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From the
Received for publication, October 11, 2001, and in revised form, November 20, 2001
Enteropeptidase, a type II transmembrane protein
of the enterocyte brush border, is sorted directly to the apical
membrane of Madin-Darby canine kidney II cells. Apical targeting
appears to be mediated by an N-terminal segment that contains a
27-amino acid residue O-glycosylated mucin-like domain
consisting of two short mucin-like repeats, A and B. Targeting signals
within these repeats were characterized by using green fluorescent
protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was
sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion
to a different O-glycosylated motif did not alter the
random secretion of GFP, and several constructs with mutations in
either repeat A or B were O-glycosylated and secreted
randomly. In addition, repeat B appears to contain an apical targeting
signal that functions in the absence of glycosylation. Density
gradient centrifugation indicated that, unlike several other apically
targeted membrane and soluble proteins, apical sorting of mucin-GFP
chimeric proteins does not appear to utilize lipid rafts.
Enteropeptidase, a serine protease localized to the brush border
of duodenal enterocytes (1), is targeted directly to the apical surface
of Madin-Darby canine kidney II (MDCK)1
cells (2). Apical sorting of
enteropeptidase may involve at least two distinct signals. One is
located in the C-terminal serine protease domain and depends on
N-glycosylation; another is proposed to be located in an
N-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites
(2). The apical targeting signals within the N-terminal region of
enteropeptidase have not been characterized structurally.
Several distinct classes of apical sorting signals have been
identified, suggesting the existence of several apical targeting mechanisms. For example, apical sorting can be mediated by
transmembrane domains (3-5), by glycosylphosphatidylinositol anchors
(6, 7), by PDZ-interacting domains (8, 9), or by N-linked oligosaccharides (10, 11). In some proteins, such as sucrase-isomaltase (12) and dipeptidyl peptidase IV (13, 14), O-linked
oligosaccharides also appear capable of mediating apical sorting. The
juxtamembrane segment of the neurotrophin receptor p75 contains
clustered O-linked oligosaccharides and is required for
apical targeting (15, 16). The O-linked
glycan-dependent apical sorting of sucrase-isomaltase also
is accompanied by association with glycosphingolipid and cholesterol-rich membrane microdomains or lipid rafts (13, 14). Inhibition of terminal To characterize apical sorting determinants within the N-terminal
segment of enteropeptidase, we employed a modified green fluorescent
protein (GFP) as a reporter (18) in transfected MDCK cell lines
expressing various chimeric enteropeptidase-GFP proteins. The results
demonstrate that either of two short O-glycosylated mucin-like repeats of enteropeptidase can confer apical targeting on a
heterologous protein. However, O-linked oligosaccharides are
not sufficient for apical targeting, and some apical targeting activity
is retained by a peptide that is not glycosylated. The apical targeting
function also does not appear to involve stable interaction with lipid rafts.
Construction of Deletion Mutants--
Plasmids
pSMAB(His)6-GFP and pS(His)6-gGFP
were derived from pdLin-BEK, which encodes the human prothrombin signal
peptide and His6 tag from pHL-BEK (19) linked to the codon
for the Ala-166 of plasmid pBEK (2). A fragment (SMAB)
encoding the human prothrombin signal peptide (S) plus His6
tag linked to two mucin-like repeats (MAB) (amino acids
166-192) of bovine enteropeptidase (20) was made by PCR (GeneAmp
reagents, PerkinElmer Life Sciences) with N-terminal primer
(5'-gacagctcgagatggcgccacgtc-3' (XhoI site
underlined) and C-terminal primer
(5'-ccgtggatcccgtggggttgccag-3' (BamHI site underlined). Fragment S(His)6 encoding the prothrombin
signal peptide and His6 tag without mucin-like repeats was
generated by PCR with the same N-terminal primer and a different
C-terminal primer (5'-cggtggatcccggctgctgtgatgatg-3'
(BamHI site underlined). The PCR fragments
SMAB(His)6 and S(His)6 were
digested with XhoI and BamHI and ligated into the
same sites of vector pEGFPN1 (GenBankTM accession number
U55762, CLONTECH Laboratories, Palo Alto, CA) to
generate pSMAB(His)6-GFP and
pS(His)6-gGFP, respectively. A fragment encoding the
prothrombin signal peptide (S) was generated from template
pS(His)6-gGFP with primers
5'-gacagctcgagatggggtcaaag-3' (XhoI site
underlined) and 5'-caggatcctgcctgtgcacaaggc-3'
(BamHI site underlined), digested with XhoI and
BamHI, and ligated into pEGFPN1 to generate pS-GFP. A
fragment encoding mucin repeats (MAB) was made similarly
from template pSMAB(His)6-GFP with primers 5'-tagggatccagcctctttggagaat-3' (BamHI site
underlined) and 5'-gctcctcgcccttgctcacca-3'. The fragment
(MAB) was digested with BamHI and ligated into
the BamHI site of pS-GFP to create plasmid
pSMAB-GFP.
Complementary pairs of oligonucleotides were synthesized
encoding mucin-like repeat A (MA) and repeat B
(MB). The MA oligonucleotides were
5'-ttccgggatccagcctctttggagaatttctctacgataagtcctgcaacaacgtcacgggatccaccg-3' and
5'-cggtggatcccgtgacgttgttgcaggacttatcgtagagaaattctccaaagaggctggatcccggaa-3' (BamHI sites underlined). Oligonucleotides encoding
MB were
5'-ttccgggatccagaaaagctaacaaccagcattcctctggcaaccccacgggatccaccg-3' and
5'-cggtggatcccgtggggttgccagaggaatgctggttgttagcttttctggatcccggaa-3' (BamHI sites underlined). After annealing, the
double-stranded fragments were digested with BamHI and
ligated into the BamHI site of pS-GFP to give
pSMA-GFP and pSMB-GFP, respectively.
Mutagenesis--
Point or clustered mutations were generated
with the QuikChangeTM site-directed mutagenesis kit
(Stratagene, La Jolla, CA) according to the manufacturer's
instructions, using pSMA-GFP or pSMB-GFP as the
template and an oligonucleotide primer that contains the desired
mutation. All product plasmids were sequenced (BigDye cycle DNA
sequencing kit, PE Applied Science, Foster, CA) to confirm the accuracy
of the construction.
Transfection--
MDCK II cells (American Type Culture
Collection) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Invitrogen) as
described (2). Cells growing in 6-well tissue culture plates were
washed with phosphate-buffered saline and incubated with 5 µg of
plasmid DNAs premixed (1:6 w/v) with 30 µl of PerFect lipid (pfx-2,
Invitrogen) in serum-free Dulbecco's modified Eagle's medium. After
5 h, fetal bovine serum was added to 10%. After an additional
18 h, cultures were split 1:50 and cultured in 48-well plates for
selection in 0.5 mg/ml geneticin (Invitrogen). Positive clones were
identified by fluorescence microscopy and Western blotting with
anti-GFP monoclonal antibody (MMS-118P, Berkeley Antibody Co.,
Richmond, CA).
Analysis of the Polarity of Protein Secretion--
MDCK cells
expressing various chimeric proteins were cultured on Transwell filters
(pore size 0.4 µm; Corning Costar Corp., Cambridge, MA) until a tight
monolayer was formed according to transmembrane resistance (2). Cells
were washed three times on both sides of the membrane with 2 ml of
phosphate-buffered saline and cultured with 2 ml of serum-free medium
(OPTI-MEM, Invitrogen) added to both apical and basolateral
compartments. After 24 h, medium was collected from the apical and
basolateral chambers, and 0.1% (v/v) protease inhibitor mixture
(4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A,
trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, bestatin, leupeptin, and aprotinin, from Sigma) was added. Cell debris
was removed by centrifugation, and the supernatants were concentrated
10-fold by ultrafiltration (Centricon-10; Amicon, Beverly, MA) and
stored at Glycosidase Digestions--
Glycosidases were obtained from
Oxford Glycosciences (Bedford, MA). Samples of concentrated conditioned
medium (40 µl) were heated to 100 °C for 5 min in 20 mM sodium citrate phosphate, pH 5.5, 0.2% SDS, and 5%
Analysis of Raft Association--
Detergent-insoluble
glycosphingolipid-enriched raft domains were prepared by cell lysis in
ice-cold Triton X-100 (for 10 min without agitation) and then sucrose
density gradient centrifugation as described previously (2).
Proteins in the fractions were precipitated with 10% trichloroacetic
acid on ice for 30 min, and pellets were resuspended in 200 µl of 0.2 N NaOH. Samples (20 µl) of trichloroacetic
acid-concentrated fractions were analyzed by SDS-PAGE and
immunoblotting with either monoclonal anti-GFP IgG (1:10,000) or
monoclonal anti-caveolin IgG (1:10,000; C37120, Transduction
Laboratories, Lexington, KY) as described above.
Targeting Activity of the Enteropeptidase Mucin
Domain--
Previous studies suggested that apical sorting of
enteropeptidase in MDCK cells depends on a signal near the N terminus,
between amino acid residues 50-197 (2). This juxtamembrane region
contains four potential N-glycosylation sites and a mucin
domain consisting of two tandem O-glycosylated Ser/Thr-rich
repeats (Fig. 1A) (20, 21).
Potential targeting signals within these mucin repeats were
characterized by using green fluorescent protein (GFP) as a reporter
(Fig. 1B) (18). Control constructs (GFP and
His6-gGFP) were secreted randomly (Fig.
2 and Table
I). Constructs with both mucin repeats
(MAB-GFP and His6-MABGFP) were
secreted apically (Fig. 2A and Table I), indicating that the
enteropeptidase mucin domain can direct apical targeting of a
heterologous protein.
Digestions with neuraminidase and O-glycanase showed that
the mucin repeats were extensively O-glycosylated (Fig.
2B). Secreted MAB-GFP and
His6MAB-GFP had apparent masses of 53 and 56 kDa, respectively (Fig. 2B, lanes 5 and
7). Enzymatic removal of O-linked oligosaccharides reduced the apparent mass of each protein to 34 and 36 kDa (Fig. 2B, lanes 6 and 8), and
these values are similar to the calculated masses of 34.5 and 36.6 kDa,
respectively, for the polypeptides alone. The specificity of these
glycosidases suggests that the oligosaccharide structures consist
almost exclusively of monosialylated or disialylated structures, which
are related to
Sia
The number of mucin repeats in enteropeptidase varies considerably,
with one repeat in human, two in bovine and porcine, three in rat, and
four in mouse enteropeptidase (21). Such heterogeneity suggests that
targeting signals could reside in single mucin repeats. Therefore, the
behavior of chimeric proteins containing mucin repeat A or B was
examined. When stably expressed in MDCK cells, constructs containing
either mucin repeat were secreted apically (Fig.
3A and Table I), indicating
that both repeats possess functional targeting signals. Glycosidase
digestions demonstrated that each mucin repeat was
O-glycosylated (Fig. 3B) and not
N-glycosylated (data not shown).
Glycosylation and Targeting--
The enteropeptidase mucin repeats
contain 11 Ser/Thr residues that could be
O-glycosylated (Fig. 1A) (20). Replacement
of all Ser/Thr residues by alanine abolished the targeting activity of
mucin repeat A, whether present in one or three copies (Fig. 4 and Table I). Mucin repeat B had
reduced but significant apical targeting activity after all Ser/Thr
residues were mutated, suggesting that features of the amino acid
sequence may contribute to targeting (Fig. 4 and Table I). Glycosidase
digestions confirmed that none of these constructs had
N-linked or O-linked oligosaccharides (data not
shown).
The role of O-linked glycosylation was investigated further
by restoring selected Ser/Thr residues, singly or in clusters. For mucin repeat A, all constructs that contained any Ser/Thr residues were O-glycosylated and constructs
MAS172A/T173A/S175A-GFP and MAT173A-GFP lost
apical targeting activity (Fig. 5 and
Table I). These data suggest that O-glycosylation is not
sufficient for apical targeting and that a central cluster of Ser/Thr
residues may be important for apical targeting of
MA-GFP. As observed for mucin repeat A, all mucin
repeat B constructs that contained Ser/Thr residues were
O-glycosylated, including
MB-T184A/T185A/S186A-GFP, which has only one Thr residue
(Fig. 6). Mutation of Thr-185 alone randomized the targeting of MB-T185A-GFP, whereas all other
mutants tested retained apical targeting similar to that of
MB-GFP (Fig. 6 and Table I). Therefore,
O-glycans appear not to be required, but the modification of
selected Ser/Thr residues can inhibit or potentiate the apical
targeting of MB-GFP.
MAB-GFP Does Not Associate with Lipid
Rafts--
Sphingolipid and cholesterol-rich membrane microdomains, or
lipid rafts, have been proposed to participate in the delivery of many
apically sorted proteins (23), but enteropeptidase appears to be an
exception. Bovine enteropeptidase, a type II transmembrane protein, was
targeted to the apical membrane of transfected MDCK cells, but
association with lipid rafts could not be demonstrated (2). Similar
results were obtained for secreted MAB-GFP (Fig. 7), which contains a subset of the apical
targeting signals found in full-length enteropeptidase. Triton X-100
lysates were prepared from MDCK cells expressing GFP and
MAB-GFP, and the extracts were fractionated by sucrose
gradient centrifugation (2). Caveolin, a marker for lipid rafts, was
recovered in low-density fractions 4-5 near the top of the gradient
(Fig. 7). In contrast, GFP (Fig. 7A) and MAB-GFP
(Fig. 7B) were recovered in denser fractions at the bottom
of the gradient. Therefore, MAB-GFP is secreted apically but does not appear to associate intracellularly with lipid rafts.
The results presented here show that small mucin-like peptides of
12-15 amino acid residues, derived from bovine enteropeptidase, can
direct the apical secretion of GFP. O-glycosylation
contributes to the efficiency of apical targeting, but no specific
O-glycan appears to be required. For mucin repeat B, some
apical targeting activity persists after all glycosylation sites are
eliminated by mutagenesis (Table I). Several O-glycosylated
peptides have no targeting activity in this system, indicating that
glycosylation alone is not sufficient. Some apical membrane and
secreted proteins associate with lipid rafts, but this is not the case
for transmembrane or soluble variants of enteropeptidase (2) Apically
secreted mucin-GFP chimeric proteins also did not associate with rafts (Fig. 7). Therefore, enteropeptidase mucin-like domains contain relatively compact apical targeting signals that depend on both O-glycosylation and amino acid sequence context but appear
to function independent of lipid rafts. This appears to be the first example of an O-glycosylated peptide that can confer apical
targeting on a randomly sorted heterologous protein.
Previous studies have implicated O-glycans in the apical
targeting of several proteins, although the evidence is indirect and
mixed. Sucrase-isomaltase (12) and the neurotrophin receptor (16) are
membrane proteins with O-glycosylated stalk regions adjacent
to their transmembrane domains. Both are targeted to the apical surface
of MDCK cells, and deletion of their stalk domains abolishes apical
targeting (12, 16). These data suggest that O-glycosylation
mediates apical targeting, but other studies suggest that sorting
signals reside elsewhere. Replacement of the N-terminal transmembrane
domain with a cleaved signal peptide causes the random secretion of
sucrase-isomaltase despite the presence of the
O-glycosylated stalk, suggesting that membrane association
contributes to targeting and the stalk region is not sufficient (12).
The replacement of Gln-117 by Arg, at a location near the N terminus of
the isomaltase domain, causes random delivery to the apical and
basolateral membranes of MDCK cells and suggests that features of
sucrase-isomaltase distinct from its stalk region are necessary for
targeting (24). Furthermore, O-glycosylation does not
correlate with the apical targeting of aminopeptidase N and
lactase-phlorizin hydrolase, which are apical membrane proteins with
O-glycosylated stalk regions near their transmembrane
domains. Deletion of these stalk regions does not affect their apical
targeting in MDCK cells (25, 26).
These various studies indicate that O-glycosylation can
contribute to apical targeting but is not sufficient and sometimes is
not necessary. Enteropeptidase mucin-like domains exhibit similar properties, in that their apical targeting activity is diminished by
mutagenesis of only certain O-glycosylated residues
(e.g. Thr-173 or Thr-185, Table I). Such residues might bear
oligosaccharides that are particularly potent targeting signals,
possibly disialylated species. Clustered O-linked
oligosaccharides might be required for targeting, and disruption of a
central member of the cluster could be sufficient to impair targeting.
Although the accumulating data do not exclude a direct targeting
function for O-glycans, other models appear to be equally
plausible. For example, O-glycans could play an indirect
role, supporting a primary targeting signal that resides elsewhere in
the protein (27). Alternatively, features of protein and
oligosaccharide structure could collaborate to form a complete
targeting signal that contains both carbohydrate and peptide
determinants, in which case these structures could be spatially close
together. Any of these models would be compatible with the observation
that short O-glycosylated peptides can target GFP for apical secretion.
The association of membrane proteins with lipid rafts often correlates
with their delivery to apical cell surfaces but, as noted for the
proposed relationship between O-glycosylation and targeting,
many exceptions are known. Sucrose-isomaltase, aminopeptidase N,
aminopeptidase A, and dipeptidyl peptidase IV associate mainly with
lipid rafts (12, 24, 28), whereas lactase-phlorizin hydrolase (29),
maltase-glucoamylase (28), and (full-length) enteropeptidase (2) do
not, yet all are apical membrane enzymes of the intestinal brush
border. Similar variability has been reported for apically secreted
proteins, natural or engineered. Thyroglogulin (30) and soluble
prohormone convertase 2 (31) have been recovered bound to lipid rafts,
whereas clusterin (32) and the ectodomains of placental alkaline
phosphatase and the neurotrophin receptor have not (33). Similarly, the
mucin-like domains of enteropeptidase direct apical secretion of GFP in
the absence of a demonstrable association with lipid rafts (Fig. 7).
These results indicate that stable binding to lipid rafts is not a
requirement for apical protein sorting. Weaker associations that are
not preserved during the isolation of rafts might contribute to
targeting, and different experimental approaches would be required to
identify them.
Apical targeting can occur with or without stable raft association,
glycosylation, or membrane anchoring. However, the apical targeting of
specific proteins has been found to depend on one or more of these
particular features. This heterogeneity may reflect the existence of
multiple apical sorting pathways that employ distinct signals.
Alternatively, the apparent diversity of signals could reflect an
indirect role of various protein structures, such as oligosaccharides,
in the stabilization of a single class of sorting determinant that
could be protein-based (27). These classes of mechanism need not be
mutually exclusive. Studies of the relatively simple targeting signals
present in enteropeptidase mucin-like domains may facilitate the
evaluation of these models.
*
This work was supported in part by National Institutes of
Health Grant DK50053.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 correspondence should be addressed: Howard Hughes
Medical Inst., Washington University School of Medicine, 660 S. Euclid
Ave., Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax:
314-454-3012; E-mail: esadler@im.wustl.edu.
Published, JBC Papers in Press, November 27, 2001, DOI 10.1074/jbc.M109857200
The abbreviations used are:
MDCK cells, Madin-Darby canine kidney II cells;
MA and MB, mucin-like repeat A and B, respectively;
MAB, both
mucin-like repeats A and B;
GFP, green fluorescent protein.
Mucin-like Domain of Enteropeptidase Directs Apical Targeting in
Madin-Darby Canine Kidney Cells*
and Department of Pathology and Immunology and
the § Howard Hughes Medical Institute, Departments of
Medicine and Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,3-sialylation with
benzyl-2-acetamido-2-deoxy--D-galactopyranoside (GalNAc--O-benzyl) blocks the apical transport of several
brush border-associated glycoproteins, including dipeptidylpeptidase IV
(14) and the mucin MUC1 (17), suggesting that the terminal structures
of certain O-linked oligosaccharides or mucin-like domains
may direct apical targeting in some polarized cells. In general, these
studies of O-glycosylation-dependent targeting have depended on the abolition of targeting by mutagenesis or metabolic
inhibitors, and none has directly identified a signal that is
sufficient for apical targeting.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Samples were analyzed by SDS-PAGE on 5-15%
Tris-glycine gels (Bio-Rad) under reducing conditions and transferred
by electroblotting onto nitrocellulose membranes (pore size 0.45 µm,
Bio-Rad). Membranes were incubated with monoclonal anti-GFP antibody
(1:10,000 dilution in 2% nonfat milk in Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) at room temperature overnight. After washing, the membrane was incubated with peroxidase-conjugated rabbit anti-mouse IgG (1:10,000 dilution in 2% nonfat dry milk in
Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) (Dako
Corp., Carpinteria, CA) at room temperature for 2 h. Bound
antibody was detected with the chemiluminescent ECL detection system
(Amersham Biosciences) and XAR5 film (Eastman Kodak Co.). Films
were exposed for several different times to obtain density signals
within the linear response range. After optical scanning, signals were
quantitated with NIH Image 1.6.1 (developed at the National Institutes
of Health and available on the Internet at
rsb.info.nih.gov/nih-image/).
-mercaptoethanol and cooled to room temperature. Detergent Nonidet
P-40 (Roche Molecular Biochemicals) was added to a final concentration
of 2%, and the samples were incubated at 37 °C for 18 h
without or with 1 milliunit/microliter Streptomyces
plicatus endoglycosidase H. Reactions with
peptide-N-glycosidase F (PNGase F) were prepared similarly
except that the buffer was 20 mM sodium phosphate, pH 7.5, 50 mM EDTA, 5% -mercaptoethanol, and 0.2% SDS, and the
amount of added enzyme was 0.5 unit. To analyze proteins for
O-glycosylation, samples of concentrated conditioned media
(40 µl) were digested with Arthrobacter ureafaciens neuraminidase (0.5 milliunit/microliter) at 37 °C for 1 h in
100 mM sodium acetate, pH 5.0, followed by digestion with
O-glycanase (0.05 milliunit/microliter) in 100 mM sodium citrate phosphate, pH 6.0, and 100 µg/ml bovine
serum albumin at 37 °C for 16 h. Digested samples were analyzed
by SDS-PAGE and Western blotting with monoclonal anti-GFP IgG as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Enteropeptidase mucin repeats and chimeric
proteins. A, structure of enteropeptidase and its mucin
repeats. Full-length enteropeptidase has a transmembrane segment
(TM), mucin repeats, two low density lipoprotein
receptor-like repeats (LDLR), two complement component
C1r/s repeats, a MAM domain, a macrophage
scavenger receptor-like domain (MSCR), and a serine protease
domain with active site residues His, Asp, and Ser. The segment between
the TM domain and first LDLR repeat comprises residues 50-197,
includes two mucin repeats (A and B), and has
four potential N-glycosylation sites (filled
diamonds). Mucin repeats A and B comprise
residues 166-192 and include one potential N-glycosylation
site (underlined). B, schematic structure of
chimeric proteins. Constructs contain a prothrombin signal peptide and
GFP, between which are inserted combinations of the following motifs:
His6 tag, mucin repeat A (MA), mucin
repeat B (MB), or both mucin repeats
(MAB).
View larger version (50K):
[in a new window]
Fig. 2.
Targeting and glycosylation of chimeric
proteins. A, transfected MDCK cell lines expressing the
indicated proteins were grown on Transwell filters, and samples from
the apical (Ap) and basolateral (Bl) compartments
were analyzed by gel electrophoresis and Western blotting as described
under "Experimental Procedures." The mass of marker proteins (kDa)
is indicated at the left. A representative experiment is
shown. Data from at least three independent experiments are summarized
in Table I. B, the indicated proteins were treated with (+)
or without ( ) neuraminidase and O-glycanase and analyzed
by gel electrophoresis and Western blotting.
Polarity of enteropeptidase-GFP chimeric protein secretion
2-3Gal1-3(Sia2-6)GalNAc-O-Ser/Thr (22).
Although mucin repeat A contains a potential N-glycosylation
site (Asn-Phe-Ser), further digestion with N-glycanase or
endoglycosidase H did not affect the mass of MAB-GFP and
His6-MABGFP, indicating that this site is not
utilized (data not shown). As expected, GFP (27.5 kDa) was not
glycosylated (Fig. 2B, lanes 1 and 2).
However, His6-gGFP was O-glycosylated,
presumably on one or more of the Ser/Thr residues flanking the
His6 tag sequence. The removal of O-linked
oligosaccharides reduced the apparent mass from 33-37 to 30 kDa (Fig.
2B, lanes 3 and 4), which is similar
to the calculated mass of 29.6 kDa for the polypeptide alone. Despite
this O-glycosylation, His6-gGFP was secreted
randomly (Table I).
View larger version (56K):
[in a new window]
Fig. 3.
Targeting activity of individual mucin
repeats. The indicated proteins were analyzed for the polarity of
protein secretion (left) and sensitivity to digestion with
neuraminidase and O-glycanase (right), as
described under Fig. 2.
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[in a new window]
Fig. 4.
Effect of mutating all
O-glycosylation sites. Codons for all Ser/Thr
residues were changed to Ala in mucin repeat A
(MAmall) or mucin repeat B (MBmall),
and the corresponding sequences were inserted between a prothrombin
signal peptide and GFP. One construct contained three copies of the
mutated MAmall repeat (MAmall×3). The polarity
of protein secretion was assessed as described under Fig. 2.
View larger version (60K):
[in a new window]
Fig. 5.
Mutation of selected Ser/Thr residues in
mucin repeat A. The amino acid sequence inserted before GFP is
given in Table I for the indicated constructs. The polarity of protein
secretion (Panel A) and sensitivity to digestion with
neuraminidase and O-glycanase (Panel B) were
assessed as described under Fig. 2.
View larger version (64K):
[in a new window]
Fig. 6.
Mutation of selected Ser/Thr residues in
mucin repeat B. The amino acid sequence inserted before GFP is
given in Table I for the indicated constructs. The polarity of protein
secretion (Panel A) and sensitivity to digestion with
neuraminidase and O-glycanase (Panel B) were
assessed as described in the Fig. 2 legend.
View larger version (52K):
[in a new window]
Fig. 7.
Relationship of chimeric MAB-GFP
to lipid rafts. MDCK cells expressing GFP (Panel A) or
MAB-GFP (Panel B) were lysed with ice-cold 1%
Triton X-100, and the lysate was subjected to sucrose density gradient
centrifugation. Fractions were collected from the top (fraction
1) to the bottom (fraction 10) of each gradient and
analyzed by Western blotting for GFP or caveolin as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Hermon-Taylor, J.,
Perrin, J.,
Grant, D. A. W.,
Appleyard, A.,
and Magee, A. I.
(1977)
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