2 MECHANISMS FOR THE RECAPTURE OF EXTRACELLULAR GM(2) ACTIVATOR PROTEIN - EVIDENCE FOR A MAJOR SECRETORY FORM OF THE PROTEIN

The G(M2) activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo func tion is to act as a substrate specific cofactor for the hydrolysis of G(M2) ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at ne utral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another mo re recent report demonstrated that deficient human cells could recaptu re nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mann ose-6-phosphate pathway is likely the major intracellular, biosyntheti c route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Ad ditionally, we show that there exists a second lower affinity recaptur e pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphi ngolipids in the plasma membrane. Finally, we document that the pool o f newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules make up the secreted forms of the protein in normal human fibroblasts.