Abstract
Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.
Highlights
Human ␣1-antitrypsin (␣1-AT)1 is a member of the serine protease inhibitor family, which has a tertiary structure composed of three -sheets and several ␣-helices [1]. ␣1-AT is synthesized as a glycoprotein of 394 amino acids in the liver and is secreted into the blood
The loop-sheet polymerization appears to be an important contributing factor for the secretion blockage of the variant ␣1-AT. This implication is based on the finding that the secretion blockage of the Z or Siiyama was efficiently suppressed in the Xenopus oocyte system either by a thermostable mutation (Phe51 3 Leu: F51L) that enhanced the closure of A -sheet or by mutations that decreased the loop mobility [9]
As observed previously [13], the folding of the nascent Z polypeptides was blocked at an intermediate state, but the presence of the F51L mutation (T1) converted some (40 –50%) of the Z-type polypeptides into the native state
Summary
Human ␣1-antitrypsin (␣1-AT) is a member of the serine protease inhibitor family, which has a tertiary structure composed of three -sheets and several ␣-helices [1]. ␣1-AT is synthesized as a glycoprotein of 394 amino acids in the liver and is secreted into the blood. The loop-sheet polymerization appears to be an important contributing factor for the secretion blockage of the variant ␣1-AT. One possible mechanism for the polymerization of the mutant ␣1-AT is that the kinetic trap during folding results in the accumulation of intermediates with a high tendency to polymerize Another possible mechanism is that the stability of the already folded structure is lost, resulting in a conformational change needed for polymerization. To test these possibilities here, we systematically investigated the folding, stability, and the secretion of the Z and Siiyama variants of ␣1-AT, using in vitro folding assay and yeast secretion system. The defect of Siiyama is much more difficult to rescue than the Z type defect, which is presumably due to another kind of defect, i.e. stability loss
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