin 3 and M antitrypsin at the reactive loop with proteinase and antitrypsin Mmalton polymers and peptide the of the peptide from reactive loop-cleaved of antitrypsin on to be contained high molecular polymers that were in of plasma. monomeric antitrypsin, the reactive center loop-cleaved antitrypsin must be an component of the The conformation and of antitrypsin in were by unfolding on transverse urea gradient gel Monomeric antitrypsin Mmalton with a similar to that of native M and Z antitrypsin M and Mmalton antitrypsin at the reactive center loop with proteinase to unfold in M that the A β-sheet had been by insertion of the reactive loop M and Mmalton antitrypsin polymers formed by at and antitrypsin Mmalton polymers isolated from the plasma were to unfolding in M The antitrypsin Mmalton polymers isolated from the plasma into the gel than M and Mmalton antitrypsin polymers formed by at This the and that antitrypsin Mmalton polymers shorter and hence of molecular than M and Mmalton antitrypsin polymers formed in of antitrypsin Mmalton and polymers isolated from plasma. in the for M antitrypsin polymers formed by M antitrypsin at for were with antitrypsin Mmalton polymers isolated from plasma were the polymers were to be antitrypsin by the of antitrypsin which were by and not monomeric antitrypsin Mmalton less readily than Z antitrypsin, but more readily than M antitrypsin, at and The polymers similar to Z antitrypsin polymers formed Monomeric antitrypsin Mmalton an antithrombin reactive center loop peptide at a than M or Z antitrypsin for M antitrypsin for Z antitrypsin and for Mmalton This that the or the gap between strands 3 and 5 of the A sheet or that the reactive center loop a conformation that peptide the of insertion of an antithrombin reactive center loop peptide into M, Z, and Mmalton antitrypsin. in was with an of a of peptide at M Z and Mmalton antitrypsin at the 9, contained 5 of native M, Z, or Mmalton protein the reactive center loop The inhibitory conformation requires the partial insertion of the loop into the A W. Huber R. Curr. Opin. Struct. Biol. 1991; 1: 45-52Crossref Scopus (85) Google Scholar, P.E. Leslie A.G.W. Finch J.T. Carrell R.W. J. Mol. Biol. 1991; 221: 941-959Crossref PubMed Scopus (403) Google Scholar, 16Mast A.E. Enghild J.J. Salvesen G. Biochemistry. 1992; 31: 2720-2728Crossref PubMed Scopus (187) Google Scholar), and it is that of this may the loop and the inhibitory with serine Mmalton had an normal association and with and with and for M D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google and with and with and for M D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google and was to form SDS-stable of the reactive loop with proteinase at and proteinase at between Mmalton and native M antitrypsin not of the A sheet not the insertion of the reactive center loop peptide cleavage as antitrypsin Mmalton was to the S to R to transition antitrypsin Mmalton was to at in of and this was by the insertion of the reactive loop peptide into the A M and Mmalton antitrypsin at for in M a monomeric not that have as the latent D.A. Wardell M.R. Carrell R.W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). vitro that Z antitrypsin was to more readily than antitrypsin which in turn more readily than M antitrypsin. this in and to show that antitrypsin Mmalton is retained at the same point of processing as Z antitrypsin, with an M antitrypsin were in Xenopus M antitrypsin from the was of the antitrypsin. Z and Mmalton antitrypsin had of and The were on at for each and on each the was the of The retained Z and Mmalton antitrypsin were to with endoglycosidase to a likely the endoplasmic We have that Z antitrypsin forms in the liver of Z by a unique loop-sheet D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar). loop-sheet polymers have been isolated from the plasma of an is a for the Siiyama D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar). This → is in the and results in the formation of K. Nukiwa T. K. H. K. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The other that to the formation of is which has been Mmalton E. H. T. K. J. 1990; Google Scholar), and C. G. M. G. G. F. Invest. 1994; Google Scholar). We here that as D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar), this point also the formation of loop-sheet associated findings for the R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 28Eldering E. Verpy E. Roem D. Meo T. Tosi M. J. Biol. Chem. 1995; 270: 2579-2587Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, D. D. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; PubMed Scopus Google Scholar, J. Carrell R.W. Wardell M.R. 1994; Google Scholar) that the polymerization occurs the of the reactive loop of one molecule to the than the sheet of the next. the findings with the of a and change in conformation of the variant antitrypsin that by the inhibitor in the complex with the P.E. Carrell R.W. Nature Struct. Biol. 1995; 2: PubMed Scopus Google Scholar). The structure of this conformation has not been but is to the that it a partial of the loop I. K. Biochemistry. 1993; PubMed Scopus Google Scholar) with release of strand 1C of the E. Verpy E. Roem D. Meo T. Tosi M. J. Biol. Chem. 1995; 270: 2579-2587Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Mmalton was isolated from the plasma of an Mmalton/QO (null) bolton as that and Mmalton polymers were as an inhibitor of and were to unfolding on transverse M urea gradient These that polymers result from the of the reactive loop of one molecule with the β-pleated sheet of a second. an interaction the P1-P1′ of the reactive center the protein but also stabilizes the A sheet of the Mmalton formed chain polymers in vitro but the polymers isolated from plasma were in This from the plasma findings in the Siiyama in which the polymers were formed by of antitrypsin Siiyama The findings for a variant of Rouen VI → D. D. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; PubMed Scopus Google Scholar), which forms short chain These polymers were at by the of the molecule to the latent We that antitrypsin Mmalton a transition to the latent form and hence result in short it was that the variant antitrypsin be into the latent this conformation was not in the monomeric or from plasma. The to the the of Mmalton polymers in was by the that a of the in the polymers isolated from plasma had been at the reactive center This is in with a mechanism in which polymerization of antitrypsin Mmalton occurs as a result of a transition to the locking conformation that is to the P.E. Carrell R.W. Nature Struct. Biol. 1995; 2: PubMed Scopus Google Scholar). The of a exposed reactive center loop with a strand from the sheet the formation of loop-sheet to polymerization. and in the of the endoplasmic this the formation of chain polymers as in the the of the of polymerization by cleavage of the exposed loop of the The of the peptide from the reactive loop-cleaved component of the plasma polymers is and This peptide is to the of the molecule of serpins in vitro and be by The of the peptide from the component of the Mmalton polymers that may cleavage of the locking conformation in which strand 1C release has with the Z and Siiyama antitrypsin Mmalton is The of antitrypsin Mmalton in plasma is in the monomeric has normal inhibitory and forms SDS-stable with Mmalton has the same as antitrypsin with proteinase and proteinase This with Z antitrypsin, which has inhibitory D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar, F. J. Clin. Invest. PubMed Scopus Google Scholar, A. Potempa J. A. Travis J. FEBS Lett. 1990; PubMed Scopus Google Scholar) and is not to cleavage at D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). Monomeric antitrypsin Mmalton forms chain polymers on in vitro at a than M antitrypsin, but at a than Z antitrypsin of findings with a molecular that the of variants of antitrypsin the endoplasmic reticulum of the We the is a for overinsertion of the reactive loop in the inhibitor molecule to a conformational change with release of strand The consequent formation of loop-C polymers in on the of the Polymerization is more in the Siiyama in which the plasma antitrypsin is D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar), and less in the Z which is in plasma as the the of its to partial insertion of its D.A. Evans D.L. Carrell R.W. Biochemistry. 1993; PubMed Scopus Google Scholar). Mmalton in a normal but its of formation of that its loop is likely to be and to homologue peptide The in of the molecular of the antitrypsin variants is by the of Z as with Mmalton in the Xenopus oocyte Z and but not M, antitrypsin were retained the oocyte at a stage likely to be in the endoplasmic the of antitrypsin Mmalton was that of Z antitrypsin, the of loop-sheet These and other R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, D. D. Carrell R.W. Wardell M.R. J. Clin. Invest. 1994; PubMed Scopus Google Scholar, J. Carrell R.W. Wardell M.R. 1994; Google Scholar), with the of Eldering E. Verpy E. Roem D. Meo T. Tosi M. J. Biol. Chem. 1995; 270: 2579-2587Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), favor the that mutations that to overinsertion of the reactive loop and a likely sheet mechanism of not the of a sheet We L. of of for peptide acid and N-terminal sequence We to the T. has