Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2.
The highly conserved polypeptide 7B2 and the subtilisin-related prohormone convertases PC1/PC3 and PC2 are broadly distributed in neurons and endocrine cells and are localized to secretory granules. We recently showed that recombinant 7B2 is in vitro a potent inhibitor of PC2 activity, but not of PC1/PC3, and that newly synthesized 7B2 is transiently associated with proPC2 in vivo. In the present study, in vitro mutagenesis was used to identify the region within the 7B2 sequence responsible for the inhibition of PC2. Mutant proteins were produced in a prokaryotic expression system and their effects on PC1/PC3 and PC2 activities were studied by two different in vitro enzyme assays. None of the 7B2 mutant proteins inhibited PC1/PC3 activity. Truncation studies revealed that a short segment within the COOH-terminal portion of 7B2 is critical for its inhibitory effect on PC2. This segment contains a pair of basic amino acid residues which may represent a recognition motif for PC2. Single amino acid substitutions within this Lys171-Lys172 site strongly diminished and a double mutation abolished the inhibitory potency of 7B2. Our results indicate that, although amino acid residues directly surrounding this dibasic pair also contribute to PC2 inhibition, the Lys171-Lys172 site is particularly important for the ability of 7B2 to inhibit PC2.
van Horssen
A M
AM
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands.
van den Hurk
W H
WH
Bailyes
E M
EM
Hutton
J C
JC
Martens
G J
GJ
Lindberg
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051859:Neuroendocrine Secretory Protein 7B2; D009490:Neurosecretory Systems / Q000378:metabolism*; D010907:Pituitary Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045706:Proprotein Convertase 2; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D017931:DNA Primers; D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones; D011994:Recombinant Proteins; C497678:SCG5 protein, human; D013381:Subtilisins; D045706:Proprotein Convertase 2