Mertens G, Van der Schueren B, van den Berghe H, David G
Center for Human Genetics, University of Leuven, Belgium.
J Cell Biol. 1996 Feb;132(3):487-97. doi: 10.1083/jcb.132.3.487.
Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.
组织腔室中发生的几种过程受到类肝素多糖的调节。为了鉴定负责极化细胞顶端硫酸乙酰肝素表达的蛋白质，我们研究了CaCo-2细胞中细胞表面硫酸乙酰肝素蛋白聚糖表达的极性。对这些细胞顶端和基底外侧膜进行结构域特异性生物素化，确定了糖基磷脂酰肌醇连接的硫酸乙酰肝素蛋白聚糖磷脂酰肌醇蛋白聚糖是顶端硫酸乙酰肝素的主要来源。然而，这种蛋白聚糖大部分在基底外侧表面表达，这对于一种糖基化蛋白来说是一个意外发现。代谢标记和追踪实验表明，分选机制而非不同的周转率导致了磷脂酰肌醇蛋白聚糖的这种双极表达。氯酸盐处理不影响CaCo-2细胞中磷脂酰肌醇蛋白聚糖表达的极性，转染的MDCK细胞也以基本非极化的方式表达野生型磷脂酰肌醇蛋白聚糖和syndecan-4/磷脂酰肌醇蛋白聚糖嵌合体。然而，从磷脂酰肌醇蛋白聚糖核心蛋白上完全去除硫酸乙酰肝素糖基化位点，导致转染细胞中磷脂酰肌醇蛋白聚糖几乎完全靶向顶端，而两链和单链突变形式具有中间分布。这些结果表明，磷脂酰肌醇蛋白聚糖是顶端硫酸乙酰肝素表达的原因，但核心蛋白的糖基化会拮抗这种蛋白聚糖的糖基磷脂酰肌醇锚所传递的顶端分选信号的活性。这些发现的一个可能含义是，硫酸乙酰肝素糖基化可能是磷脂酰肌醇蛋白聚糖亚细胞表达的一个决定因素。或者，磷脂酰肌醇蛋白聚糖中糖基化与顶端分选的反向关系可能确保硫酸乙酰肝素持续不断地输送到顶端区室，或者硫酸乙酰肝素通过“主动”的糖基磷脂酰肌醇介导进入顶端膜区室可能需要克服硫酸乙酰肝素链的这种拮抗作用。
