Role of loop structures of neuropsin in the activity of serine protease and regulated secretion.
Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999) J. Biol. Chem. 274, 4220-4224). Little, however, is known about the roles of these loops. Thus, the present study investigated whether surface loop structures of neuropsin were essential for the generation of enzymatic activity and/or secretion of the enzyme via a regulated secretory pathway. The loops include those stabilized by six disulfide bonds or a loop C (Gly(69)-Glu(80)) and an N-glycosylated kallikrein loop (His(91)-Ile(103)) not containing a site linked by a disulfide bond. First, among the six disulfide bonds, only SS1 in loop E (Gly(142)-Leu(155)) and SS6 in loop G (Ser(185)-Gly(197)) were necessary for the catalytic efficiency of neuropsin. Second, disruptions of loop C and the N-linked oligosaccharide chain on the kallikrein loop affected the catalytic efficiency and P2 specificity, respectively. Alternatively, disruptions of loop C and the kallikrein loop enhanced the regulated secretion, whereas there was no one disruption that inhibited the secretion, indicating that there was no critical loop required for the regulated secretion among loops surrounding the substrate-binding site.
Oka
Takuya
T
Division of Structural Cell Biology and Division of Structural Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, Japan.
Hakoshima
Toshio
T
Itakura
Makoto
M
Yamamori
Saori
S
Takahashi
Masami
M
Hashimoto
Yasuhide
Y
Shiosaka
Sadao
S
Kato
Keiko
K
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D006031:Glycosylation; D007610:Kallikreins; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; C094799:KLK8 protein, human; D007610:Kallikreins; D012697:Serine Endopeptidases