Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization

Abstract

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

“…Therefore, temperature can be used as a probe of mechanism: deviations from this trend indicate effects on other characteristics of the polymerisation pathway [22,39,62]. A comparison of polymerisation half-life interpolated at 55°C (Figure 1D) and stability determined by thermal unfolding (Figure 1E, left) is shown in Figure 1E (right).…”

The structural basis for Z α 1 -antitrypsin polymerization in the liver

“…Human M and Z α 1 -antitrypsin were purified from donor plasma, and recombinant α 1 -antitrypsin was purified from Escherichia coli as previously described ( 24 , 40 ). Monoclonal antibodies were purified from hybridomas according to published methods ( 12 ) and stored in phosphate-buffered saline (PBS) with 0.02% (w/v) sodium azide.…”

Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin

“…Hexahistidine-tagged human A1AT introduced into the pQE-30 vector (QIAGEN) was expressed in the XL1-Blue strain of E. coli (Thermo Fisher Scientific), purified by nickel-affinity and ion-exchange chromatography, as described previously (43), and its purity was assessed by SDS-PAGE and nondenaturing PAGE. Plasma M, Z, and S A1AT were purified using Antitrypsin Select affinity resin (GE Healthcare) and ion-exchange chromatography, as described previously (44). Heat polymers were generated by incubating 21 μM of A1AT in phosphate saline buffer pH 7.4 (PBS) at 55°C (WT M) or 50°C (Z A1AT) for 16 hours (16), with removal of residual monomer by ion-exchange chromatography.…”