Abstract
The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%–5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo. To this end, we developed a monoclonal antibody (mAb2H2) that specifically binds to M in preference to Z A1AT, localized its epitope using crystallography to a region perturbed by the Z (Glu342Lys) substitution, and used Fab fragments to label polymers isolated from an MZ heterozygote liver explant. Glu342 is critical to the affinity of mAb2H2, since it also recognized the mild S-deficiency variant (Glu264Val) present in circulating polymers from SZ heterozygotes. Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample. These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals.
Highlights
Surface plasmon resonance (SPR) experiments using M or Z A1AT monomers that applied to a CM5 chip coated with mAb2H2 confirmed that binding was almost exclusively to the M variant (Figure 1B and Supplemental Figure 1C)
Over the 0–2 μM concentration range tested, only a small proportion of the native and cleaved forms of the Z variant were captured by the antibody, such that it was not possible to determine the KD for these samples (Figure 1B and Supplemental Figure 1C), which would be substantially greater than 2 μM or 34-fold that of M A1AT
More recently, heteropolymers of M and Z A1AT were identified in a cellular model of A1ATD in which tags were introduced for immunorecognition [21]
Summary
Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals. The secretory defect of the common severe Z A1AT mutant (Glu342Lys) is the result of protein misfolding, leading in part to intracellular degradation [2] and to the formation of ordered polymeric chains that condense and accumulate as inclusion bodies within the endoplasmic reticulum (ER) of hepatocytes [1, 3] These inclusions cause liver disease in ZZ A1AT homozygotes by impairing the ability of hepatocytes to function normally [4, 5] or to respond to stressor events [6, 7]. We have developed a conformational antibody with selectivity for M A1AT with respect to Z A1AT and used it as a sensitive molecular probe for the presence of M A1AT within polymers extracted from the liver tissue of an MZ A1AT heterozygote
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