A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4.
SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.
Tsuji
Akihiko
A
Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjima, Tokushima, 770-8506, Japan. tsuji@bio.tokushima-u.ac.jp
Kikuchi
Yayoi
Y
Sato
Yukimi
Y
Koide
Shizuyo
S
Yuasa
Keizo
K
Nagahama
Masami
M
Matsuda
Yoshiko
Y
D000236:Adenoma / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D045683:Furin / Q000037:antagonists & inhibitors; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000473:pathology; D045702:Proprotein Convertase 5 / Q000037:antagonists & inhibitors; D043484:Proprotein Convertases; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; D011498:Protein Precursors; C510564:RCN3 protein, human; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; C080654:PCSK6 protein, human; C513087:Pcsk6 protein, rat; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D045683:Furin