A novel human PACE4 isoform, PACE4E is an active processing protease containing a hydrophobic cluster at the carboxy terminus.
PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.
Mori
K
K
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minamijosanjima.
Kii
S
S
Tsuji
A
A
Nagahama
M
M
Imamaki
A
A
Hayashi
K
K
Akamatsu
T
T
Nagamune
H
H
Matsuda
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D043484:Proprotein Convertases; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D007527:Isoenzymes; D011498:Protein Precursors; C080654:PCSK6 protein, human; D043484:Proprotein Convertases; D012697:Serine Endopeptidases