Protein engineering of human alpha 1-antitrypsin gene and the study of secretion defective mutants

Wu, Ying (1991) Protein engineering of human alpha 1-antitrypsin gene and the study of secretion defective mutants. University of Southampton, Doctoral Thesis.

Abstract

The naturally occurring Z mutation in the human α1-antitrypsin (α1-AT) gene encodes a glutamic acid to lysine substitution at position 342 in the polypeptide chain. PiZ homozygotes have an increased incidence of lung and liver disease associated with a low plasma level of the protein that arises from a failure to secrete the mutant variant effectively from the site of synthesis in liver cells. The block in export of the protein may be caused either by the loss of an acidic residue Glu342 which forms a salt-bridge in α1-AT molecule, or by the introduction of a basic one at this point in the polypeptide chain. Site-directed mutagenesis has been used to construct novel α1-AT mutants, to study the effect of amino acid substitution at position 342 on the secretion of human α1-AT from Xenopus oocytes and mammalian cell lines. The results suggest that the salt-bridge interaction between residue Glu342 and Lys290 is not essential for α1-AT protein secretion. It is rather the introduction of a basic residue at this point which results in the blockage of secretion and consequently produces the intracellular accumulation of the protein. The secretory behaviour of the PiZ protein in transfected human macrophages was also studied by comparison with wild type M in different types of cell line. The results show the Z protein is normally secreted from transfected U937 cells - a cell line of human macrophage/monocyte linkage, after 4 hours metabolic labelling with ³⁵S-methionine, suggesting that human macrophages are capable of secreting Z-type α1-AT protein. As a preliminary approach to α1-AT and liver disease, the mRNA level of α1-AT in liver tissue samples from normal- and liver disease-individuals was detected by Northern blot and RNA dot blot techniques, showing that α1-AT mRNA level in the liver with primary biliary cirrhosis or Biliary atresia was at least five fold higher than the normal.

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