Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein.
The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral 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 more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of 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 makeup the secreted forms of the protein in normal human fibroblasts.
Rigat
B
B
Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Wang
W
W
Leung
A
A
Mahuran
D J
DJ
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology; D008360:Mannosephosphates / Q000494:pharmacology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D049289:G(M2) Activator Protein; D008360:Mannosephosphates; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000643:Ammonium Chloride; D005678:G(M2) Ganglioside; C027693:mannose-6-phosphate; D001619:beta-N-Acetylhexosaminidases