Department of Molecular Biosciences, University of Oslo, Blindern, Oslo, Norway.
Glycobiology. 2011 Nov;21(11):1416-25. doi: 10.1093/glycob/cwr069. Epub 2011 Jun 14.
Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (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) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.
不同类型的聚糖被认为是上皮细胞分泌途径中顶端蛋白分拣的介质，但最近的研究表明，分拣到顶端和基底外侧表面可能发生在聚糖合成完成之前。我们之前已经表明，一种蛋白聚糖 (PG) 核心蛋白可以在顶泌和基底外侧分泌途径中获得不同的糖胺聚糖 (GAG) 结构 (Tveit H、Dick G、Skibeli V、Prydz K. 2005. 一种蛋白聚糖在向 Madin-Darby 犬肾 (MDCK) 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)。现在，我们还确定了同一顶泌和基底外侧分泌途径中单个糖蛋白获得的详细 N-聚糖结构。为此，将具有两个 N-聚糖位点 (rGH-2N) 的大鼠生长激素 (rGH) 插入 rGH 部分 (NAS 和 NFT) 融合到绿色荧光蛋白 (GFP) 中，并在 MDCK 细胞中表达。通过质谱法分析免疫分离的 rGH 变体的位点占有率和 N-聚糖结构。NAS 和 NFT 位点占有率的程度不同，但在顶泌和基底外侧分泌的 rGH-2N 中是可比的。附着在每个 N-聚糖位点的聚糖存在微观异质性，但在顶泌和基底外侧途径中没有观察到主要差异。将 PG 糖胺聚糖结合蛋白聚糖的 GAG 修饰域转移到 rGH-2N 和 GFP 的融合位点允许 GAG 链聚合到新的蛋白变体上，并影响 N-聚糖的微观异质性向更酸性的聚糖，但没有改变相对位点占有率。总之，在 MDCK 上皮细胞的顶泌和基底外侧分泌途径中，表达模型蛋白获得的 N-聚糖结构没有观察到主要差异，但插入 GAG 附着域会使 N-聚糖向更酸性的结构转移。
