Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin
Mattia Laffranchi, … , David A. Lomas, James A. Irving
Mattia Laffranchi, … , David A. Lomas, James A. Irving
Published July 23, 2020
Citation Information: JCI Insight. 2020;5(14):e135459. https://doi.org/10.1172/jci.insight.135459.
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Research Article Genetics Hepatology

Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin

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.

Authors

Mattia Laffranchi, Emma L.K. Elliston, Elena Miranda, Juan Perez, Riccardo Ronzoni, Alistair M. Jagger, Nina Heyer-Chauhan, Mark L. Brantly, Annamaria Fra, David A. Lomas, James A. Irving

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Figure 1

Identification of a mAb specific for the WT M A1AT.

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Identification of a mAb specific for the WT M A1AT. (A) Anti-A1AT mAb3C1...
(A) Anti-A1AT mAb3C11 (nonconformationally selective, left panel) or mAb2H2 (right panel) antibodies were used to probe purified M (blue) and Z (red) A1AT in either the monomeric (dashed lines) or heat-induced polymeric (solid lines) forms by antigen ELISA. Recognition of the samples by mAb3C11 was approximately equal, but mAb2H2 showed a preference for the M variant. (B) Interaction between immobilized mAb2H2 and plasma-purified monomeric M (blue) or Z (red) A1AT variants in either the native or reactive loop-cleaved form. The relative maximal response above baseline was calculated from progress curves recorded at each concentration and is proportional to the mass of the material captured by the chip-bound antibody. Data are shown as ± SD (n = 3). The curves correspond with a hyperbolic function used to derive the KD values for M A1AT (solid lines); this was not possible for the Z A1AT samples due to the limited binding observed over the concentration range (dashed lines). (C) Evaluation of mAb2H2 specificity by immunofluorescence in cells. CHOK1 cells expressing either M or Z A1AT were seeded on coverslips, induced with doxycycline for 48 hours, permeabilized, and stained with anti–total A1AT mAb3C11, anti–polymer mAb2C1, or mAb2H2. Cells expressing Z A1AT showed punctate staining with mAb2C1 but no signal with mAb2H2; conversely, cells expressing M A1AT were negative to mAb2C1 and showed strong recognition by mAb2H2. Both variants were well recognized by the control mAb3C11. Scale bars: 15 μm.