Alpha-1 Antitrypsin Deficiency

Years of Progress

O brave new world

 William Shakespeare 1611

The Biochemical Story

In 1964, P Gross and collaborators published an important work showing that intratracheal administration of papain, a plant protease, induces emphysema in rats. This study, together with the Laurell and Eriksson`s discoveries, supported the concept of a proteolytic mechanism in emphysema and opened a new chapter in experimental and clinical emphysema research. In the following years, several research groups in Europe and USA demonstrated that alpha-1 antitrypsin (AAT) is an effective inhibitor of pancreatic elastase and emphasised a possible role of elastase in the pathogenesis of emphysema. Based on these developments it was generally accepted that the protease-antiprotease balance as an essential requirement for respiratory health, and that perturbations of this balance may result in the loss of lung tissue and the development of emphysema. Furthermore, this concept opened new insights into why in emphysema and proteolytic overload are more common in cigarette smokers. In a 1978 classical paper by L. Larsson and others very convincingly demonstrated that the age of onset of emphysema in Z homozygous patients who were also current or ex-smokers was very rapidly reduced.

The detection and biochemistry of AAT variants became the greatest challenge from 1963 to 1978. The molecule AAT was found to occur in many genetic variants, and Laurell together with Magne Fagerhol in Oslo assembled them as the protease inhibitor Pi-system. Jan-Olof Jeppsson, who joined the Malmö group in 1970, took the lead in tracking the structural differences of the numerous molecular Pi-variants by isoelectric focusing. The Pi-system was developed, based on the migration of the AAT variants in an electric field. The position of the migrated proteins is identified by a letter, where M is normal, while the positions of the slower-moving proteins are marked by letters before M in the alphabet and those of the faster-moving proteins are denoted by letters after M.

During the 70s Robin Carrell analysed the S variant of AAT while the Malmö group analysed the Z variant by mapping its tryptic peptides. By 1975, Carrell and co-workers were able to show that S variant abnormality is due to the substitution of a glutamic acid by a valine. Soon after this, the abnormality of Z AAT (glutamic acid substitution by lysine) was solved by Jeppsson in Malmö.

At this time, Laurell started a collaboration with Carrell in Cambridge and they presented the 3-dimensional structure of the dominating molecular variants of AAT. It become apparent that the active AAT protease inhibitory centre contained a crucial serine.  A series of publications in the late 70s demonstrated a 1000-fold more rapid rate of association between neutrophil elastase and AAT than between trypsin and AAT. Therefore, the most important function of AAT was suggested to be the control of activity of neutrophil elastase but not of the trypsin.

The Serpin superfamily The AAT sequence was completed in 1982.  It was now known where the reactive site is, where the S and Z mutations occur, and where the points of attachment of three oligosaccharide side-chains are located. The multiplicity of elecrophoretic bands of AAT was explained to be due to the presence of different glycoforms of AAT, each with variations in the antennary structure of their oligosaccharides.  The conclusions drawn in 1986 from these studies was that the mutation in Z AAT does not affect protein synthesis but causes defective secretion of the protein.

It was clear that further understanding of the biological functions of AAT required a detailed knowledge of the three-dimensional structure of AAT. Identification of the serpin family occurred with the recognition in the 1980s by Hunt and Dayhoff that ovalbumin, AAT and antithrombin III share a 30% to 50% sequence homology.  The term Serpin (an acronym for SERine Proteinase INhibitor) was later introduced as a general denominator by R Carrell and J Travis in 1985 to describe a superfamily of serine protease inhibitors.  Genetic characterisation has revealed a large group of genes belonging to the serpin family that probably arose by gene duplication of an ancestral gene. Huber and colleagues in Munich in 1984 resolved the crystal structure of the cleaved AAT for the first time. The cleaved form, with its change from a five-stranded to a six-stranded A-sheet provided an enigma and a challenge to structural biology and biochemistry as a whole because no protein had been known to undergo such a remarkable change in its folding. Later it was shown in the native form of AAT that the reactive centre loop lies outside the tertiary core of the protein to allow binding of the target protease. A large conformational change is triggered upon cleavage of the reactive centre loop by the protease. In recent years the structures of numerous serpins in a variety of different conformations have been solved by X-ray crystallography.

Although already in 1969 Sharp and co-workers had described accumulation in the liver of the Z variant of AAT, the understanding of this problem was initiated only in the 1990s when David Lomas and his group in Cambridge, showed that Z AAT forms polymers due to the insertion of the reactive site of one molecule into the opened B-sheet of the next. This gave the molecular mechanism that underlies AAT deficiency and helped explain the deficiency of other members of the serpin superfamily. A number of mutations and other changes have been reported in serpins which result in conformational instability, molecular polymerisation and loss of serpin activity.  The effects have been associated with enhanced susceptibility to inflammatory, neurodegenerative, cardiovascular diseases and cancer. These include the deficiency of antithrombin, C1-inhibitor and alpha1-antichymotrypsin in association with thrombosis, angio-oedema and emphysema. Moreover, the accumulation of mutant neuroserpin within neurones causes the novel dementia, familial encephalopathy with neuroserpin inclusion bodies. These conditions were grouped together as the serpinopathies.