From the Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany
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ABSTRACT |
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The temporal association between
O-glycosylation and processing of N-linked
glycans in the Golgi apparatus as well as the implication of these
events in the polarized sorting of three brush border proteins has been
the subject of the current investigation. O-Glycosylation
of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and
dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing
of the mannose-rich N-linked glycans is blocked by
deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the
presence of swainsonine, an inhibitor of Golgi mannosidase II. The
results indicate that removal of the outermost mannose residues by
mannosidase I from the mannose-rich N-linked glycans is
required before O-glycosylation can ensue. On the other
hand, subsequent mannose residues in the core chain impose no sterical
constraints on the progression of O-glycosylation.
Reduction or modification of N- and
O-glycosylation do not affect the transport of pro-SI, ApN,
or DPPIV to the cell surface per se. However, the polarized
sorting of two of these proteins, pro-SI and DPPIV, to the apical
membrane is substantially altered when O-glycans are not
completely processed, while the sorting of ApN is not affected. The
processing of N-linked glycans, on the other hand, has no
influence on sorting of all three proteins. The results indicate that
O-linked carbohydrates are at least a part of the sorting
mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither
O-linked nor N-linked glycans and is driven
most likely by carbohydrate-independent mechanisms.
Glycosylation of membrane and secretory glycoproteins comprises a
cascade of steps that implicate a number of oligo- and monosaccharide transferases and commences in the lumen of the endoplasmic reticulum (ER)1 concomitant with
protein translation and translocation (1-3). This initial
glycosylation event, known as N-linked core glycosylation, involves the transfer of
Glc3Man9GlcNAc2 unit to acceptor
asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr and is
catalyzed by the enzyme oligosaccharyltransferase (1-3).
N-Linked glycosylation is essential for the function,
stability, folding, intracellular transport, and secretion of
glycoproteins (4-7). Upon arrival to the cis-Golgi network,
the initial mannose-rich core chains are trimmed by mannosidases I and
II of the cis-Golgi. In the medial and
trans-Golgi formation of complex type of glycans ensues and
is terminated by the addition of sialic acid in the
trans-Golgi network (TGN) (1-3, 8, 9).
Another glycosylation event of many, but not all, membrane and
secretory glycoproteins is O-linked glycosylation at
particular serine or threonine residues. In contrast to
N-linked glycosylation, O-glycosylation is a
posttranslational event and occurs in the cis-Golgi (4, 10,
11). A consensus acceptor sequence, as the tripeptide Asn-Xaa-Ser/Thr
in N-linked glycosylation, does not exist. However,
comparison of amino acid sequences around a large number of
O-glycosylation sites of several glycoproteins revealed a
significantly increased frequency of proline residues at positions A role of O-glycosylation in the biological function of
glycoproteins and in conferring stability and protection against
proteolytic degradation has been proposed (16-18). A direct
implication of O-glycosylation in the intracellular
transport of proteins is unlikely, since acquisition of transport
competence implicates a cascade of steps that occur in the ER and
O-glycosylation is a cis-Golgi event. Consistent
with this is the finding that truncation of the highly
O-glycosylated stalk region of ApN does not affect the
transport and targeting of this enzyme (19).
At first glance N- and O-linked glycosylation
could be considered as independent events by virtue of the different
structural requirements of the glycosylation sites, the temporal aspect
(cotranslational versus posttranslational), and the
different monosaccharide composition of the individual chains. Recent
observations, however, suggested that at least an indirect association
between the two events does exist. Here, inhibition of trimming of the
mannose-rich chains of brush border lactase-phlorizin hydrolase (LPH,
EC 3.2.1.23-62) (20) by deoxymannojirimycin (dMM), an inhibitor of
cis-Golgi In an effort to analyze the role of carbohydrate modification on the
sorting of the brush border hydrolases, pro-SI, ApN, and dipeptidyl
peptidase IV (DPPIV, EC 3.4.14.5), we used inhibitors of trimming of
carbohydrate chains in the cis-Golgi and surprisingly found
that dMM has generated similar effects on three different proteins as
those observed with LPH. The O-linked glycosylation of
pro-SI, ApN, and DPPIV was substantially affected. Moreover, the
polarized sorting of SI and DPPIV, but not of ApN, was drastically altered from high sorting fidelity to the apical membrane to random distribution on both sides.
Biological Material and Cell Lines--
Human intestinal
biopsies (5-10 mg) were obtained for routine diagnosis by suction with
a pediatric Watson capsule and processed as described elsewhere (22).
The colon carcinoma Caco-2 cells (23) were cultured in Dulbecco's
modified Eagle's medium containing 0.45% glucose and supplemented
with 20% heat-inactivated fetal calf serum, 1% nonessential amino
acids, and antibiotics (Life Technologies, Inc.).
Immunochemical Reagents--
Monoclonal antibodies (mAbs)
against human small intestinal brush border membrane hydrolases were a
generous gift from Dr. H.-P. Hauri (Biocenter, Basel, Switzerland) and
Dr. E. E. Sterchi (University of Bern, Bern, Switzerland) (24).
The mouse mAb anti-ApN and anti-DPPIV were products of hybridomas HBB
3/775 and HBB 3/153, respectively. For immunoprecipitation of pro-SI, a
mixture of four different monoclonal antibodies was used (HBB 1/691,
HBB 2/614, HBB 3/705, HBB 2/219).
Biosynthetic Labeling--
Intestinal biopsy specimens were
either continuously labeled with [35S]methionine or
subjected to a pulse-chase protocol essentially as described elsewhere
(18, 22). Caco-2 cells were cultured on membrane filters (Falcon)
according to the procedure described previously for LPH in Madin-Darby
canine kidney cells (25) and were usually labeled at day 6 after
confluence. When used, dMM, an inhibitor of mannosidase I (21), and
swainsonine, an inhibitor of mannosidase II (26), were present in the
culture medium during preincubation of the tissue or cells in
methionine-deficient medium and during the pulse and chase periods
essentially as described before (20). The final concentration
of dMM was 5 mM and that of swainsonine was 4 µg/ml
(both reagents were obtained from Roche Molecular Biochemicals).
Immunoprecipitations and SDS-PAGE--
The labeled biopsy
specimens were homogenized with a Teflon-glass homogenizer in 1 ml of
25 mM Tris-HCl, pH 8.1, supplemented with 50 mM
NaCl and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, pepstatin (1 µg/ml), leupeptin (5 µg/ml), benzamidine (17.4 µg/ml), and aprotinin (1 µg/ml)) (all were from Sigma). Cell
debris and nuclei were removed by centrifugation at 1000 × g for 30 min. The supernatant was recovered and solubilized with 0.5% Triton X-100 and 0.5% sodium deoxycholate (final
concentrations) by stirring on ice for 30 min. Caco-2 cells were lysed
at 4 °C for 1 h in lysis buffer (25 mM Tris-HCl, pH
8.0, 50 mM NaCl, 0.5% Triton X-100, 0.5% sodium
deoxycholate, and the mixture of protease inhibitors mentioned above).
The solubilized cells or biopsy homogenates were spun for 1 h at
100,000 × g at 4 °C, and the supernatants were
immunoprecipitated as described by Naim et al. (18, 22). SDS-PAGE was performed according to Laemmli (27), and the apparent molecular masses were assessed by comparison with high molecular mass
markers (Bio-Rad Laboratories, Munich, Germany) run on the same gel.
Cell surface immunoprecipitations of proteins from biosynthetically
labeled Caco-2 cells on membrane filters were performed by adding the
appropriate antibody to either the apical or basolateral compartment.
The cells were extensively washed to remove excessive antibody and
lysed as described above. Protein A-Sepharose (Amersham Pharmacia
Biotech) was finally added to capture the antigen-antibody complexes.
Deglycosylation Experiments--
Treatments with endoglycosidase
H (endo H) (Roche Molecular Biochemicals) and endoglycosidase
F/N-glycopeptidase F (endo F/GF) (Sigma) were performed
according to Naim et al. (22). For elimination of
O-linked sugars, the immunoprecipitated glycoproteins were first N-deglycosylated with endo F/GF and the digestion
products were precipitated with ice-cold acetone. The protein pellets
were subjected to treatment with trifluoromethanesulfonic acid (TFMS) that cleaves O-glycosidically linked sugar chains (28) as
described (18).
O-Glycosylation of ApN and DPPIV
We have shown previously that pro-SI is an
O-glycosylated molecule, whose O-glycosylation
pattern is heterogeneous and correlates with the position of the
individual subunits, sucrase and isomaltase, that comprise this enzyme
complex (18). These data were obtained by employing a number of
approaches, including enzymatic and chemical deglycosylation and lectin
binding studies. Here, we extended these studies to ApN and DPPIV.
First we demonstrate that ApN and DPPIV are O-glycosylated
and that, as in the case of pro-SI and LPH, this glycosylation is also heterogeneous.
Enzymatic and Chemical Deglycosylation of ApN and DPPIV
ApN--
Human ApN is synthesized in intestinal cells as a 130-kDa
mannose-rich glycosylated precursor, which matures with a half time of
30-40 min to a complex glycosylated species of apparent molecular mass
of 160 kDa (24, 29). The biosynthesis and processing of ApN in other
species has been also studied (30). The full-length cDNA of the
human enzyme has been cloned (14), and the amino acid sequence of the
protein deduced from cDNA revealed a Ser/Thr-rich domain, which, as
in SI, may provide potential O-glycosylation sites. However,
no direct evidence from biosynthetic or structural analyses has been
described so far.
We first assessed the O-glycosylation pattern by employing a
strategy that combines enzymatic and chemical deglycosylation of
precursor and mature forms of ApN and compared the apparent molecular
weights of the deglycosylation products. First, the glycoprotein was
enzymatically deglycosylated of its N-linked sugars by using
the combination endoglycosidase F/N-glycopeptidase F (endo
F/GF), which removes complex and mannose-rich N-linked carbohydrates. Endo H, which cleaves mannose-rich glycans, was used
when mannose-rich glycoproteins were analyzed. In either case, the
resulting molecular form(s) were further treated with TFMS, a chemical
reagent that cleaves O-glycosidic linkages (28). A further
reduction in the apparent molecular weight of an enzymatically N-deglycosylated species provides an ample evidence for the
presence of O-linked sugars in the treated protein. The
results are shown in Fig. 1.
Immunoprecipitation of biopsy samples with mAb anti-ApN revealed a
130-kDa labeled protein after 30 min of chase. This band corresponded
to the mannose-rich precursor of ApN (denoted ApNh) as
assessed by its sensitivity to treatment with endo H and its conversion
to a 100-kDa polypeptide (Fig. 1A, lanes
1 and 2). A similar digestion product was
obtained with endo F/GF (Fig. 1A, lane
3), supporting the endo H data that the 130-kDa polypeptide
is N-glycosylated of the mannose-rich type. Treatment of the
endo F/GF product with TFMS did not produce a further shift in the size
of the 100-kDa band (Fig. 1A, lane 4),
indicating that the 130-kDa protein is not O-glycosylated.
After 4 h of chase, a predominantly labeled endo H-resistant
160-kDa ApN band could be detected, which corresponded to the complex
glycosylated mature form of ApN (Fig. 1A, lanes
5 and 6) (this form will be referred to
throughout as ApNc). When this immunoprecipitate was
treated with endo F/GF, a diffuse band pattern was obtained that,
however, could be resolved into at least two bands (Fig. 1A,
lane 7; see also Fig. 1B). Prolonged
treatment of mature ApNc with endo F/GF by using higher
concentrations of the endoglycosidase did not eliminate the blurred
band pattern (data not shown), indicating the existence of endo
F/GF-resistant oligosaccharides. Treatment with TFMS eliminated the
band heterogeneity and generated one single band, which was slightly
smaller than the lowest periphery of the diffuse band pattern. Due to
the specificity of TFMS in cleaving O-glycosidic bonds, the
result demonstrates the existence of O-linked glycans in
mature ApNc. Fig. 1B provides a clearer evidence
for the existence of several products of N-deglycosylated ApN and compares further in the same experimental sample the endo F/GF
products of the 130-kDa mannose-rich ApNh and the 160-kDa complex glycosylated ApNc species (Fig. 1B,
lane 4). In this experiment, ApN was
immunoprecipitated from a biopsy sample that was continuously labeled
for 2 h, during which time the proportion of ApNh
relative to ApNc is still high enough, thus allowing a
direct comparison of the N-deglycosylated forms of the two
species in the same experimental sample (Fig. 1B,
lane 3). Mannose-rich ApNh
immunoprecipitated from a biopsy sample labeled for 30 min served as a
control (Fig. 1B, lane 1). Endo
F/GF-treated ApN from the 2-h labeled biopsy sample revealed three
major bands (Fig. 1B, lane 4). The
lowest of these is the N-deglycosylation product of the
130-kDa form, since a similar product was obtained upon deglycosylation
of the 130-kDa ApNh alone (compare with lane
2). The other two larger products corresponded hence to the
endo F/GF-treated 160-kDa ApNc species. The smaller size of
the N-deglycosylated mannose-rich 130-kDa ApNh
(lanes 2 and 4) as compared with the
N-deglycosylated mature 160-kDa ApNc provides
additional evidence for the existence of sugar residues in mature ApN
other than the N-linked ones.
DPPIV--
The earliest detectable form of DPPIV synthesized
in intestinal epithelial cells is the mannose-rich precursor of
apparent molecular mass ~100 kDa (DPPIVh) (Fig.
2, lane 1; see
Refs. 24 and 31). This form is rapidly converted to a complex
glycosylated mature form (DPPIVc) (Fig. 2, lane
5) with a half-time of 15-25 min (24, 31). Mature
DDPIVc appears as a diffuse band of an average apparent
molecular mass of 124 kDa (this molecular mass will be referred to
throughout). Deglycosylation of the DPPIVh mannose-rich
polypeptide with endo H and endo F/GF revealed essentially similar
molecular species of an apparent molecular mass around 85 kDa (Fig. 2,
lanes 2 and 3). TFMS treatment of the
N-deglycosylated DPPIVh did not generate a
further shift in its size indicating that the 85-kDa species is devoid
of O-glycosidically linked glycans. By contrast, the mature
124-kDa DPPIVc form obtained within 4 h of chase (Fig.
2, lane 5) is heavily O-glycosylated
as assessed by the combined endo F/GF and TFMS treatments.
N-Deglycosylation generated a diffuse band of an apparent
molecular mass ranging between 90 and 110 kDa (Fig. 2, lane
7). TFMS treatment converted this heterogeneous form to a
band of an apparent molecular weight smaller than the lowest band
periphery of the N-deglycosylated product (Fig. 2,
lane 8, and compare this band with the band in lane 7). The result demonstrates that the
N-deglycosylated protein contains
O-glycosidically linked oligosaccharides, which have been
eliminated by TFMS.
Altogether, the chemical and enzymatic treatments provide an evidence
for O-glycosylation of ApN and DPPIV. Further, the observed multiple and heterogeneous band patterns in N-deglycosylated
ApN and DPPIV suggest the presence of several populations of
differently O-glycosylated species of these glycoproteins,
which could be converted to one unglycosylated species upon TFMS
treatment. The existence of several populations of N- and
O-glycosylated glycoproteins has been also reported for two
other brush border enzymes, LPH (17, 20) and SI (18). In all these
cases, a possible association between the differentiation state of
intestinal cells and the various glycoforms is favored.
Association between Processing of Mannose-rich N-Linked
Carbohydrates and O-Glycosylation
We have demonstrated before that impaired processing of
cotranslationally added mannose-rich N-linked sugars in the
cis- and medial Golgi by inhibitors of mannosidases I and II
is associated with drastic reduction, if not complete blocking, of the
O-linked glycosylation of human intestinal LPH (20). A
possible conformational interference of unprocessed N-linked
sugar chains with potential O-glycosylation sites lying in
their vicinity was proposed. This has inspired us to further examine
other heavily O-glycosylated proteins such as ApN, DPPIV,
and pro-SI, with the ultimate goal of analyzing the role of
N-linked and more importantly O-linked glycosylation on the sorting behavior of proteins in polarized cells.
We studied therefore the effects of dMM, an inhibitor of mannosidase I
(21), and swainsonine, an inhibitor of mannosidase II (26), on the
processing of pro-SI, ApN, and DPPIV in intestinal cells. The results
are depicted in Fig. 3 and a survey of
the glycoforms of the proteins analyzed is provided in Table
I.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
and +3 relative to the glycosylated residues and a marked increase of
serine, threonine, and alanine residues (12). It has been also
suggested that stretches of serine and threonine residues in
glycoproteins, such as in sucrase-isomaltase (SI, EC 3.2.1.48-10)
(13), aminopeptidase N (ApN, EC 3.4.15.1) (14), glycophorin (15), or
the low density lipoprotein receptor (16), are potential sites for
O-glycosylation.
-mannosidase I (21), was accompanied by
generation of only N- rather than N- and
O-glycosylated forms of LPH (17). In this very example, it
was proposed that conformational alterations in LPH due to persisting
mannose-rich type of glycosylation in the presence of dMM may render
potential O-glycosylation sites in LPH inaccessible to Golgi
transferases. It is not clear, however, whether the observed effect
with LPH may be generalized to other O-glycosylated proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
N- and
O-glycosylation of ApN. A, biopsy
specimens were pulse-labeled with [35S]methionine for 15 min and chased for the indicated times with cold methionine. Detergent
extracts of the labeled tissues were immunoprecipitated with mAb
anti-ApN. The immunoprecipitates were treated or not treated with endo
H, endo F/GF, and endo F/GF followed by TFMS. The proteins were
subjected to SDS-PAGE on 6% slab gels and fluorography. B,
biopsy specimens were continuously labeled with
[35S]methionine for 30 or 120 min, followed by
solubilization and immunoprecipitation with mAb anti-ApN. The isolated
proteins were treated or not treated with endo F/GF and subjected to
SDS-PAGE on 6% slab gels and fluorography.
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Fig. 2.
N- and
O-glycosylation of DPPIV. Biosynthetic labeling,
immunoprecipitation, and deglycosylations were performed as described
in Fig. 1A, except that mAb anti-DPPIV was used in the
immunoprecipitations instead of mAb anti-ApN.
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Fig. 3.
Effects of dMM and swainsonine on the
glycosylation patterns of pro-SI, ApN, and DPPIV. Biopsy samples
were biosynthetically labeled with [35S]methionine for
6 h in the presence or absence of 5 mM dMM and 4 µg/ml swainsonine. Detergent extracts of the labeled tissues were
immunoprecipitated with mAbs directed against pro-SI (upper
panel), ApN (middle panel), and DPPIV
(lower panel). Part of the immunoprecipitates was
treated with endo H, another part was treated with endo F/GF, and one
part was untreated. Treated and non-treated samples were subjected to
SDS-PAGE on 5% (pro-SI), and 6% (ApN and DPPIV), followed by
fluorography.
Glycoforms of the brush border proteins pro-SI, ApN, and DPPIV
Effects of dMM on Pro-SI, ApN, and DPPIV
Fig. 3 shows the molecular forms of pro-SI, ApN, and DPPIV immunoprecipitated from a normal intestinal biopsy specimen that has been labeled for 6 h. In each case, a mannose-rich and a complex glycosylated polypeptides were detected, as has been shown previously for pro-SI (18) and above for ApN and DPPIV. For pro-SI these forms are denoted pro-SIh (mannose-rich 210 kDa) and pro-SIc (complex glycosylated 245 kDa) (18). For ApN and DPPIV, these forms have been described above (ApNh, ApNc, and DPPIVh and DPPIVc; see Figs. 1 and 2). Treatment of the biopsy specimen with 5 mM dMM during the labeling period has resulted in the generation of single glycosylated forms of pro-SI (pro-SIdMM), ApN (ApNdMM), and DPPIV (DPPIVdMM). Pro-SIdMM and ApNdMM had apparent molecular weights almost similar to their mannose-rich pro-SIh and ApNh counterparts, respectively. The glycoform of DPPIV was a diffuse band of an average apparent molecular around 100 kDa (denoted DDPIVdMM) and is therefore significantly smaller than complex glycosylated DPPIVc (124 kDa). All three glycoforms were sensitive to endo H, indicating their predominant mannose-rich type glycosylation. The endo H-resistant proportions of the sugar content in pro-SIdMM, ApNdMM, and DPPIVdMM were eliminated by endo F/GF, as assessed by the shift in the apparent molecular weight upon treatment with the endoglycosidase (in the case of pro-SIdMM, this shift was slight). N-Deglycosylation of pro-SIdMM and ApNdMM revealed in each case a single band pattern that corresponded to the lowest band of the endo F/GF forms of the control glycoforms pro-SIc and ApNc. For DPPIVdMM, endo F/GF treatment resulted in the generation of a substantially less diffuse band than that of DPPIVc and which had also a reduced apparent molecular weight. Since the endo F/GF-forms of pro-SIc, ApNc, and DPPIVc, which are O-glycosylated (Ref. 18, and see above), are substantially larger than those of their pro-SIdMM, ApNdMM, and DPPIVdMM counterparts, our results demonstrate a drastic reduction in O-linked glycosylation of pro-SI, ApN, and DPPIV in the presence of dMM.
Effects of Swainsonine on pro-SI, ApN, and DPPIV
The same type of analysis was performed with swainsonine. A marked effect on the glycosylation patterns of pro-SI, ApN, and DPPIV was obtained with this inhibitor, albeit to a lesser extent than that revealed with dMM (Fig. 3). Two glycoforms of pro-SI and ApN were revealed, the mannose-rich pro-SIh and ApNh and the slightly endo H-sensitive species, a 235-kDa pro-SI (denoted thereafter pro-SIswa) and a 145-kDa ApN (denoted ApNswa). In the case of DPPIV, a diffuse band was obtained, the lower periphery of which runs at the same apparent molecular weight as mannose-rich DPPIVh. DPPIVswa was also slightly sensitive to endo H and had an apparent molecular weight of approximately 115 kDa (denoted DPPIVswa). That all three glycoforms were only partially sensitive to endo H is indicative of a processing of the mannose-rich N-linked glycans to complex type. However, the marked difference in the apparent molecular weight of pro-SIswa, ApNswa, and DPPIVswa as compared with the corresponding control complex glycosylated pro-SIc, ApNc, and DPPIVc indicates that complete processing of the N-linked glycan units has not occurred. Treatment of the 235-kDa pro-SIswa, the 145-kDa ApNswa, and the 115-kDa DPPIVswa with endo F/GF generated N-deglycosylation products similar in size to the completely N-deglycosylated products of wild type pro-SIc, ApNc, and DPPIVc, and these products are O-glycosylated (Ref. 18 and see above). The result indicates therefore that, in contrast to dMM, swainsonine treatment of intestinal cells had no effect on the processing of O-linked chains in pro-SI, ApN, and DPPIV.
In summary, the data shown here with three different proteins demonstrate that inhibition of mannose-rich chains processing by dMM has induced substantial effects on the O-linked glycosylation, while swainsonine effects are restricted to N-linked glycosylation. These results are supported by the data obtained with LPH (20).
Effects of Variations in the O- and N-Linked Glycosylation Patterns on the Polarized Sorting of Pro-SI, ApN, and DPPIV
Pro-SI, ApN, and DPPIV are apically sorted proteins in intestinal cells. In Caco-2 cells, pro-SI and ApN follow predominantly the direct sorting pathway to the apical membrane, while DPPIV follows both the direct and the transcytotic pathways (32). The significant effects of dMM on the O-glycosylation and swainsonine on the N-linked complex glycosylation patterns of pro-SI, ApN, and DPPIV have inspired us to investigate these events in the context of polarized sorting of these proteins.
For this purpose, we performed cell surface immunoprecipitations of
pro-SI, ApN, and DPPIV using Caco-2 cells that have been cultured on
membrane filters and biosynthetically labeled for 6 h in the
presence or absence of dMM and swainsonine. Immunoprecipitation of
pro-SI, ApN, and DPPIV was performed with antibodies added to either
the apical or basolateral compartments. As expected (32), Fig.
4A shows that
pro-SIc, ApNc, and DPPIVc purified from non-treated Caco-2 cells were mainly immunoprecipitated from the
apical surface (left panels). However, the
sorting behavior of the corresponding glycoforms of these proteins
generated in the presence of dMM was different. While the sorting of
ApN was not affected, the dMM glycoforms of pro-SI and DPPIV were found on both sides of the cell, the apical and basolateral (Fig.
4A, middle vertical
panels). Almost 42% of pro-SIdMM was found at the basolateral membrane as compared with 7% of pro-SIc,
and the delivery of DPPIV to the basolateral membrane shifted from 8% to almost 40% in the presence of dMM (Fig. 4B). This
indicates that elimination or reduction of O-glycosylation
has resulted in abolishment of the high fidelity of polarized sorting
of pro-SIdMM and DPPIVdMM to the apical
surface. On the other hand, modulation of N-linked
glycosylation in the presence of swainsonine remained without effect on
the sorting behavior of pro-SIswa, ApNswa, and DPPIVswa, since these glycoforms were predominantly
transported to the apical membrane as their wild type counterparts
(Fig. 4, A, right panels, and
B). The results clearly demonstrate that alterations in the
O-glycosylation patterns of pro-SI and DPPIV had strong
influence on their polarized sorting in epithelial cells, while
modification of N-linked glycosylation in the Golgi apparatus is not an essential event in the sorting process. The sorting
of ApN is neither affected by O-linked nor
N-linked glycosylation.
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DISCUSSION |
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N- and O-glycosylations exert various
fundamental roles that relate to biological function, processing,
folding, trafficking, and sorting of membrane and secretory proteins. A
large number of these proteins are both N-glycosylated at
particular Asn residues and O-glycosylated at either Ser or
Thr residues (1-4). These glycosylation types are the outcome of
distinct temporal events, and are structurally different (3, 4,
10-12). A direct or an indirect linkage between these processes has
not been so far demonstrated, except for brush border LPH (20). Here it
could be demonstrated that O-glycosylation does not proceed
to completion, or is perhaps blocked, as a result of impaired
processing or complete inhibition of the processing of the first
mannose residues in the cis-Golgi by dMM, an inhibitor of
cis-Golgi -mannosidase I (21). This rather unexpected finding
appeared initially to represent a special case of LPH. We postulated
then that the unprocessed mannose-rich chains in the presence of dMM
may sterically hinder the addition of glycan chains to neighboring
potential O-glycosylation Ser or Thr residues. The present
report, however, clearly documents that this phenomenon is not
restricted to LPH alone. Three structurally and functionally different
proteins of the brush border membrane have been examined with
inhibitors of the processing of mannose-rich chains. In all these
cases, inhibition by dMM of the processing of the first four mannose
residues of the mannose-rich sugar chains results in a substantial
impairment of O-glycosylation. How do these results
accommodate with the role of glycosylation in general and with our
knowledge of the structure of the analyzed brush border proteins in particular?
All the proteins analyzed are extensively N- and O-glycosylated. The potential N-glycosylation sites vary from 16 for pro-SI (1827 amino acids) (13, 18) and LPH (1927 amino acids) (34), to 10 for ApN (966 amino acids) (14), and 9 for DPPIV (766 amino acids) (35) and are almost evenly spread all over the ectodomains of these proteins. Many of the potential O-glycosylation sites are found in stalked regions, which are located in close proximity to the membranes and also among potential N-glycosylation sites (13, 14, 19, 34, 35). By virtue of the large number of N-linked glycosylation sites, it is likely that a particular structure of the mannose-rich N-linked glycans, which exist earlier in the biosynthesis than O-glycosidically linked sugars, elicit specific constraints on the processing of the O-linked sites. The results obtained with two different inhibitors of mannosidases I and II of the cis-Golgi compartment strongly suggest that the accessibility of potential O-glycosylation sites to galactosyl- or N-acetyl-galactoseaminyltransferases requires a reduced number of mannose residues of neighboring N-linked chains. A completely unprocessed N-linked mannose-rich chain in the presence of dMM may sterically reduce or hinder the glycosylation of a neighboring potential O-glycan site. Processing of the mannose-rich chains by mannosidase I appears to be sufficient for O-glycosylation to ensue, since inhibition of mannosidase II and subsequently further processing steps by swainsonine remains without marked effects on normal O-glycosylation of the four brush border proteins. Since the drastic impairment in O-linked glycosylation is only observed when mannosidase I, but not mannosidase II, is inhibited, our data suggest that the outermost four mannose residues cleaved by mannosidase I (21) are critically important or may be rate-limiting in determining the extent of O-glycosylation. Furthermore, the results suggest that O-glycosylation does not commence before processing of the mannose-rich chains by mannosidase I is achieved.
One main aspect of the present work is that related to the implication of glycosylation, in particular O-glycosylation in the trafficking and polarized sorting of the brush border proteins pro-SI, ApN, and DPPIV. The observation that O-glycosylation could be substantially affected in the presence of dMM, i.e. when particular mannose residues are not cleaved, opens up the possibility to investigate in a simple and straightforward set up the role of O-glycosylation in sorting. Recent studies have demonstrated that carbohydrates, N- or O-linked, carry sorting information to the apical membrane (36-38). However, a general concept on the significance of the glycosylation events in polarized sorting cannot be drawn, in particular since only a few studies have addressed the role of N- and O-glycosylation of proteins naturally occurring in epithelial cells. In addition, it is not obvious how glycosylation correlates with sorting and what carbohydrate structures are implicated in the sorting event. It is clear that modifications in the N-linked carbohydrate structure of glycoproteins from mannose-rich to complex type precede polarized sorting in the trans-Golgi network. In addition, many proteins acquire O-linked glycosylation prior to transport to the TGN. Should carbohydrates play a role in the sorting at all, one may expect that the complex type as well as O-glycosylated may be involved. Our paper shows that modification of the N-linked glycosylation is not effective in the context of apical sorting of a number of heavily N-glycosylated brush border enzymes. By contrast, elimination or reduction of O-glycan units in the presence of dMM affects the sorting behavior drastically. In their predominantly mannose-rich type of glycosylation, pro-SIdMM and DPPIVdMM follow a random pattern of delivery to the apical and basolateral membranes. It is obvious that O-linked glycans are directly implicated in apical sorting. On the other hand, the sorting signal of ApN appears not to involve sugar chains, since ApNdMM and ApNswa are delivered with similar fidelity to the apical membrane as their fully glycosylated wild type ApNc counterpart. This result supports data, in which deletion of the potentially O-glycosylated Ser/Thr-rich stalk domain of ApN has no effects on the sorting behavior of the mutant (19) and indicates that sorting motifs other than O-linked glycans are responsible for apical targeting of ApN. The strong role of O-glycans in the sorting of pro-SI and DPPIV does not exclude, however, that the sorting of these proteins may implicate additional structural motifs that may function to tune up the apical sorting of these proteins. This may explain why a slightly more apical than basolateral delivery of pro-SIdMM and DPPIVdMM still occurs despite the removal of O-glycans. One possible mechanism of apical sorting has been described for the pig species of pro-SI, ApN, and DPPIV (39), which involves association of these proteins with glycolipid microdomains or rafts. If O-glycosylation is implicated in this mechanism, then at least the apical delivery of ApN would implicate different sorting signals and mechanism, since ApNdMM, which is largely devoid of O-linked glycans, is targeted correctly to the apical membrane.
Unlike basolateral sorting, apical sorting appears to implicate signals of different types, structure, and location, emphasizing the diversity of mechanisms responsible for this process. It has been proposed that the heavily O-glycosylated stalked region of the neurotrophin receptor is implicated in its sorting to the apical membrane, since deletion of this region leads to a mistargeting of the mutant protein to the basolateral membrane and suggesting the existence of a suppressed basolateral signal in this protein (38). A similar situation does not prevail with pro-SIdMM and DPPIVdMM, suggesting that except for apical sorting signals no other motifs are present in these proteins. Due to the presence of GalNAc residues in O-linked carbohydrates, these chains could be considered as an exquisite candidate for binding specific sorting elements. The leguminous lectin-like VIP 36 protein, for example, is a resident of the Golgi apparatus that has been located to glycolipid rafts and is presumably involved in the sorting of some GPI-anchored proteins to the apical membrane (40).
Our results do not exclude a partial O-glycosylation occurring at Ser or Thr residues in SI, ApN, and DPPIV in the presence of dMM. The slight differences in the electrophoretic patterns obtained upon endo F/GF treatment of DPPIVdMM as compared with DPPIV (and to a lesser extent those of SIdMM and ApNdMM) suggest that some O-glycosylation still occurs. Nevertheless, an interaction with potential sorting elements, may not be as avid as with completely processed O-chains to ensure a high sorting fidelity.
While O-glycans are strongly implicated in apical sorting of
pro-SI and DDPIV, partial modification of the N-glycan units to a hybrid type in the presence of swainsonine remains without significant effect on the sorting of these proteins. This is direct evidence that N-linked complex type of glycosylation
per se is not an essential factor for correct sorting to the
apical membrane. Moreover, it is not clear and also experimentally not
established yet, how N-linked mannose-rich residues would
mediate polarized sorting, particularly since many proteins are
targeted to the apical membrane regardless of their
N-glycosylation pattern, such as those shown in this report.
One possible explanation is that these residues participate in early
sorting processes between the ER and Golgi, for instance via binding
leguminous lectin-like proteins, such as ERGIC-53 (41-43), whereby
later sorting of these proteins from proteins with strong basolateral
signals constitutes an indirect discriminatory process. This mechanism
may apply only to a limited class with no or preferentially weak apical
sorting signals. Meanwhile, the sorting of many apical proteins, most notably those of the intestinal brush border membrane, appears to take
place via specific sorting elements (19, 25, 44-46). Perhaps the most
important role of the N-linked glycans in these proteins is
to achieve correct folding (4, 5) and presumably to impose and
stabilize particular secondary structures that may act as sorting
motifs and interact with sorting receptors (47). Since the initial and
most critical folding of glycoproteins occurs in the ER, the type of
N-glycosylation, mannose-rich or complex, plays an indirect
role, if any, in the sorting event as our results clearly demonstrate.
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ACKNOWLEDGEMENTS |
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We thank Dr. Hans-Peter Hauri, Biozentrum, University of Basel and Dr. Erwin Sterchi, Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland for generous gifts of monoclonal antibodies.
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FOOTNOTES |
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* This work was supported by Grant Na 331/1-2 from the Deutsche Forschungsgemeinschaft, Bonn/Germany (to H. Y. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed. Tel.:
49-511-953-8780; Fax: 49-511-953-8585; E-mail:
hnaim{at}biochemie.tiho-hannover.de.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; TGN, trans-Golgi network; SI, sucrase-isomaltase (all forms); pro-SI, uncleaved sucrase-isomaltase; ApN, aminopeptidase N; LPH, lactase-phlorizin hydrolase; DPPIV, dipeptidyl peptidase IV; dMM, deoxymannojirimycin; mAb, monoclonal antibody; endo H, endoglycosidase H; endo F/GF, endoglycosidase F/N-glycopeptidase F; TFMS, trifluoromethanesulfonic acid; VIP36, 36-kDa vesicular integral membrane protein.
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REFERENCES |
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