The role of a cathepsin D-like activity in the release of Galβ1-4GlcNAc α2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response

Abstract

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose: N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.