Secreted site-1 protease cleaves peptides corresponding to luminal loop ofsterol regulatory element-binding proteins

We describe a permanent line of Chinese hamster ovary cells transfected wit h a cDNA encoding a truncated form of Site-1 protease (S1P) that is secrete d into the culture medium in an enzymatically active form. S1P, a subtilisi n-like protease, normally cleaves the luminal loop of sterol regulatory ele ment-binding proteins (SREBPs). This cleavage initiates the two-step proteo lytic process by which the NH2-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate trans cription of genes involved in the biosynthesis and uptake of cholesterol an d fatty acids. Truncated S1P (amino acids 1-983), produced by the transfect ed Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Li ke native S1P, this truncated protein undergoes normal autocatalytic proces sing after residue 137 to release an NH2-terminal propeptide, thereby gener ating an active form, designated S1P-B. Prior to secretion, truncated S1P-B , like native S1P-B, is cleaved further after residue 186 to generate S1P-C , which is the only form that appears in the culture medium. The secreted e nzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates i n a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresp onds to the internal propeptide cleavage site that generates S1P-B as descr ibed in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secrete d enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiolog ic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leu cine when the RSVL sequence is contained within a 16-residue peptide corres ponding to the central portion of the SREBP-2 luminal loop. The catalytic a ctivity of S1P(983)-C differs from that of furin/prohormone convertases, tw o related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after ly sine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby function s to control lipid biosynthesis and uptake in animal cells.