N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain
Anders Moen
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Tilahun T Hafte
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Heidi Tveit
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Wolfgang Egge-Jacobsen
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Kristian Prydz
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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:
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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)