Robledo Y, Marigómez I, Angulo E, Cajaraville M P
Department of Zoology & Animal Cell Biology, School of Science & Technology, University of the Basque Country, P.O. BOX 644, Bilbo, Basque Country, Spain.
Cell Tissue Res. 2006 May;324(2):319-33. doi: 10.1007/s00441-005-0125-9. Epub 2006 Feb 1.
Our aim was to contribute to the understanding of the synthesis, maturation and activation of lysosomal enzymes in an invertebrate cellular model: the endo-lysosomal system (ELS) of mussel digestive cells. The activities of 5'-nucleotidase (AMPase), arylsulphatase (ASase) and acid phosphatase (AcPase), which are transported towards acidic compartments as membrane proteins, were localised by enzyme cytochemistry. AcPase activity was found within large heterolysosomes and residual bodies. ASase was located in endosomes, endolysosomes and heterolysosomes. AcPase and ASase activities were recorded within small vesicles and cisterns of the trans-Golgi network. Conversely, AMPase activity was primarily found in microvilli and apical vesicles and, less conspicuously, in lysosomes and the cis-side of the Golgi and the cis-Golgi network (CGN). In order to understand the processes of synthesis and maturation of these lysosomal enzymes, selected glycoconjugates were localised after lectin cytochemistry. N-acetylglucosamine, mannose and fucose residues were almost ubiquitous in the ELS, as were galactose residues, which were apparently less abundant. N-acetylglucosamine residues occurred in the inner membrane co-localised with mannose residues within the lysosomal and pre-lysosomal acidic compartments. Based on these results, glycosylation and sorting pathways are proposed for both soluble and membrane enzymes. Unlike in mammalian cells, O-glycosylation is fully completed in the CGN, mannose addition in N-glycosylation extends beyond the CGN and galactose addition is fully achieved at the intermediate side. Sorting of soluble lysosomal enzymes, as in crustaceans, is mediated by the indirect transport of membrane-linked proteins with GlcNAc1-P6Man residues that are removed in endolysosomes and heterolysosomes.
我们的目标是通过一种无脊椎动物细胞模型——贻贝消化细胞的内溶酶体系统（ELS），来促进对溶酶体酶的合成、成熟和激活过程的理解。作为膜蛋白被转运至酸性区室的5'-核苷酸酶（AMPase）、芳基硫酸酯酶（ASase）和酸性磷酸酶（AcPase）的活性，通过酶细胞化学方法进行了定位。AcPase活性在大型异溶酶体和残余小体中被发现。ASase位于内体、内溶酶体和异溶酶体中。AcPase和ASase活性在反式高尔基体网络的小泡和扁平囊泡中被记录到。相反，AMPase活性主要在微绒毛和顶端小泡中被发现，在溶酶体以及高尔基体顺面和顺式高尔基体网络（CGN）中则不太明显。为了理解这些溶酶体酶的合成和成熟过程，在凝集素细胞化学之后对选定的糖缀合物进行了定位。N-乙酰葡糖胺、甘露糖和岩藻糖残基在ELS中几乎无处不在，半乳糖残基也是如此，不过其含量明显较少。N-乙酰葡糖胺残基出现在溶酶体和前溶酶体酸性区室内与甘露糖残基共定位的内膜中。基于这些结果，提出了可溶性酶和膜酶的糖基化及分选途径。与哺乳动物细胞不同，O-糖基化在CGN中完全完成，N-糖基化中的甘露糖添加超出了CGN，半乳糖添加在中间面完全实现。与甲壳类动物一样，可溶性溶酶体酶的分选是由带有GlcNAc1-P6Man残基的膜连接蛋白的间接转运介导的，这些残基在内溶酶体和异溶酶体中被去除。
