Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters.
Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study I have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake, and that each tetrameric, holoenzyme molecule has sufficient carbohydrate for an average of 10 Man8GlcNAc2 oligosaccharides. None of the oligosaccharides or glycopeptides from the lysosomal enzymes bind to an immobilized PMR, but those with 2 PDE show slight interaction. Competition of 125I-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Furthermore, the uptake of a lysosomal enzyme isolated from a mutant strain (modA), which produces oligosaccharides with only 1 but not 2 PDE, is about 10-fold less than the uptake of wild-type enzyme which has predominantly 2 PDE. Complete denaturation of 125I-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes. The conformation of the protein may be important in orienting the oligosaccharides in a favorable position for binding to the PMR.
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D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D004023:Dictyostelium / Q000201:enzymology*; D005347:Fibroblasts / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000032:analysis / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000032:analysis
D002241:Carbohydrates; D006020:Glycopeptides; D007457:Iodine Radioisotopes; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C034304:cation-dependent mannose-6-phosphate receptor; D006596:Hexosaminidases; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase