C - Chemistry – Metallurgy – 12 – P
Patent
C - Chemistry, Metallurgy
12
P
C12P 21/06 (2006.01) C07K 14/48 (2006.01) C12N 9/64 (2006.01)
Patent
CA 2249648
Using RT-PCR and degenerate oligonucleotides derived from the active site residues of subtilisin-kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat and mouse type-I membrane-bound proteinase called subtilisin-kexin-isozyme-1 (SKI-1). Computer data bank searches reveals that human SKI-1 was previously cloned but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is widely distributed, e.g., in thymus, in adrenal, submaxillary and pituitary glands, in skin, teeth, ribs, intestines, cortex of the kidney and brain, cerebellum, retina, and hippocampus. Cleavage specificity studies show that SKI-1 generates a 28 kDa product from the 32 kDa brain-derived neurotrophic factor precursor (proBDNF) cleaving at an RGLT~SL bond. In HK293 cells, proSKI-1 processed in two steps at the level of the endoplasmic reticulum and a large fraction of the membrane-bound enzyme is released into the medium. Immunocytochemical analysis shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of proBDNF.
Chretien Michel
Marcinkiewicz Mieczyslaw
Seidah Nabil G.
Goudreau Gage Dubuc
Institut de Recherches Cliniques de Montreal/ircm
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