The effect of mannose6-phosphate on the turnover of cell surface glycosaminoglycans.
Human fibroblasts (SL66) were cultured in medium containing 35SO4(2-) to label the glycosaminoglycans (GAGs). After washing, the labeled cells were chased in the presence or absence of mannose6-phosphate (M6P) and the GAGs were analyzed in terms of three arbitrary fractions: 1, Extracellular (soluble medium), 35S radioactivity higher in cultures without M6P than in cultures with M6P. 2, Pericellular (cell surface-associated), 35S radioactivity lower in cultures without M6P than in cultures with M6P. 3, Intracellular (residue within the intact cell), no difference in 35S radioactivity between the two sets of cultures. In addition, when the 35S-labeled GAGs from corresponding cellular compartments derived from cultures with and without M6P were digested with pronase and chondroitin ABC lyase, and then compared by chromatography on Sepharose CL-6B, distinct molecular differences in both the extracellular and pericellular fractions were observed. Several lines of evidence indicate that the effect of M6P on the turnover of 35S-labeled GAGs in our assay system reflects disruption of cell surface lysosomal enzyme activity. For example, when the experiment was performed with I cells, which lack enzymes carrying the M6P marker, no difference was seen in cultures with or without M6P. The addition of lysosomal enzymes derived from normal human fibroblasts to 35SO4-labeled I cells, however, resulted in the turnover of pericellular GAGs and this effect was inhibited by M6P. These results suggest that one possible function of cell surface receptors recognizing the M6P moiety of lysosomal enzymes is to anchor certain of these enzymes proximate to their substrates at the cell surface.
D000643:Ammonium Chloride / Q000494:pharmacology; D002451:Cell Compartmentation; D002452:Cell Count; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts; D006025:Glycosaminoglycans / Q000378:metabolism*; D006600:Hexosephosphates / Q000494:pharmacology*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008360:Mannosephosphates / Q000494:pharmacology*
D006025:Glycosaminoglycans; D006600:Hexosephosphates; D008360:Mannosephosphates; D000643:Ammonium Chloride; C027693:mannose-6-phosphate; D002809:Chondroitin Sulfates; D006867:Hydrolases