Gonzalez-Noriega A, Grubb J H, Talkad V, Sly W S
J Cell Biol. 1980 Jun;85(3):839-52. doi: 10.1083/jcb.85.3.839.
Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta-glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.
成纤维细胞对酸性水解酶的吸附性胞饮作用取决于酶上的磷酸甘露糖识别标记以及细胞表面的高亲和力胞饮受体。在本研究中，发现附着的成纤维细胞表面与β-葡萄糖醛酸酶的结合具有饱和性，且可被6-磷酸甘露糖（Man-6-P）抑制。在中性pH条件下，细胞结合的β-葡萄糖醛酸酶解离非常缓慢，但通过将pH降低至6.0以下或暴露于Man-6-P可大大加速解离。最大细胞表面结合量与观察到的酶胞饮速率的比较表明，胞饮受体大约每5分钟被替换或重新利用一次。酶胞饮作用在数小时内不受新蛋白质合成抑制的影响，这表明存在大量的细胞内受体和/或内化受体的重新利用。用氯喹处理正常人成纤维细胞有三种作用：（a）大大增强了带有摄取识别标记的新合成酸性水解酶的分泌，（b）使细胞表面的酶结合位点减少，（c）抑制外源酶的胞饮作用。在I型细胞病成纤维细胞中只观察到了第三种作用，并且这些细胞对此作用的敏感性也低于对照细胞。这些观察结果与酸性水解酶的运输模型一致，该模型提出，将新合成的酸性水解酶输送到溶酶体需要酶上有磷酸甘露糖识别标记，以及细胞内受体将受体结合的酶分隔到小泡中以便运输到溶酶体。该模型解释了氯喹如何通过提高溶酶体内pH值来破坏新合成酸性水解酶的细胞内途径以及细胞表面胞饮受体摄取外源酶的途径。
