a1-antitrypsin mutations : an investigation of their effects on protein processing and secretion

Abstract

The mechanism of loop sheet polymerization has been shown to have an important role in the formation of aggregates of α1AT that occur in the liver and lungs of patients with α1AT deficiency. Crystallographic analysis and predictive molecular modelling have interpreted the effects of point mutations known to cause a clinical deficiency in terms of their ability to promote loop sheet insertion.

This study examines the relative secretion of a number of both existing deficiency variants (Z, Sii, S, F and I), and novel mutations (termed P5 V, Q & S and Tyr³⁸→Phe/Cys), compared to M α1AT, following injection of mRNA into Xenopus oocytes and protein analysis using immunoprecipitation and SDS-PAGE. The results show that the secretion of Z (16%), Sii (13%), F (53%), P5S (53%), I (26%) and S (49%), along with Tyr³⁸→Phe (48%), is reduced compared to M (63%). Secretion of the other 2 P5 mutants V (62%) & Q (60%) shows no sig. difference (p<0.01) to M. This demonstrates the importance of mutations affecting specific regions of the molecule - particularly the A β-sheet (Z, Sii), the reactive centre loop (F and P5S) and the shutter domain (I, S and Tyr³⁸→Phe), on the secretion of α1AT.

The form of α1AT within oocytes following mRNA injection was also examined and compared between Z, Sii, F & I variants and M α1AT. Oocyte secretory pathway was isolated and α1AT protein content analysed using Native-PAGE and immunoblotting. Results indicated the presence of high MW forms of α1AT within the pathway of oocytes injected with Z, Sii and I mRNA, but not those injected with M and F mRNA. This suggests that the blockade in secretion occurring with Xenopus oocytes, similar to that seen in liver cells, is due to the formation of high MW aggregates of α1AT protein.

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