Molecular characterization of the new defective P_bresciaalpha1-antitrypsin allele

Abstract

Alpha1-antitrypsin (α 1 AT) deficiency is a hereditary disorder associated with reduced α 1 AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the α 1 AT gene (SERPINA1) causing α 1 AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective α 1 AT allele (c.745G>C) coding for a mutant α 1 AT (Gly225Arg), named P brescia . The P brescia α 1 AT allele was first identified in combination with the rare defective M würzburg allele in an 11-year-old boy showing significantly reduced serum α 1 AT level. Subsequently, the P brescia allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the P brescia mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M α 1 AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z α 1 AT and suggests that the mutation present in the P brescia α 1 AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe α 1 AT deficiency in the plasma and toxic protein-overload in the liver.

A Novel Monoclonal Antibody to Characterize Pathogenic Polymers in Liver Disease Associated with α1-Antitrypsin Deficiency†

“…Given the homology to the highly polymerogenic His338Arg variant of neuroserpin, it is likely that neuroserpin mutants form polymers with a similar structure to those formed by a 1 -antitrypsin. 23 Our new mAb 2C1 similarly recognized polymers formed by the Siiyama (Ser53Phe) 26 and Brescia (Gly225Arg) 27 mutants that are also located within the shutter region of a 1 -antitrypsin.…”

“…The 2C1 antibody was used to assess polymers formed by two other shutter domain mutants of a 1 -antitrypsin: Siiyama (Ser53Phe) 26 and Brescia (Gly225Arg). 27 These were transiently expressed in COS-7 cells, in parallel to M, Z, and H334D a 1 -antitrypsin. The cell lysates were assessed by SDS and nondenaturing PAGE followed by western blot analysis and also by sandwich ELISA with the 2C1 mAb.…”

Polymers of Z α1-antitrypsin are secreted in cell models of disease

“…M α 1 -AT is readily secreted and can be detected by immunofluorescence within the Golgi compartment [5,7,[9][10][11]. Although polymers of Z and other mutant variants of α 1 -AT are found in the culture medium of expressing cells, they have never been shown to co-localise with Golgi-resident proteins [5,7,10,11].…”

“…Although polymers of Z and other mutant variants of α 1 -AT are found in the culture medium of expressing cells, they have never been shown to co-localise with Golgi-resident proteins [5,7,10,11]. This may be due to very low levels of these proteins transiting the Golgi at steady state, and so we used a temperature block by culturing cells at 20°C, which reduces the exit of secretory proteins from the Golgi apparatus without affecting ER to Golgi transport [12].…”

Population Genetic Screening for Alpha1-Antitrypsin Deficiency in a High-Prevalence Area

“…Through this procedure, 18 additional AATD-related variants were detected: 2 subjects (0.2%) were compound heterozygotes for the two mutations Z and P brescia [18,19], 9 (1.1%) were P brescia heterozygotes, 4 (0.5%) M wurzburg [20] heterozygotes, 2 (0.2%) I [21] heterozygotes and 1 (0.1%) P lowell [22] heterozygote (fig. 1; table 1).…”