SHEDDING OF THE LYMPHOCYTE L-SELECTIN ADHESION MOLECULE IS INHIBITED BY A HYDROXAMIC ACID-BASED PROTEASE INHIBITOR - IDENTIFICATION WITH ANL-SELECTIN-ALKALINE PHOSPHATASE REPORTER

Expression of the L-selectin adhesion molecule can be rapidly down-mod ulated by regulated proteolysis at a membrane-proximal site, The L-sel ectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors, We have deve loped an L-selectin-alkaline phosphatase reporter, consisting of the e ctodomain of human placental alkaline phosphatase fused to the membran e-proximal cleavage, transmembrane, and cytoplasmic domains of L-selec tin, to aid in the screening for L-selectin secretase inhibitors, A hy droxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited r elease of the L-selectin-alkaline phosphatase reporter in a dose-depen dent manner, The hydroxamic acid-based peptide was also found to inhib it wild type L-selectin down-regulation from the surfaces of phorbol m yristate acetate-activated peripheral blood lymphocytes and phytohemag glutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selecti n proteolysis. Lymphocyte L-selectin was not down-regulated when co-cu ltured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane- bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, a lthough L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane -bound TNF-alpha.