Alpha<SUB>1</SUB>-antitrypsin is the most abundant proteinase inhibitor in plasma and the archetypal member of a large family of serine proteinase inhibitors known as the serpins. All serpin members have a common molecular architecture which consists of three β-sheets surrounded by eight α-helixes with an exposed mobile reactive centre loop. In this thesis I show that it is possible to induce and purify an inactive latent α<SUB>1</SUB>-antitrypsin conformation which can be reactivated after denaturation and refolding. An inactive latent component was also shown to exist in commercial α<SUB>1</SUB>-antitrypsin pasteurised for use as replacement therapy in patients with α<SUB>1</SUB>-antitrypsin deficiency. Another consequence of the mobile reactive centre loop is the formation of inactive loop-sheet polymers which form when the reactive centre loop of one molecule is inserted into the β-sheet of another. This interaction accounts for the retention of Z (Glu<SUP>342</SUP>→Lys) α<SUB>1</SUB>-antitrypsin in the endoplasmic reticulum of hepatocytes with accompanying plasma deficiency. I show here that the plasma deficiency associated with the common S α<SUB>1</SUB>-antitrypsin allele (Glu <SUP>264</SUP>→Val), which is present in 19% of individuals of Spanish descent, can also be attributed to loop-sheet polymerisation. The structural basis of this loop-sheet linkage was clarified by the solving here of the crystal structures of intact wildtype and a thermostable mutant of α<SUB>1</SUB>-antitrypsin. Both structures show the reactive centre loop in a canonical conformation that docks perfectly with its cognate proteinase. The rest of the reactive centre loop is held in a β-strand conformation via a stabilising salt bridge with the body of the molecule. In this conformation the reactive loop can be readily modelled into the A β-sheet of a second molecule, providing strong support for A β-sheet linkage as the mechanism for loop-sheet polymerisation. Finally, loop-sheet polymers of α<SUB>1</SUB>-antitrypsin have also been shown to occur within the small airways and alveoli of the lungs of Z α<SUB>1</SUB>-antitrypsin homozygotes. This findings provides another mechanism for the inactivation of α<SUB>1</SUB>-antitrypsin, thereby exacerbating the proteinase-antiproteinase imbalance in the lung disease emphysema.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598809 |
| Date | January 1999 |
| Creators | Elliott, P. R. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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