Am J Physiol Renal Physiol 296: F459 –F469, 2009.
First published October 29, 2008; doi:10.1152/ajprenal.90340.2008.
Review
Role of N-glycosylation in trafficking of apical membrane proteins
in epithelia
Olga Vagin,1,2 Jeffrey A. Kraut,2,3,4 and George Sachs1,2
1
Department of Physiology, David Geffen School of Medicine at University of California; 2Membrane Biology Laboratory,
University of California; 3Division of Nephrology, Veterans Administration Greater Los Angeles Health Care System;
and 4David Geffen School of Medicine at University of California, Los Angeles, California
Submitted 2 June 2008; accepted in final form 27 October 2008
apical sorting; apical membrane retention; H-K-ATPase ␤-subunit; lectin
vectorial transport that requires
polarized distribution of transporters and receptors to apical or
basolateral membrane domains. These domains are separated
by tight junctions that connect neighboring cells in the epithelial monolayer and act as diffusion barriers to prevent mixing
of apical and basolateral membrane components (22). Asymmetric distribution of plasma membrane proteins is accomplished by their sorting into apical and basolateral containers in
the trans-Golgi network (TGN) and/or endosomes followed by
vectorial transport of these containers and insertion and retention of the proteins in the appropriate plasma membrane
domains. Sorting depends on recognition of apical and basolateral sorting signals within the proteins by cellular sorting
machinery (20, 58, 59, 75, 76).
Numerous studies have indicated that both O- and N-glycans
attached to the extracellular domains of some membrane proteins are important for apical location of these proteins. This
review will focus on the role of N-glycans in polarized distribution of plasma membrane proteins in epithelia. The role of
O-glycans in apical sorting has been described in several recent
excellent reviews (18, 71) and will not be discussed further
here.
A putative role of N-glycans as apical sorting signals was
postulated more than 10 years ago (24, 31). However, this
hypothesis remains controversial primarily because N-glycans
EPITHELIAL CELLS CARRY OUT
Address for reprint requests and other correspondence: O. Vagin, Dept. of
Physiology, David Geffen School of Medicine at Univ. of California, Bldg.
113, Rm. 324, 11301 Wilshire Blvd., Los Angeles, CA 90073 (e-mail: olgav
@ucla.edu).
http://www.ajprenal.org
are important for the processes that precede or follow the actual
sorting event, such as protein folding, quality control, endoplasmic reticulum (ER)-associated degradation, ER-to-Golgi
trafficking, and retention of glycoproteins in the apical membrane. Therefore, merely examining the effect of altering the
number or the nature of N-linked glycans on the relative
abundance of the glycoprotein in the apical membrane, as has
been done in many of the studies reported, does not allow one
to distinguish between effects on apical sorting and these other
processes. Additionally, the mechanisms by which sorting
information encoded by N-glycans is recognized by cellular
sorting machinery have not been defined. Two lectins have
been identified as potential sorting receptors that recognize
N-glycans as apical sorting signals (15, 17, 34, 35). However,
it is not clear how glycan-lectin interactions that must occur in
the luminal compartments of TGN/endosomes are converted
into a cascade of cytoplasmic events resulting in apical delivery of N-glycosylated proteins.
In the present review, we discuss the roles of N-glycans in
apical sorting and the processes that precede and follow this
event. Furthermore, we summarize the evidence for the importance of glycoprotein clustering in membrane microdomains
and putative roles of glycan-binding proteins in apical distribution of N-glycosylated proteins.
Normal N-Glycosylation of Membrane Proteins
All membrane glycoproteins are synthesized by ribosomes
attached to the ER and become inserted into the ER membrane
via a protein-conducting channel, the translocon. Glycoproteins acquire the N-linked glycans during the process of transF459
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Vagin O, Kraut JA, Sachs G. Role of N-glycosylation in trafficking of apical
membrane proteins in epithelia. Am J Physiol Renal Physiol 296: F459 –F469,
2009. First published October 29, 2008; doi:10.1152/ajprenal.90340.2008.—Polarized distribution of plasma membrane transporters and receptors in epithelia is
essential for vectorial functions of epithelia. This polarity is maintained by sorting
of membrane proteins into apical or basolateral transport containers in the transGolgi network and/or endosomes followed by their delivery to the appropriate
plasma membrane domains. Sorting depends on the recognition of sorting signals
in proteins by specific sorting machinery. In the present review, we summarize
experimental evidence for and against the hypothesis that N-glycans attached to the
membrane proteins can act as apical sorting signals. Furthermore, we discuss the
roles of N-glycans in the apical sorting event per se and their contribution to folding
and quality control of glycoproteins in the endoplasmic reticulum or retention of
glycoproteins in the plasma membrane. Finally, we review existing hypotheses on
the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36
and galectin-3, as putative apical sorting receptors.
Review
F460
N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
contains five mannose residues. Following the addition of
GlcNAc and further mannose trimming, GnT-II catalyzes formation of the complex-type N-glycans in which GlcNAc is
substituted for both ␣3- and ␣6-linked mannose residues. The
actions of GnTs-III, -IV, -V, and -VI underlie the variability in
the number of branches in N-glycans: GnTs-IV, -V, and -VI
promote branching of N-glycans, whereas GnT-III stops
branching. The latter enzyme adds the bisecting GlcNAc to
hybrid or complex N-glycans that prevents the downstream
action of the branching enzymes (Fig. 1 and Table 1). Many of
the other glycosyltransferases, which compete with each
other for the N-glycan substrate in the trans-Golgi, facilitate
elongation of the GlcNAc-substituted chains by adding
various monosaccharides. Thus individual branches of the
mature N-glycan can vary in both their length and carbohydrate composition. Most frequently, individual branches are
elongated by addition of galactose and sialic acid residues
catalyzed by galactosyltransferases and sialyltransferases.
Individual branches can also be elongated by addition of
multiple sialic acid residues or polylactosamine sequences
(series of repeated galactose-GlcNAc disaccharides).
The diversity of N-glycans in various glycoproteins is a
result of the interplay of several factors. The particular set of
glycosyltransferases expressed in a specific cell type is one of
the most important. This difference in expression of glycosyltransferases likely explains the finding that N-glycans of
Fig. 1. Simplified schema showing the pathways and enzymes involved in synthesis of high-mannose, hybrid, and complex N-glycans in the Golgi.
High-mannose N-glycans imported from the endoplasmic reticulum (ER) to the Golgi can remain unchanged or can undergo various transformations under the
influence of Golgi mannosidases and glycosyltransferases. Six different N-acetylglucosamine-glycosyltransferases (GnTs) present in the Golgi (I–VI) can add
N-acetylglucosamine (GlcNAc) residues to the 3-mannosyl core of N-glycans (green rectangle) and thereby produce diverse carbohydrate structures. The addition
of GlcNAc residues by GnT-I, -II, -IV, -V, and -VI, but not by GnT-III, allows elongation of the chains, referred to as “antennae.” Elongation of the chains is
accomplished by addition of monosaccharide linkages catalyzed by other various glycosyltransferases (dashed arrows). GnT-I is a critical regulatory enzyme for
formation of both hybrid and complex-type N-glycans. GnT-II acts downstream of GnT-I to catalyze the formation of the complex-type N-glycans. GnTs-IV,
-V, and -VI promote branching of N-glycans by adding GlcNAc into the positions shown by different shades of blue. Individual N-glycans can be modified by
one or more of these enzymes, resulting in N-glycans that contain from 1–5 antennae. In contrast, GnT-III (red) stops branching by adding a bisecting GlcNAc
to any of the N-glycans, thereby preventing the downstream action of GnTs-II, -IV, -V, and -VI. In the example depicted, the effect of this enzyme on the product
of GnT-I results in the formation of a 1-antennary N-glycan of the hybrid type. Branching also can be prevented by exposure of cells to inhibitors of the Golgi
mannosidase I and II, deoxymannojirimycin (dMM) and swainsonine, resulting in preservation of the high-mannose or hybrid-type structure of N-glycans,
respectively. A modified version of this figure is in Ref. 89.
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location and elongation of the polypeptide chain. First, while
the protein continues to be associated with the translocon, a
14-saccharide core is transferred from the dolichol phosphate
precursor to the N-glycosylation site of the nascent membrane
protein, an asparagine (Asn) residue within a consensus sequence of Asn-X-Thre/Ser (X is any amino acid residue except
Pro). Next, terminal glucose residues of this core are trimmed
by ER glucosidases. Then, after removal of one mannose
residue by the ER ␣-mannosidase I, the glycoproteins are
exported to the Golgi.
Some glycoproteins that are trafficked to the plasma membrane move through the Golgi and post-Golgi vesicular compartments to this destination without further processing. These
glycans are high-mannose N-glycans (Fig. 1). However, the
structure of the majority of N-glycans is altered further in the
Golgi. After removal of one, two, or three mannose residues by
Golgi mannosidases, various glycosyltransferases catalyze
branching and elongation of the carbohydrate chains, producing hybrid or complex N-glycans (Fig. 1 and Table 1) (1, 39).
This rebuilding of the N-glycans is initiated by addition of
N-acetylglucosamine (GlcNAc) residues to the N-glycan core
structure by one or more of the six different N-acetylglucosamine-glycosyltransferases (GnTs) located in the cis-Golgi
and medial Golgi.
GnT-I initiates formation of hybrid N-glycans by attachment
of GlcNAc to the ␣3 arm of the core at the stage when the core
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F461
N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
Table 1. Roles of N-glycan-specific enzymes in processing and maturation of membrane glycoproteins
Enzyme
Location
Effect
Glucosidases
ER
␣-Mannosidases I
GnT-I
GnT-I, Golgi ␣-mannosidase II and GnT-II
GnT-IV
GnT-IV, GnT-V, and GnT-VI
GnT-III
Sialyltransferase, galactosyltransferases,
and other glycosyltransferases
ER and Golgi
Medial Golgi
Medial Golgi
Medial Golgi
Medial and trans-Golgi
Medial and trans-Golgi
Medial and trans-Golgi
Removal of terminal glucose residues from oligosaccharide
core and formation of high-mannose glycoproteins
Trimming of high-mannose N-glycans
Formation of hybrid-type glycoproteins
Formation of complex-type glycoproteins
Branching of N-glycans in hybrid-type glycoproteins
Branching of N-glycans in complex-type glycoproteins
Prevention of branching of N-glycans
Elongation of branches in hybrid and complex N-glycans
ER, endoplasmic reticulum.
Role of N-Glycans in Protein Folding, Stability, and Quality
Control in the ER
Folding of proteins in the ER is initiated cotranslationally
and is completed posttranslationally. Heat shock proteins, such
as GRP78/BIP, act as chaperones facilitating the normal folding of proteins and their delivery from the ER to the Golgi.
However, N-linked glycans also play a major role in folding
and quality control of newly synthesized glycoproteins, helpAJP-Renal Physiol • VOL
ing to ensure that only properly folded proteins are delivered
from the ER to the Golgi and thence to their final destination.
N-linked glycans can contribute to proper folding both
directly and indirectly. Addition of bulky polar carbohydrate
chains can modify the properties of the polypeptide (7, 70).
Glycosylation can affect the local secondary structure of proteins by stabilizing the conformation of residues proximal to
the glycosylation site (42). An interaction of N-glycan with the
polypeptide chain can induce a ␤-turn structure (7). Also,
glycoproteins are more stable and more resistant to proteolysis
than their corresponding unglycosylated counterparts (42, 46).
For example, the normally fully glycosylated wild-type ␤-subunit of the apical transport enzyme, the gastric H-K-ATPase, is
more resistant to proteolysis by trypsin (13) or proteinase K (5)
than its glycosylation-deficient mutants. Resistance to proteolysis has been attributed to restricted access of the protease to
cleavage sites within the protein after it achieves its normal
configuration.
N-glycans also can promote folding and quality control
indirectly by serving as recognition “tags,” or sorting signals,
that allow glycoproteins to interact with a variety of lectins,
glycosidases, and glycosyltransferases (37, 39, 40, 77)
(Table 2). Immediately after coupling the oligosaccharide core
to the asparagine of the N-glycosylation consensus site, the two
outermost terminal glucose residues are removed sequentially
by glucosidase I and glucosidase II. The monoglucosylated
form of N-linked glycan binds to calnexin, the membranebound lectin, or to calreticulin, the homologous soluble lectin.
Calnexin and calreticulin are associated with ERp57, a thioldisulfide oxidoreductase that is involved in disulfide bond
formation that influences protein folding (32, 33, 40). When
glucosidase II removes the remaining glucose, the glycoprotein
dissociates from calnexin and calreticulin.
The protein now meets one of three possible fates. If properly folded, it moves to the Golgi. Exit from the ER may be
assisted by mannose-binding lectins, such as ERGIC-53,
VIP36, and VIPL (2, 62, 83). If the glycoprotein is incompletely folded, it becomes a substrate for a luminal ER glucosyltransferase that reglucosylates the glycans located in improperly folded regions. Via these reformed glycans, the glycoprotein again binds to calnexin, calreticulin, and ERp57 and
other folding factors to achieve its correctly folded state (40).
If, despite repeated cycles of deglycosylation-reglucosylation,
a glycoprotein fails to fold properly, it is retained in the ER and
eventually degraded. Targeting of misfolded proteins to the
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the Na-K-ATPase ␤1-subunit and E-cadherin have a more
complex structure in the rabbit intestinal cells than in rabbit
renal cells (89).
Moreover, the expression of various glycosyltransferases
can change during cell or tissue development, resulting in
alterations of glycosylation of a particular protein. For
example, downregulation of the enzymes GnT-IV and
GnT-V, upregulation of GnT-III, and a decrease in the
complexity of N-glycans of the Na-K-ATPase ␤1-subunit
and E-cadherin are observed as dispersed Madin-Darby
canine kidney (MDCK) cells in culture are transformed into
a confluent monolayer (89).
In addition, the presence of certain amino acid motifs or
residues in a glycoprotein can affect the nature of its glycosylation. For instance, scattered basic residues and a peptide loop
adjacent to the glycosylation site of lysosome-resident acid
hydrolases are recognized by a GlcNAc-phosphate transferase
in the Golgi. This results in the formation of a mannose
6-phosphate on the termini of high-mannose N-linked glycans,
binding to the mannose-6-phosphate receptor, and targeting to
lysosomes (30, 45).
The spatial location of N-glycosylation sites in a protein
tertiary structure can also affect the type of N-glycans formed.
Certain N-glycans are more accessible to the Golgi glycosyltransferases and are therefore more likely to be processed to the
complex N-glycans than others. As a result, particular glycosylation sites in a protein may be occupied by complex Nglycans and other sites by high-mannose or hybrid N-glycans.
As an example, the mature Eag1 potassium channel has two
N-glycans, but one of the N-glycans is complex while the other
is high-mannose (61). Also, in the bovine lysosomal ␣-mannosidase, three of the six N-glycosylation sites carry only
high-mannose N-glycans while the other three sites are occupied by various types of N-glycans (21). These studies indicate
that the nature of N-glycan processing is dependent on the
position of a particular N glycosylation site within the protein.
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N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
Table 2. Putative roles of N-glycans in folding, quality control, apical sorting, and plasma membrane retention of
glycoproteins arising from their interactions with lectins
Function
Lectin
Location
Reference(s)
Folding and quality control of glycoproteins
Targeting misfolded proteins to ERAD
Protein export from the ER to the Golgi
Sorting of glycoproteins to lysosomes
Clustering and stabilization of glycoproteins in lipid rafts
Apical sorting of glycoproteins in polarized cells
Plasma membrane retention of glycoproteins
Calnexin and calreticulin
EDEM
ERGIC53, ERGL, and VIP36
MPR
VIP36
VIP36 and Galectin-3
Unknown lectins
ER
ER
ER and ERGIC
TGN and endosomes
Cis-Golgi and medial Golgi
TGN and endosomes
Plasma membrane or extracellular surface
32, 33
57
2, 62, 83
30, 45
23
15, 17, 34, 35
N/A
ERAD, ER-associated degradation.
AJP-Renal Physiol • VOL
acts as a chaperone to ensure that the ␣-subunit is properly
folded so it can exit the ER.
Role of N-Glycans in Post-Golgi Trafficking and Retention
in the Apical Membrane
N-glycans are required for polarized apical distribution of
many membrane proteins in epithelia. Removal of N-glycans
from many apical proteins has been shown to decrease their
abundance in the apical membrane. For example, mutagenic
removal of N-glycosylation sites in the gastric H-K-ATPase
␤-subunit (90), bile salt export pump (56), and glycine transporter 2 (51, 94) significantly decreased their apical content
and increased their intracellular accumulation (Table 3). Similarly, prevention of N-glycosylation by tunicamycin resulted
in complete intracellular retention of the normally apically
located membrane-bound enteropeptidase (95) (Table 3).
One explanation for these findings is that normal N-glycosylation is required for apical sorting of these glycoproteins.
Alternatively, since N-glycosylation is necessary for proper
folding of proteins for them to exit the ER, the lack of
N-glycans could result in ER retention, which of course would
prevent trafficking to any membrane. To account for this, it is
necessary to subtract the fraction of the protein retained in the
ER from the total cellular pool of the protein. The majority of
mature glycoproteins that traverse the Golgi carry complex
N-glycans, whereas those residing in the ER are of the highmannose type. They can be distinguished from each other after
treatment with EndoH, since high-mannose and hybrid glycans
are sensitive to, whereas complex N-glycans are resistant to,
cleavage by EndoH (Fig. 2). Only the quantity of mature,
EndoH-resistant protein should be considered when the percentage of glycoprotein that is delivered to the apical membrane is calculated.
Using this approach, the abundance of glycosylation-deficient mutants and wild-type H-K-ATPase ␤-subunit present in
the apical membrane, as determined using surface-selective
biotinylation, was compared after normalization for the total
cellular amount of the mature subunit (90). The relative quantity of the subunit in the apical membrane was greatly decreased after mutation of N1, N3, N5, or N7, but not after
mutation of N2, N4, or N6. By contrast, these mutations had no
effect on the relative abundance of this subunit in the basolateral membrane (90). These findings indicate that N-glycans at
N1, N3, N5, and N7 are important for apical membrane
localization of this ␤-subunit.
Similarly, comparison of a wild-type and a series of glycosylation-deficient mutants of the sialomucin endolyn expressed
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ER-associated degradation (ERAD) is facilitated by lectin
EDEM (57). ER retention and subsequent ERAD are critical
for normal cellular function because they prevent accumulation
of dysfunctional proteins in the cell (39, 40, 77). The contribution of N-glycans to the complex ER quality control system
is emphasized by some of the functional abnormalities that
arise in the presence of defective N-glycosylation (52, 64, 65).
In some glycoproteins with multiple N-glycosylation sites,
individual N-glycans are not of equal importance in mediating
the processes of folding, ER retention, and export from the ER
to the Golgi. For example, only one of the six glycans of the
apically located protein, influenza virus hemaglutinin, is essential for folding and exit from the ER (38).
Studies of the ␤-subunit of the gastric H-K-ATPase expressed in LLC-PK1 cells have also shown that mutation of
some of its seven N-glycosylation sites affect its exit from the
ER, while mutations of others have little impact. The normally
glycosylated ␤-subunit is distributed between the plasma membrane and the intracellular compartments. At steady state,
⬃40% of the protein is detected in the ER, and the remaining
60% is distributed among the plasma membrane, Golgi, TGN,
and endosomes (90). Removal of the seventh N-glycosylation
site, N7, leads to virtually total retention of the subunit in the
ER. Mutation of N1 increases the amount retained in the ER to
75%. Similarly, mutation of N3 and N5 also significantly
increases the relative amount of the ER-resident fraction, but to
a lesser extent. However, mutation of N2, N4, or N6 does not
appreciably alter the quantity of protein retained in the ER.
On the other hand, for some glycoproteins, the number of
N-glycans rather than a specific N-glycan is important for their
normal folding and production of fully functional glycoproteins. Mutagenic removal of one or two of the four N-glycosylation sites from the apical bile salt export pump (ABCB11)
did not significantly affect its stability, trafficking, or function
(56). However, removal of three or all four N-glycosylation
sites dramatically increased the rate of proteasome-dependent
degradation and resulted in significant intracellular retention of
the protein. Comparison of mutants of ABCB11 with various
combinations of mutated glycosylation sites showed that presence of at least two N-glycans is required for normal folding
and trafficking of the protein (56).
In oligomeric proteins, N-glycans can be important for
subunit assembly and/or correct folding of the assembled
complex. For example, studies using several different cell lines
have shown that the catalytic ␣-subunits of either the Na-KATPase or the gastric H-K-ATPase are unable to exit the ER
when expressed without their corresponding glycosylated
␤-subunits (29). These observations suggest that the ␤-subunit
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N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
F463
Table 3. Effect of impairment of N-glycosylation on localization of apical membrane proteins in polarized epithelia
Apical Glycoprotein
Number of N-Glycans
Cell Type
Association
With Lipid
Rafts
Enteropeptidase
3
MDCK
No
Bile acid export pump (ABCB11)
4
MDCK
Not tested
Method Used to Impair
N-Glycosylation
Exposure to
tunicamycin
Mutagenesis of 1 or
2 sites
Mutagenesis of 3 or
4 sites
Mutagenesis of each
individual site;
mutagenesis of
2 sites
7
LLC-PK1
Not tested
Tail-anchored chimeric protein
2
MDCK
No
Mutagenesis of both
sites
Glycine transporter 2 (GLYT2)
4
MDCK
No
Mutagenesis of all
3 sites
Membrane dipeptidase (MDP)
2
MDCK, Caco-2
Yes
Sialomucin endolyn
8
MDCK
Not tested
NPP3
Not specified
MDCK, Caco-2
Yes
Chimeric Fc receptor
Not specified
MDCK
Not tested
p75(NTR)
1
MDCK
No
(SPNT)
3
MDCK
Not tested
Sodium-sulfate cotransporter (NaSi-1)
1
OK, LLC-PK1,
and MDCK
No
M2 muscarinic acetylcholine receptor
3
MDCK
No
Mutagenesis of all
4 sites
Mutagenesis of both
sites
Exposure to
tunicamycin;
mutagenesis of
1-8 sites
Exposure to
tunicamycin
Exposure to
tunicamycin
Exposure to
tunicamycin
Mutagenesis of all
3 sites
Exposure to
tunicamycin;
mutagenesis of a
single site
Mutagenesis of all
3 sites
Reference(s)
Intracellular retention*
95
No change
56
Intracellular retention*
Increased retention in ER;
increase in rate of
endocytosis; decrease
in ratio of apical to
basolateral abundance
Decrease in ratio of
apical to basolateral
abundance
Decrease in ratio of
apical to basolateral
abundance
Intracellular retention*
Decrease in ratio of
apical to basolateral
abundance
Decrease in ratio of
apical to basolateral
abundance
90
10
51, 94
67
72
Intracellular retention*
19, 53
Retention in Golgi
31
No change
93
No change
50
No change
74
No change
12
N-glycosylation was prevented by mutation of the consensus N-glycosylation site (s) of the individual protein, or by treatment of cells with tunicamycin, a
general inhibitor of N-glycosylation. OK, opposum kidney; MDCK, Madin-Darby canine kidney. *Presize intracellular compartments were not identified.
in MDCK cells has shown that only two specific sites (N68 and
N74) of the eight N-glycosylation sites are important for apical
distribution of this protein (72). Disturbance of N-glycosylation of several other membrane proteins, such as the tailanchored chimeric protein (10), glycine transporter 2 (51, 94),
membrane dipeptidase (67), ecto-nucleotide pyrophosphatase/
phosphodiesterase NPP3 (19, 53), and chimeric Fc receptor
(31) reduced their apical expression compared with their basolateral expression (Table 3). However, the relative importance of specific N-glycans in apical localization of these
proteins has not been determined.
Further evidence for the role of N-glycans in apical distribution of proteins has been derived from studies in which the
effect of addition of N-glycans to various proteins on their
localization was examined. A truncated occludin and a chimeric ERGIC-53 residing in the Golgi in their nonglycosylated
forms were redistributed to the apical membrane after addition
of N-glycans (31) (Table 4). Similarly, addition of N-glycans
to chimeric non-raft-associated transmembrane protein and
tail-anchored chimeric protein, both residing on the basolateral
AJP-Renal Physiol • VOL
membrane, caused them to be redistributed to the apical membrane (6, 10) (Table 4). Finally, the sequential addition of one
to five N-glycans to the basolaterally located Na-K-ATPase
␤1-subunit, a protein with three N-glycans, caused an increasing fraction of this subunit to be distributed to the
apical membrane of HGT-1 cells (91). The mutant of NaK-ATPase ␤1 which contained eight N-glycans in a pattern
identical to that of the homologous isoform, the Na-KATPase ␤2-subunit (which normally resides on the apical
membrane in HGT-1 cells), was predominantly detected on
the apical membrane (91).
These data are consistent with an important role for the
N-glycans in promoting distribution of a number of proteins to
the apical membrane of polarized cells. However, the presence
of N-glycans on a protein does not ensure it will be distributed
to the apical membrane. Several N-glycosylated proteins, including but not restricted to the transferrin receptor (63), liver
sodium-dependent bile acid transporter (87), and E-cadherin
(54, 55) are located only on the basolateral membrane of
polarized cells.
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H-K-ATPase ␤-subunit
Effect on Protein Distribution
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N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
β
β
β
Furthermore, several proteins such as transmembrane neurotrophin receptor p75 (93), the sodium-dependent purineselective nucleoside transporter SPNT (50), the sodium-sulfate
cotransporter Na,Si-1 (74), and M2 muscarinic acetylcholine
receptor (12) are N-glycosylated, but normal N-glycosylation
is not necessary for their apical localization. Presumably, these
proteins contain other intrinsic sorting signals that are sufficient to ensure their apical distribution. It has been shown that
the O-glycosylated stem region of p75 acts as the apical sorting
signal (93). The M2 muscarinic acetylcholine receptor contains
apical sorting information in its third intracellular loop (12).
However, the apical sorting signals for SPNT and Na,Si-1
remain to be identified. It is unclear why N-glycans are important for the apical delivery of some N-glycosylated proteins,
but not for others. This remains fertile ground for further
studies.
N-glycans are required for both apical membrane delivery
and retention of glycoproteins in the apical membrane. Many
glycoproteins require intact N-glycans to be located on the
apical membrane. One possible explanation is that N-glycans
act as apical sorting signals and thus promote apical membrane
delivery of the proteins. However, the quantity of protein on
the apical membrane depends on the rate of both its delivery to
this location and endocytosis of the protein.
As noted above, the mutations of N1, N3, N5, or N7
significantly decreased the relative apical content of the H-K
ATPase ␤-subunit (90). Mutations of N1, N3, or N5 also
increased the rate of endocytosis of the ␤-subunit. The rate of
endocytosis of the N7 mutant could not be measured due to the
very low apical content of the mutant. Mutations of N2, N4, or
N6 did not change either the relative apical content or endocytosis. None of the mutations affected the rate of degradation
(90). The decrease in the relative apical content of the H-K
ATPase ␤-subunit N1, N3, and N5 mutants could be due to
impaired apical membrane delivery and/or enhanced endocytosis. The apical content of all three mutants was decreased to
a greater extent than could be expected from an increase in the
rate of endocytosis. For example, in the N1 mutant, the apical
content was decreased sixfold, but the rate of endocytosis was
increased only threefold. This indicates that the apical membrane delivery from the TGN/endosomes was also affected by
the mutation.
Therefore, the presence of specific N-glycans is required for
both apical delivery and membrane retention of the H-KATPase ␤-subunit. The rate of delivery of a protein from the
TGN/endosomes to the apical membrane depends on the efficiency of protein sorting into apical transport containers and
the rate of delivery of these containers to the apical membrane.
Table 4. Effect of addition of N-glycans on polarized distribution of proteins in the plasma membrane of epithelial cells
Protein
Na-K-ATPase ␤1-subunit
Truncated occludin
Chimeric ERGIC-53
Chimeric non-raft-associated
transmembrane protein
Tail-anchored chimeric protein
Number of
N-Glycans Added
Cell Type
1-5*
2*
2*
2*
HGT-1
MDCK
MDCK
MDCK
2†
MDCK
Protein Location Before
Addition
Ratio of Apical to
Basolateral Abundance
Reference
Basolateral membrane
Golgi
Golgi
Mostly intracellular;
basolateral
membrane
Basolateral membrane
Increased
Increased
Increased
Increased
91
31
31
6
Increased
10
*N-glycosylation sites in the protein extracellular domains were engineered by mutagenesis. †Twenty-one-amino acid sequence of bovine opsin containing
2 N-glycosylation sites was added to the COOH terminus of the extracellular domain of a tail-anchored protein.
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Fig. 2. H-K-ATPase ␤-subunits expressed on the apical surface of LLC-PK1 cells are complex-type glycosylated, while the intracellular nonmature fraction
contains high-mannose-type subunits. The YFP-linked ␤-subunit of the H-K-ATPase (YFP-␤) stably expressed in LLC-PK1 cells is detected in total cell lysates
in 2 forms: EndoH resistant and EndoH sensitive. Only the EndoH-resistant complex-type form reaches the apical surface, as detected by apical surface
biotinylation. The intracellular form of YFP-␤ is converted by EndoH to the deglycosylated protein that has the same gel mobility as YFP-␤ in cell lysates
prepared after exposure of the cells to tunicamycin, the inhibitor of N-glycosylation,. The gel mobility of the EndoH-sensitive form of YFP-␤ is the same as that
of YFP-␤ from the cells exposed to dMM (preserves the high-mannose structure of glycoproteins) and greater than that of YFP-␤ from the cells exposed to
swainsonine (preserves the hybrid structure of glycoproteins). These results indicate that the intracellular fraction of YFP-␤ contains high-mannose N-glycans
and likely represents the ER-resident form. Symbols for monosaccharide residues and abbreviations correspond to those in the legend to Fig. 1.
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N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
AJP-Renal Physiol • VOL
In contrast, the apical localization of gp114 (recently identified as a dog homolog of CEACAM proteins) (27) in MDCK
cells was not affected by dMM (48), indicating that terminal
chains are not important for apical distribution of this protein.
Consistently, exposure of LLC-PK1 cells expressing the H-KATPase ␤-subunit to dMM or swainsonine led to only a slight
decrease in the relative apical content of the H-K-ATPase
␤-subunit, while mutation of one of the seven N-glycosylation
sites, either N1, N3, N5, or N7, resulted in a severalfold
greater reduction in relative apical abundance (90). On the
other hand, the rate of endocytosis of the ␤-subunit was
significantly higher in cells exposed to swainsonine than
with any of the single site-deficient mutants (90). These data
suggest that the core region sugars play the major role in
apical delivery of the ␤-subunit, whereas the terminal
monosaccharide residues are important for stabilization and
retention of the ␤-subunit in the apical membrane.
Putative Apical Sorting Machinery
that Recognizes N-Glycans
Search for apical sorting receptors. The mechanism by
which the presence of N-glycans can facilitate apical sorting of
glycoproteins is poorly understood. The ␮1B-subunit of the
AP-1B adaptor protein complex binds specifically to basolateral sorting motifs present in the cytoplasmic domains of
basolateral proteins, promoting their placement into the basolateral transport containers and subsequent delivery to the
basolateral membrane (25, 26, 28). It seems logical that a
similar coupling between a sorting receptor and N-glycans
would be used by the cell for delivery of glycoproteins to the
apical membrane. Since N-glycans are composed of carbohydrate moieties, a priori the apical sorting receptor must be a
lectin, i.e., a carbohydrate-binding protein.
One potential apical sorting receptor, the mannose-binding
lectin VIP36 (43, 44, 79), is predominantly localized in ERGIC
and the cis-Golgi (36, 83), and to a lesser extent in the TGN
and plasma membrane (34, 82). VIP36 plays a role in the
export of glycoproteins from the ER (36, 83) (Table 2). VIP36
was also found to promote apical sorting of several glycoproteins. The apical membrane content of this lectin in MDCK
cells was found to be twice as high as the basolateral content,
and the plasma membrane glycoproteins recognized by VIP36
were also twofold enriched in the apical membrane compared
with the basolateral membrane (34). Overexpression of VIP36
in MDCK cells increased the relative apical content of both
VIP36 and VIP36-recognized glycoproteins. In contrast, the
overproduction of a mutant version of VIP36, which has no
lectin activity, had no effect. Furthermore, VIP36 has been
shown to bind to clusterin and ␣-amylase and to increase the
relative apical abundance of these proteins (34, 35). Consistent
with a specificity of VIP36 for high-mannose-type glycans (43,
44, 79), both clusterin and ␣-amylase carry not only complextype N-glycans but also the high-mannose-type N-glycans (34,
35). However, since the majority of mature glycoproteins
contain predominantly complex-type N-glycans, VIP36 is unlikely a universal apical sorting receptor.
A galactose-binding lectin, galectin-3, is another potential
apical sorting receptor. This lectin was detected in raft-independent apical carrier vesicles in MDCK cells (15). Galectin-3
has been found to directly interact with three apical glycopro-
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The mutation of N-glycosylation sites in a cargo protein is
unlikely to affect export and transport of the containers. Thus
the decreased rate of delivery of the glycosylation-deficient
mutants to the apical membrane is consistent with the hypothesis that N-glycans facilitate apical sorting. However, it is not
clear whether N-glycans per se serve as apical sorting signals
or whether they facilitate recognition of other apical sorting
signals present in a glycoprotein by providing an optimal
conformation of the protein. Also, it is possible that N-glycans
facilitate association of glycoproteins with other proteins that
contain apical sorting information, thus promoting cosorting of
glycoproteins into vesicles destined for the apical membrane.
It is remarkable that the same N-glycans (N1, N3, and N5)
of the H-K-ATPase ␤-subunit that are important for ER-toGolgi trafficking are also important for apical delivery and
membrane retention of the protein (90). These results suggest
that, similar to ER-to-Golgi trafficking, both apical delivery
and apical membrane retention of the H-K-ATPase ␤-subunit
are lectin dependent and only specific N-glycans are accessible
to these lectins. Reduced retention of the mutants in the apical
membrane might result from disruption of the interaction of
specific N-glycans of the H-K-ATPase ␤-subunit with an
unidentified integral or extracellular lectin at the apical membrane, which acts to anchor the protein in the membrane. As a
consequence, the rate of constitutive endocytosis of the mutant
subunit would increase. Interactions of lectins with N-glycans
attached to other membrane proteins have been shown to be
important for their retention in the plasma membrane in several
different cell types (11, 47, 64, 65, 68).
The majority of mature glycoproteins are of the complex
type. The complex-glycosylated proteins contain the threemannosyl core (Fig. 1, green rectangle) and terminal chains
that are added during rebuilding of high-mannose N-glycans in
the Golgi. To examine whether the core region, the terminal
chains, or both are necessary for delivery and membrane
retention of apical proteins, various inhibitors of N-glycan
processing have been employed. Treatment of cells with these
inhibitors prevents trimming and further terminal glycosylation
of high-mannose N-glycans. This results in the absence of
either all (dMM) or most (swainsonine) of the terminal sugars
normally present in the complex N-glycans (Fig. 1). Similar to
dMM, kifunesine, the inhibitor of the ER ␣-mannosidase 1,
abolishes addition of terminal chains to the high-mannose
glycans (not shown).
Treatment of MDCK cells expressing endolyn with dMM or
kifunesine decreased the relative apical abundance, biosynthetic apical delivery, and postendocytic apical delivery of the
protein to the same extent as tunicamycin (72, 73). These
results suggest that the terminal chains rather than the residues
of the core region of N-glycans are important for apical sorting
of endolyn. However, endolyn contains not only N-glycans,
but also O-glycans. It has been shown that disruption of
N-glycan processing by dMM dramatically inhibited O-glycosylation of several proteins that contain both N- and Oglycosylation sites (60). Therefore, the possibility that dMM or
kifunesine affected apical distribution of endolyn due to the
impairment of its O-glycosylation cannot be excluded. In
accord with the latter possibility, benzyl-N-acetyl-␣-D-galactosaminide that inhibits elongation of both O- and N-glycans
also impaired apical polarity of endolyn (72).
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AJP-Renal Physiol • VOL
natural GPI-anchored glycoprotein, membrane dipeptidase
(67, 88). Removal of a GPI anchor excluded the protein from
lipid rafts but did not impair its apical distribution. On the other
hand, removal of N-glycans from the protein resulted in basolateral sorting but did not affect the protein association with
rafts (67, 88).
Oligomerization of apical glycoproteins appears to increase
their affinity for rafts. For example, clustering of gp114 molecules in the plasma membrane induced by interaction with the
gp114-specific antibodies increases association of gp114 with
lipid rafts, as judged by increased detergent resistance of the
glycoprotein (92). It has been postulated that intracellular
multivalent lectins could cluster apical glycoproteins in rafts in
vivo and thus facilitate their apical delivery (41, 81, 92).
However, direct evidence for the importance of N-glycans in
clustering of apical N-glycosylated proteins in lipid rafts has
not been presented. Furthermore, several pieces of evidence
suggest that the presence of N-glycans does not facilitate raft
association. First, many N-glycosylated apical proteins are not
associated with lipid rafts (Table 3). Second, removal of
N-glycans did not affect raft association of the membrane
dipeptidase, while removal of a GPI anchor excluded this
protein from lipid rafts (67, 88). Finally, apical pyrophosphatase/phosphodiesterase NPP3, but not its basolateral homolog, NPP1, associates with rafts, even though both NPP3
and NPP1 contain N-glycans. Specific positively charged
amino acid residues in the cytoplasmic tail rather than Nglycans have been shown to be important for association of
NPP3 with lipid rafts (19, 53).
Moreover, lectins that cluster N-glycosylated proteins in
lipid rafts have not been found. Lectins of the galectin family
were considered as candidates. They usually are multivalent
and, therefore, binding of one lectin molecule to N-glycans that
belong to different glycoprotein molecules can organize glycoproteins in clusters, bundles, arrays, and lattices (8). Indeed,
galectin-3 causes clustering of apical glycoproteins, lactasephlorizin hydrolase, transmembrane neurotrophin receptor
p75, or gp114 (15, 17). However, none of these three proteins
are present in rafts. Also, galectin-3 does not bind to sucraseisomaltase, the apical glycoprotein that associates with lipid
rafts (15). These results indicate that galectin-3 does not play a
role in clustering of apical glycoproteins in lipid rafts.
Another lectin of the same family, galectin-4, is a major
component of lipid rafts (14, 16). Depletion of galectin-4
impairs formation of rafts and affects apical sorting of several
proteins in HT-29 cells. However, galectin-4 binds with high
affinity to glycolipids but not glycoproteins (16).
Recent studies have shown that association of apical proteins
with rafts occurs not only in the TGN (9) or late compartments
of the Golgi apparatus (86) but also in the cis-Golgi or even in
the ER (3, 78). It is possible that the mannose-binding lectin
VIP36 facilitates apical sorting of glycoproteins by stabilizing
them in lipid rafts in the early steps of the glycoprotein
processing in the ER or cis-Golgi, when the mannose residues
are exposed at the termini of the carbohydrate chains. This
hypothesis is in agreement with the fact that VIP36 was
originally isolated from rafts (23), as well as with the data
showing that overexpression of VIP36 increases apical distribution of two raft-associated N-glycosylated proteins, clusterin
and ␣-amylase (34, 35).
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teins, lactase-phlorizin hydrolase, transmembrane neurotrophin
receptor p75, and gp114 (15, 17). This interaction is competitively inhibited by lactose, suggesting that galectin-3
binds to the glycans attached to these proteins. Lactasephlorizin hydrolase and transmembrane neurotrophin receptor p75 contain both N- and O-glycans, whereas gp114 has
only N-glycans Carbohydrate-dependent binding of galectin-3 to p75 requires the presence of the O-glycosylated
stalk domain that does not contain putative N-glycosylation
sites (15, 17). These data suggest that galectin-3 can bind to
both N- and O-glycans.
Depletion of galectin-3 from MDCK cells prevents formation of the high-molecular-weight clusters containing lactasephlorizin hydrolase, transmembrane neurotrophin receptor p75
or gp114 and results in missorting of all three apical membrane
proteins to the basolateral membrane (15, 17). These findings
suggest that galectin-3 cross-links glycoproteins to form large
galectin-3-containing clusters in the TGN membranes. These
clusters presumably bud from the TGN to form apical transport
containers (15, 17).
Galectin-3 binds to galactose, a terminal residue that might
be exposed at the carbohydrate chain termini of the mature
complex N-glycans (69). Consistently, the efficiency of coimmunoprecipitation of gp114 with galectin-3 and formation of
the high-molecular-weight clusters containing gp114 and galectin-3, as well as apical distribution of gp114 are severely
impaired in galactosylation-deficient MDCKII-RCAr cells (15,
17, 48). However, the apical localization of gp114 in wild-type
MDCK cells is not affected by the mannosidase I inhibitor
dMM, which prevents formation of hybrid and complex Nglycans and thus abolishes the insertion of the galactose residues into N-glycans (48). The explanation for these contradictory experimental observations is unknown.
Relationship between N-glycans and lipid rafts. Significant
evidence has accumulated to suggest involvement of lipid
rafts in sorting and delivery of proteins to the apical membrane. Lipid rafts are rigid membrane microdomains enriched in glycolipids, sphingolipids, and cholesterol embedded in the fluid membrane (81). In 1997, Simons and Ikonen
(84) postulated that newly synthesized apical proteins accumulate in these microdomains in the TGN, while basolateral
proteins are excluded from these lipid rafts. Small lipid rafts
are then clustered, forming bigger rigid membrane domains
that then bud from the TGN as apical transport containers
(41, 81).
Certain apical integral glycoproteins, including influenza
viral proteins hemaglutinin and neuraminidase, associate with
lipid rafts via their transmembrane domains (4, 80). Although transmembrane domains of these proteins are important for both raft association and apical sorting, the signals
for raft association and apical sorting are not identical (4,
49, 80). Raft association appears necessary, but not sufficient, for apical sorting of hemaglutinin (4, 49, 80). In
contrast, raft association is not absolutely required for apical
sorting of neuraminidase (4, 80).
GPI-anchored proteins associate with lipid rafts due to the
favorable packing of the GPI anchor into ordered domains
of the membrane (85). Stable association with rafts is
required for apical sorting of several chimeric GPI-anchored
proteins (18, 66, 75, 81, 85). However, raft association is
neither necessary nor sufficient for apical sorting of a
Review
N-GLYCANS AND TRAFFICKING OF APICAL MEMBRANE PROTEINS
Conclusions and Future Directions
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-077149, DK-58333, and DK-053642.
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N-glycans are attached to the majority of membrane proteins. Besides the direct effect of N-glycans on the conformation and intrinsic properties of proteins, N-glycans play a role
in intracellular trafficking that arises from specific interactions
of N-glycans with intracellular lectins. The best studied of
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lectins that are crucial to folding and quality control of newly
synthesized glycoproteins and their export from the ER to
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Growing experimental evidence also suggests that N-glycans attached to a number of apical proteins are required for
their apical distribution (Tables 3 and 4). Recent studies
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potential apical sorting receptors: the mannose-binding lectin
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will be identified.
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retention in the apical membrane. As a consequence, defects in
N-glycosylation can lead to cellular abnormalities in different
tissues. Further investigations may reveal previously unsuspected roles of N-glycans in trafficking of glycoproteins which
will provide insight into normal epithelial development and the
pathogenesis of various diseases.
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