Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).
The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.
Milla
M E
ME
Department of Biochemistry, Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. mmilla@mail.med.upen.edu
Leesnitzer
M A
MA
Moss
M L
ML
Clay
W C
WC
Carter
H L
HL
Miller
A B
AB
Su
J L
JL
Lambert
M H
MH
Willard
D H
DH
Sheeley
D M
DM
Kost
T A
TA
Burkhart
W
W
Moyer
M
M
Blackburn
R K
RK
Pahel
G L
GL
Mitchell
J L
JL
Hoffman
C R
CR
Becherer
J D
JD
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human