Mammalian subtilisin kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization

Using reverse transcriptase-PCR and degenerate oligonucleotides derived fro m the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, ra t, and mouse type I membrane-bound proteinase called subtilisin/kexin isozy me-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstr ates that SKI-1 mRNA is present in most tissues and cells. Cleavage specifi city studies show that SKI-1 generates a 28-kDa product from the 32-kDa bra in-derived neurotrophic factor pre cursor, cleaving at an RGLT down arrow S L bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI- 1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) dif fering by the nature of their N-glycosylation. Late along the secretory pat hway some of the membrane bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows t hat SKI-1 is present in the Golgi apparatus and within small punctate struc tures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca 2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.