Edit Szepessy1, Miklós Sahin-Tóth. 1. Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University, Boston, Mass., USA.
Abstract
BACKGROUND: The archetypal mammalian elastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene. AIMS: In this study, we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1. METHODS: Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovine milk protein. RESULTS: Surprisingly, recombinant ELA2B was completely devoid of proteolytic activity, while ELA2A readily hydrolyzed all three test substrates. Furthermore, ELA2A formed an SDS-resistant complex with alpha1-antitrypsin, whereas ELA2B did not bind covalently to the inhibitor. Finally, chimeras and point mutations engineered between ELA2A and ELA2B revealed that multiple evolutionary mutations inactivated ELA2B. CONCLUSIONS: The results indicate that ELA2B is not an elastase enzyme and confirm that ELA2A is the major elastase in the human pancreas. 2006 S. Karger AG, Basel and IAP
BACKGROUND: The archetypal mammalianelastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene. AIMS: In this study, we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1. METHODS: Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovinemilk protein. RESULTS: Surprisingly, recombinant ELA2B was completely devoid of proteolytic activity, while ELA2A readily hydrolyzed all three test substrates. Furthermore, ELA2A formed an SDS-resistant complex with alpha1-antitrypsin, whereas ELA2B did not bind covalently to the inhibitor. Finally, chimeras and point mutations engineered between ELA2A and ELA2B revealed that multiple evolutionary mutations inactivated ELA2B. CONCLUSIONS: The results indicate that ELA2B is not an elastase enzyme and confirm that ELA2A is the major elastase in the human pancreas. 2006 S. Karger AG, Basel and IAP
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