Protein core-dependent glycosaminoglycan modification and glycosaminoglycan-dependent polarized sorting in epithelial Madin-Darby canine kidney cells
Protein core-dependent glycosaminoglycan modification and glycosaminoglycan-dependent polarized sorting in epithelial Madin-Darby canine kidney cells
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DOI：
10.1093/glycob/cwq180
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发表时间：
2011-04-01
期刊：
影响因子：
4.3
通讯作者：
Tveit, H.
中科院分区：
文献类型：
--
作者：
Hafte, T. T.;Fagereng, G. L.;Tveit, H.
与绿色荧光蛋白 (GFP) 融合的蛋白聚糖丝甘氨酸 (SG) 主要从极化上皮 Madin-Darby 犬肾 (MDCK) 细胞单层的顶端表面分泌，但分泌的小部分基底外侧携带更强烈的硫酸化糖胺聚糖 (GAG) 链 (Tveit H、Dick G、Skibeli V、Prydz K. 2005。 蛋白聚糖在到达 Madin-Darby 犬肾细胞的顶端和基底外侧表面的过程中经历不同的修饰。 J Biol Chem 280：29596-29603）。为了研究 SG 中具有 GAG 附着位点的结构域是否足以驱动顶端蛋白质分选和 (ii) 独立产生在顶端和基底外侧途径中观察到的硫酸化差异，将 SG 的 GAG 结构域融合到大鼠生长激素 (rGH) 和 GFP 的连接处，并在 MDCK 细胞中表达，rGH 部分有或没有两个 N-糖基化位点。两种变体都获得了硫酸软骨素 GAG 链，并主要分泌到顶端培养基，其程度与仅具有两个 N-糖基化位点的 rGH-GFP 相同，并且与缺乏糖基化位点的未分选变体不同。将 GAG 附着结构域从 SG 转移到新的 rGH 环境消除了在顶端和基底外侧分泌途径中观察到的 SG 硫酸化强度和位置的差异。因此，这些差异是由 GAG 附着域之外的元件编码的。
The proteoglycan serglycin (SG) fused to green fluorescent protein (GFP) is secreted predominantly from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cell monolayers, but the minor fraction secreted basolaterally carries more intensely sulfated glycosaminoglycan (GAG) chains (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem 280: 29596-29603). To investigate whether the domain with GAG attachment sites in SG (i) is sufficient to drive apical protein sorting and (ii) independently generates the sulfation differences observed in the apical and basolateral pathways, the GAG domain of SG was fused into the junction of rat growth hormone (rGH) and GFP and expressed in MDCK cells, either with or without two N-glycosylation sites in the rGH part. Both variants acquired chondroitin sulfate GAG chains and were secreted predominantly to the apical medium, to the same extent as rGH-GFP with two N-glycosylation sites only, and different from the nonsorted variant lacking glycosylation sites. Transfer of the GAG attachment domain from SG to the new rGH context abolished the differences in sulfation intensity and positions observed for SG in the apical and basolateral secretory routes. Thus, these differences are coded by elements outside the GAG attachment domain.