Investigating the proteolytic function of A Disintegrin And Metealloproteinase 33 (ADAM33) and its role in asthma pathogenesis
Investigating the proteolytic function of A Disintegrin And Metealloproteinase 33 (ADAM33) and its role in asthma pathogenesis
Aims: The aims of this study were to characterise the proteolytic activity of ADAM33 and to establish a role for ADAM33 mediated proteolysis that may be relevant to the development of asthma.
Methods: The pro and metalloproteinase domains of ADAM33 and a proteolytically inactive mutant form were expressed as secreted recombinant proteins in insect cells. A purification strategy using a series of chromatographic steps was devised to recover ADAM33 proteins from the culture medium. The activity of purified ADAM33 proteins was assessed using the α2-Macroglobulin protease biding assay and a FRET peptide cleavage assay. The sensitivity of the ADAM33 activity to various biochemical parameters and inhibitors were evaluated. The functional relevance of the proteolytic activity of ADAM 33 was investigated by treating primary epithelial and endothelial cells with ADAM 33. To search for a substrate for ADAM33 a novel PNA-FRET peptide library was screened for cleaved sequences. These were used to identify potentially relevant substrates.
Results: ADAM33 transfected insect cells secreted high levels of ADAM33 proteins. A 5-step purification procedure involving Con A affinity chromatography followed by IMAC, the cation exchange chromatography, then IMAC concentration of the sample, and lastly gel filtration produced highly pure ADAM33 protein preparations. The purified wild-type ADAM protein was shown to be catalytically active and as expected the mutant exhibited no activity. ADAM33 was sensitive to inhibition by a hydroxamate compound (3R)-(+)-[2-(4-methoxybenzenesulfonyl)]-1,2,3,4-tetrahydroisoquinoline-3-hydroxymate and also the MMP activity regulator TIMP-3. ADAM33 was found to induce endothelial cell differentiation in vitro. The screening of the PNA-FRET peptide library highlighted a number of potential substrates for ADAM33.
The ability of ADAM33 to induce endothelial cell differentiation, suggests that ADAM33 cleaves a substrate that has an important role in angiogenesis and airway remodelling.
University of Southampton
Pang, Yun Yun
2068c95e-104b-4412-96a6-a043504f088b
2007
Pang, Yun Yun
2068c95e-104b-4412-96a6-a043504f088b
Pang, Yun Yun
(2007)
Investigating the proteolytic function of A Disintegrin And Metealloproteinase 33 (ADAM33) and its role in asthma pathogenesis.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Aims: The aims of this study were to characterise the proteolytic activity of ADAM33 and to establish a role for ADAM33 mediated proteolysis that may be relevant to the development of asthma.
Methods: The pro and metalloproteinase domains of ADAM33 and a proteolytically inactive mutant form were expressed as secreted recombinant proteins in insect cells. A purification strategy using a series of chromatographic steps was devised to recover ADAM33 proteins from the culture medium. The activity of purified ADAM33 proteins was assessed using the α2-Macroglobulin protease biding assay and a FRET peptide cleavage assay. The sensitivity of the ADAM33 activity to various biochemical parameters and inhibitors were evaluated. The functional relevance of the proteolytic activity of ADAM 33 was investigated by treating primary epithelial and endothelial cells with ADAM 33. To search for a substrate for ADAM33 a novel PNA-FRET peptide library was screened for cleaved sequences. These were used to identify potentially relevant substrates.
Results: ADAM33 transfected insect cells secreted high levels of ADAM33 proteins. A 5-step purification procedure involving Con A affinity chromatography followed by IMAC, the cation exchange chromatography, then IMAC concentration of the sample, and lastly gel filtration produced highly pure ADAM33 protein preparations. The purified wild-type ADAM protein was shown to be catalytically active and as expected the mutant exhibited no activity. ADAM33 was sensitive to inhibition by a hydroxamate compound (3R)-(+)-[2-(4-methoxybenzenesulfonyl)]-1,2,3,4-tetrahydroisoquinoline-3-hydroxymate and also the MMP activity regulator TIMP-3. ADAM33 was found to induce endothelial cell differentiation in vitro. The screening of the PNA-FRET peptide library highlighted a number of potential substrates for ADAM33.
The ability of ADAM33 to induce endothelial cell differentiation, suggests that ADAM33 cleaves a substrate that has an important role in angiogenesis and airway remodelling.
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Published date: 2007
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Local EPrints ID: 466379
URI: http://eprints.soton.ac.uk/id/eprint/466379
PURE UUID: 6480b80c-b2c0-4e3a-93b9-46e37b298023
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Date deposited: 05 Jul 2022 05:12
Last modified: 16 Mar 2024 20:40
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Author:
Yun Yun Pang
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