Xiaojin Li1, Liliana Pérez, Huizhou Fan. 1. Xiaojin Li, Liliana Pérez, Huizhou Fan, Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, United States.
Abstract
AIM: To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding. METHODS: Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS: Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE. CONCLUSION: The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.
AIM: To determine if the cytotail of the principal sheddase tumornecrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding. METHODS: Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS: Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600YTACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600YTACE. CONCLUSION: The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.
Authors: M E Milla; M A Leesnitzer; M L Moss; W C Clay; H L Carter; A B Miller; J L Su; M H Lambert; D H Willard; D M Sheeley; T A Kost; W Burkhart; M Moyer; R K Blackburn; G L Pahel; J L Mitchell; C R Hoffman; J D Becherer Journal: J Biol Chem Date: 1999-10-22 Impact factor: 5.157
Authors: Peter W Janes; Nayanendu Saha; William A Barton; Momchil V Kolev; Sabine H Wimmer-Kleikamp; Eva Nievergall; Carl P Blobel; Juha-Pekka Himanen; Martin Lackmann; Dimitar B Nikolov Journal: Cell Date: 2005-10-21 Impact factor: 41.582
Authors: M L Moss; S L Jin; M E Milla; D M Bickett; W Burkhart; H L Carter; W J Chen; W C Clay; J R Didsbury; D Hassler; C R Hoffman; T A Kost; M H Lambert; M A Leesnitzer; P McCauley; G McGeehan; J Mitchell; M Moyer; G Pahel; W Rocque; L K Overton; F Schoenen; T Seaton; J L Su; J D Becherer Journal: Nature Date: 1997-02-20 Impact factor: 49.962
Authors: P Reddy; J L Slack; R Davis; D P Cerretti; C J Kozlosky; R A Blanton; D Shows; J J Peschon; R A Black Journal: J Biol Chem Date: 2000-05-12 Impact factor: 5.157
Authors: Patricia E Gonzales; Ariel Solomon; Ann B Miller; M Anthony Leesnitzer; Irit Sagi; Marcos E Milla Journal: J Biol Chem Date: 2004-04-20 Impact factor: 5.157
Authors: A Amour; P M Slocombe; A Webster; M Butler; C G Knight; B J Smith; P E Stephens; C Shelley; M Hutton; V Knäuper; A J Docherty; G Murphy Journal: FEBS Lett Date: 1998-09-11 Impact factor: 4.124