Activator Proteins for Glycosphingolipid Hydrolysis by Endoglycoceramidases

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, 11, and 111) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264,9510-9619). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. ”777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator I1 in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and PI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase 11 in the absence of detergents in a concentration-dependent manner. Interestingly, activator I1 stimulated the activity of EGCase I1 much more than that of EGCase I on using asialo-GMl as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase 11. Besides activator 11, strain “777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator 11. Following the addition of activator 11, EGCase I1 hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator I1 in place of Triton X100 for stimulating EGCase I1 activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.