Roff C F, Wozniak R W, Blenis J, Wang J L
Exp Cell Res. 1983 Apr 1;144(2):333-44. doi: 10.1016/0014-4827(83)90412-3.
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.
人成纤维细胞（SL66）在含有35SO4(2-)的培养基中培养，以标记糖胺聚糖（GAGs）。洗涤后，将标记的细胞在有或没有甘露糖6-磷酸（M6P）的情况下进行追踪，并根据三个任意部分对GAGs进行分析：1. 细胞外（可溶性培养基），在没有M6P的培养物中35S放射性高于有M6P的培养物。2. 细胞周（细胞表面相关），在没有M6P的培养物中35S放射性低于有M6P的培养物。3. 细胞内（完整细胞内的残留物），两组培养物之间35S放射性没有差异。此外，当用链霉蛋白酶和软骨素ABC裂解酶消化来自有和没有M6P的培养物的相应细胞区室的35S标记的GAGs，然后通过琼脂糖CL-6B柱层析进行比较时，在细胞外和细胞周部分都观察到明显的分子差异。几条证据表明，在我们的检测系统中，M6P对35S标记的GAGs周转的影响反映了细胞表面溶酶体酶活性的破坏。例如，当用缺乏携带M6P标记的酶的I细胞进行实验时，在有或没有M6P的培养物中没有观察到差异。然而，将源自正常人成纤维细胞的溶酶体酶添加到35SO4标记的I细胞中，导致细胞周GAGs的周转，并且这种作用被M6P抑制。这些结果表明，识别溶酶体酶M6P部分的细胞表面受体的一种可能功能是将某些这些酶锚定在细胞表面与其底物相邻的位置。
