N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Glycobiology, Nov 2011

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.

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N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Anders Moen 0 Tilahun T Hafte 0 Heidi Tveit 0 Wolfgang Egge-Jacobsen 0 Kristian Prydz 0 0 Department of Molecular Biosciences, University of Oslo , Box 1041, Blindern, 0316 Oslo , Norway The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: - 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. 280:2959629603) of epithelial MadinDarby 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 1To whom correspondence should be addressed: Tel: +47-22854611; Fax: +47-22854443; e-mail: (W.E.-J.); Tel: +47-22856753; Fax: +47-22854443; e-mail: (K.P.) 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. Introduction The biological functions of glycoproteins are strongly influenced by their glycans, but the details of the carbohydrate structure is difficult to elaborate, since post-translationally added glycans cannot be deduced directly from genomic information, may be branched and are variable with tissue and developmental stage, even for the same protein core. The immature N-glycan structures are important to quality-control procedures for glycoprotein folding in the endoplasmic reticulum (Ellgaard and Helenius 2003; Anelli and Sitia 2008). The mature structure of N-glycans eventually obtained during glycoprotein passage through the Golgi apparatus can influence the residence time at the cell surface and the signaling potential of trans-membrane receptors (Partridge et al. 2004; Lau and Dennis 2008). Although less abundant than N-glycans, O-glycans of the mucin and glycosaminoglycan (GAG) types are important contributors to signaling processes and are in some instances essential to development and growth (Singh and Hollingsworth 2006; Schaefer and Schaefer 2010). N-Linked and O-linked glycans have been shown to contribute to polarized sorting of glycoproteins to the apical surface of MadinDarby canine kidney (MDCK) epithelial cells (Scheiffele et al. 1995; Yeaman et al. 1997; Gut et al. 1998; Benting et al. 1999; Kolset et al. 1999), but not all glycan modification sites have shown the same potential in this respect (Su et al. 1999), even when localized to the same protein (Kitagawa et al. 1994; Potter et al. 2004). An important question is whether particular details of the glycan structures mediate apical sorting or whether the glycan structures produced merely result from the glycosylation regimes encountered in the secretory pathway. In the classical view, apical and basolateral proteins pass through the same Golgi environment, to the trans-Golgi network, the Golgi terminus at the trans side, before segregation for exit in direction of the apical or basolateral surfaces, or toward endosomes. According to this model, apical and basolateral proteins are subjected to the same processing machinery prior to their disjunction and departure from the trans-Golgi network. During the last few years, however, this view has been challenged (Alfalah et al. 2005; Tveit et al. 2005; Vuong et al. 2006; Prydz et al. 2008) by indications of segregation early in the secretory pathway of apically and basolaterally destined proteins in epithelial MDCK cells. In Drosophila imaginal disk cells, where individual Golgi (...truncated)


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Anders Moen, Tilahun T Hafte, Heidi Tveit, Wolfgang Egge-Jacobsen, Kristian Prydz. N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain, Glycobiology, 2011, pp. 1416-1425, 21/11, DOI: 10.1093/glycob/cwr069