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The molecular pathology of serine proteinase inhibitors: secretion of novel genetically engineered variants of human a1-Antitrypsin

The molecular pathology of serine proteinase inhibitors: secretion of novel genetically engineered variants of human a1-Antitrypsin
The molecular pathology of serine proteinase inhibitors: secretion of novel genetically engineered variants of human a1-Antitrypsin
The hepatic secretory protein a1-antitrypsin, a member of the serpin family of serine proteinase inhibitors, is the most abundant proteinase inhibitor in human plasma. a1-Antitrypsin plasma deficiency variants include the common Z variant (Glu342→Lys) and the rarer Siiyama (Ser53→Phe) and MMalton (Phe52 deleted) a1-antitrypsins. These variants form hepatic inclusion bodies within the endoplasmic reticulum and may predispose individuals to the lung disease emphysema, and to the development of liver cirrhosis. It has been proposed that the accumulation of these abnormal proteins occurs by a common mechanism of loop-sheet polymerization, with the insertion of the reactive centre loop of one molecule into a β-pleated sheet of another.

Oligonucleotide-directed mutagenesis using the polymerase chain reaction (PCR) was used to reconstruct these mutations. In vitro transcription of the PCR templates produced messenger RNA that was microinjected into Xenopus oocytes to investigate the biosynthesis, glycosyiation, and secretion of normal (M) and abnormal variants. All three deficiency variants duplicated the secretory defect seen in hepatocytes. Digestion with Endoglycosidase H localised all three deficiency variants to a pre-Golgi compartment, suggesting a common site for the accumulation of nonsecreted inhibitor.

Two complementary approaches were taken to investigate in detail the loop-sheet polymerization process. This involved: (a) mutations in the hydrophobic core underlying the A sheet to prevent loop insertion and (b) mutations in the loop which would restrict loop mobility and impede insertion into the A sheet, a necessary prerequisite to polymerization and protein aggregation.

The non-inhibitory serpin ovalbumin is unable to undergo the conformational change, typical of inhibitory serpins, because of sequence differences in the reactive centre loop. Mutants of ovalbumin were constructed in an attempt to increase loop mobility and induce inhibitory activity, and also to investigate the association between loop mobility and secretory capacity. a1-Antitrypsin and ovalbumin mutants were also constructed by expression-PCR with the 5'-primer incorporating a SP6 RNA polymerase promoter, to allow direct transcription without the need for cloning into a transcription vector. These transcripts translated efficiently in vitro but not in oocytes.

A decrease in the intracellular accumulation of a1-antitrypsin Z and Siiyama variants was achieved with the mutations designed to restrict loop entry into the A sheet, and lends supports to the principle of loop-sheet polymerization.
University of Southampton
Sidhar, Sanjiv Kumar
47307f47-4870-4465-aaa9-e779ade70874
Sidhar, Sanjiv Kumar
47307f47-4870-4465-aaa9-e779ade70874
Foreman, Richard
c3c1ed19-ec2a-431d-bb57-e3dfb86049a4

Sidhar, Sanjiv Kumar (1994) The molecular pathology of serine proteinase inhibitors: secretion of novel genetically engineered variants of human a1-Antitrypsin. Doctoral Thesis, 238pp.

Record type: Thesis (Doctoral)

Abstract

The hepatic secretory protein a1-antitrypsin, a member of the serpin family of serine proteinase inhibitors, is the most abundant proteinase inhibitor in human plasma. a1-Antitrypsin plasma deficiency variants include the common Z variant (Glu342→Lys) and the rarer Siiyama (Ser53→Phe) and MMalton (Phe52 deleted) a1-antitrypsins. These variants form hepatic inclusion bodies within the endoplasmic reticulum and may predispose individuals to the lung disease emphysema, and to the development of liver cirrhosis. It has been proposed that the accumulation of these abnormal proteins occurs by a common mechanism of loop-sheet polymerization, with the insertion of the reactive centre loop of one molecule into a β-pleated sheet of another.

Oligonucleotide-directed mutagenesis using the polymerase chain reaction (PCR) was used to reconstruct these mutations. In vitro transcription of the PCR templates produced messenger RNA that was microinjected into Xenopus oocytes to investigate the biosynthesis, glycosyiation, and secretion of normal (M) and abnormal variants. All three deficiency variants duplicated the secretory defect seen in hepatocytes. Digestion with Endoglycosidase H localised all three deficiency variants to a pre-Golgi compartment, suggesting a common site for the accumulation of nonsecreted inhibitor.

Two complementary approaches were taken to investigate in detail the loop-sheet polymerization process. This involved: (a) mutations in the hydrophobic core underlying the A sheet to prevent loop insertion and (b) mutations in the loop which would restrict loop mobility and impede insertion into the A sheet, a necessary prerequisite to polymerization and protein aggregation.

The non-inhibitory serpin ovalbumin is unable to undergo the conformational change, typical of inhibitory serpins, because of sequence differences in the reactive centre loop. Mutants of ovalbumin were constructed in an attempt to increase loop mobility and induce inhibitory activity, and also to investigate the association between loop mobility and secretory capacity. a1-Antitrypsin and ovalbumin mutants were also constructed by expression-PCR with the 5'-primer incorporating a SP6 RNA polymerase promoter, to allow direct transcription without the need for cloning into a transcription vector. These transcripts translated efficiently in vitro but not in oocytes.

A decrease in the intracellular accumulation of a1-antitrypsin Z and Siiyama variants was achieved with the mutations designed to restrict loop entry into the A sheet, and lends supports to the principle of loop-sheet polymerization.

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Published date: November 1994

Identifiers

Local EPrints ID: 432065
URI: http://eprints.soton.ac.uk/id/eprint/432065
PURE UUID: 4214b795-e102-4831-9032-b278bdc2b8dc

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Date deposited: 28 Jun 2019 16:30
Last modified: 08 Jul 2019 16:30

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Contributors

Author: Sanjiv Kumar Sidhar
Thesis advisor: Richard Foreman

University divisions

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