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J. Biol. Chem., Vol. 276, Issue 26, 23506-23510, June 29, 2001
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From the
Received for publication, April 27, 2001, and in revised form, May 4, 2001
The impaired sorting profile to the apical
membrane of human intestinal sucrase-isomaltase is the underlying cause
in the pathogenesis of a novel phenotype of intestinal congenital
sucrase-isomaltase deficiency. Molecular characterization of
this novel phenotype reveals a point mutation in the coding region of
the sucrase-isomaltase (SI) gene that results in an amino acid
substitution of a glutamine by arginine at residue 117 of the
isomaltase subunit. This substitution is located in a domain revealing
features of a trefoil motif or a P-domain in immediate vicinity of the
heavily O-glycosylated stalk domain. Expression of the
mutant SI phenotype in epithelial Madin-Darby canine kidney cells
reveals a randomly targeted SI protein to the apical and basolateral
membranes confirming an exclusive role of the Q117R mutation in
generating this phenotype. Unlike wild type SI, the mutant protein is
completely extractable with Triton X-100 despite the presence of
O-glycans that serve in the wild type protein as an apical
sorting signal and are required for the association of SI with
detergent-insoluble lipid microdomains. Obviously the
O-glycans are not adequately recognized in the context of
the mutant SI, most likely due to altered folding of the P-domain that
ultimately affects the access of the O-glycans to a
putative sorting element.
The composition and function of the plasma membrane of polarized
cells are maintained by a complex intracellular traffic moving cell
surface glycoproteins between organelles. This requires the recognition
and sorting of different classes of proteins not only during
biosynthesis, but also during distributive events to the diverse
cellular compartments (1). Oftentimes altered and defective intracellular trafficking of proteins due to single point or deletion mutations in the coding region of the gene result in pathological disorders (2, 3). Investigating the molecular basis of naturally occurring mutant protein phenotypes in diseases constitutes therefore a
powerful means to unravel the molecular mechanisms underlying intracellular protein transport and sorting. A small intestinal disorder directly associated with a folding mutant and defective intracellular protein transport is congenital sucrase-isomaltase deficiency (CSID).1 CSID is
an autosomal recessive disease that is characterized by an absent
sucrase activity within the sucrase-isomaltase (SI) enzyme complex,
while the isomaltase activity can vary from absent to normal. The
disease is clinically manifested as an osmotic-fermentative diarrhea
upon ingestion of di- and oligosaccharides (4). Several phenotypes of
the disease have been characterized on the basis of cellular
mislocalization or aberrant function of the SI mutant protein. SI is a
type II membrane-bound glycoprotein of the intestinal brush border
comprising two strongly homologous subunits, sucrase and isomaltase
(5-8). These two domains originate from a large polypeptide precursor,
pro-SI, by tryptic cleavage occurring in the intestinal lumen and
ultimately maintain a strong association through noncovalent ionic
interactions (9, 10). This enzyme complex is a heavily N-
and O-glycosylated protein (10). Particularly O-glycosylation is critical for targeting of SI to the
apical membrane through direct association in detergent-insoluble lipid rafts (11).
In this paper we describe a novel phenotype of CSID and the
corresponding mutation, which results in a random distribution of the
SI protein at the apical and basolateral membrane. Here a point
mutation, an adenine to glutamine at nucleotide 412 in the coding
region of the isomaltase subunit, results in a substitution of
glutamine to arginine at amino acid residue 117. Defects in polarized
protein sorting implicated in the pathogenesis of diseases have been
rarely observed, perhaps because such defects have lethal consequences
during early stages of development. Abnormalities in protein
trafficking in polycsytic kidney disease and cystic fibrosis have been
shown to be associated with mutations in the coding regions of the
corresponding genes (3, 12). The mutation identified in this report,
however, is to our knowledge the first of its kind, in which aberrant
polarized sorting of an intestinal protein is disease-associated. By
establishing the functional details of this mutation and its
implication in the polarized sorting of SI, the structure and function
of the sorting elements possibly interacting within the region
harboring the mutation could be elucidated.
Processing of Biopsy Samples--
CSID in a 4-year-old patient
with acidic diarrhea was suggested by a sucrose tolerance test while
under total parenteral nutrition. Biopsy specimens were obtained and
immediately frozen in liquid nitrogen for enzyme activity measurements
and RNA preparation.
Isolation and Mutagenesis of SI cDNA--
Cloning of the SI
cDNA from the patient's mucosal cells followed the same strategy
as described previously (13). Messenger RNA was isolated using
Dynabeads© Oligo(dT) (Dynal, Oslo, Norway), and cDNA
was synthesized with the First Strand cDNA Synthesis
Kit© (Amersham Pharmacia Biotech, Freiburg,
Germany) using random hexamer nucleotide primers. For each PCR reaction
Mutation of A to G was confirmed by sequencing, and the plasmid
obtained was denoted phSIA/G.
Transfection and Metabolic Labeling of MDCK Cells--
MDCK
cells were co-transfected with phSIA/G and the neomycin
resistance vector pcDNA3 (Invitrogen, Groningen, The Netherlands) using the calcium phosphate procedure as described previously (15). Stable cell lines expressing wild type or mutant SI were selected
by using 0.25 mg/ml active G418-containing selection medium (Life
Technologies, Inc.). Isolated clones were screened by metabolic
labeling and immunoprecipitation as described below. In control
experiments the MDCK-SIWT cell line that expresses wild
type SI was used (16). MDCK cells were biosynthetically labeled with 80 µCi of L-35S-RedivueTM
PRO-MIXTM (Amersham Pharmacia Biotech, Freiburg, Germany)
as described by Naim and co-workers (14) either continuously or by
employing a pulse-chase protocol. Here, cells were pulse-labeled for 30 min and chased for different periods of time with cold methionine. MDCK
cells were also biosynthetically labeled in the presence or absence of
benzyl-GalNAc
(benzyl-N-acetyl- Immunoprecipitations--
Biosynthetically labeled cells were
solubilized for 1 h at 4 °C in lysis buffer (25 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 0.5% Triton X-100,
0.5% sodium deoxycholate and a mixture of protease inhibitors
(all from Sigma, Deisenhofen, Germany) containing 1 µg/ml pepstatin,
5 µg/ml leupeptin, 17.4 µg/ml benzamidine, and 1 µg/ml
aprotinin). Usually 1 ml of ice-cold lysis buffer was used for each
100-mm culture dish (~2-4 × 106 cells). The
detergent extracts were centrifuged for 1 h at 100,000 × g at 4 °C, and the supernatants were immunoprecipitated
according to Naim et al. (10) with four epitope-specific
mAbs directed against sucrase, isomaltase, or SI. These were
products of the following hybridomas: HBB 1/691, HBB 2/614, HBB 2/219,
and HBB 3/705 (19) generously provided by Dr. H. P. Hauri
(Biocenter, Basel, Switzerland) and Dr. E. E. Sterchi (University
of Bern, Bern, Switzerland).
Cell surface immunoprecipitations of wild type and mutant SI antigens
from MDCK cells grown on membrane filters were performed as described
previously (11). Here, MDCK cells were labeled for 4 h with 100 µCi of [35S]methionine (Amersham Pharmacia Biotech,
Freiburg, Germany). Wild type or mutant SI expressed at the cell
surface was immunoprecipitated from intact cells by addition of mAb
anti-SI to either the apical or basolateral compartments. The cells
were solubilized in the lysis buffer described above, and the
antigen-antibody complex was captured by protein A-Sepahrose (Amersham
Pharmacia Biotech, Freiburg, Germany).
Association of Wild Type and Mutant SI with Lipid
Microdomains--
The association of SI with
sphingolipid/cholesterol-rich microdomains was assessed in detergent
extractability assays using Triton X-100 essentially as
described previously (11). Here, MDCK cells containing either
wild type or mutant SI were biosynthetically labeled for 4 h and
solubilized for 2 h at 4 °C in a lysis buffer containing 1%
Triton X-100, 25 mM Tris-HCl, pH 8.0, 50 mM
NaCl. The detergent extracts were centrifuged at 100,000 × g for 1 h at 4 °C, and the supernatant or the
detergent-soluble fraction was retained for immunoprecipitation. The
pellet containing the detergent-insoluble proteins was dissolved by
boiling in 1% SDS for 10 min. Thereafter a 10-fold volume of a buffer
containing 1% Triton X-100 was added, and these extracts as well as
the Triton X-100-soluble fraction were immunoprecipitated with the
mixture of mAb anti-SI antibodies mentioned above that recognize native and denatured forms of SI (19).
Other Procedures--
Washing, digestion of immunoprecipitates
with endo- Clinical assessment of the sucrose malabsorption was achieved by
the hydrogen breath test and confirmed by measuring the enzymatic activities of sucrase and isomaltase in intestinal biopsy samples. Both
enzymes revealed drastic reduction in their activities with that of
sucrase being 2 IU/mg and of isomaltase 5 IU/mg of protein. In
comparison, the normal activity of sucrase and that of isomaltase in
the small intestine range from 40 to 60 IU/mg of protein. Another disaccharidase in the small intestine, lactase-phlorizin hydrolase, revealed 42 IU/mg of protein, which is in the normal range, indicating that the sucrose malabsorption is restricted to the sucrase-isomaltase complex. The next step was therefore to analyze the molecular basis and
the corresponding mechanisms leading to this defect. Due to the limited
amount of the biopsy specimen, we were not able to investigate the
biosynthesis and processing of the SI complex in organ culture as has
been performed with other cases of CSID (8, 22). However, the tissue
available was adequate for the isolation of the cDNA encoding the
patient's SI by reverse transcriptase-PCR following a similar
strategy to that described previously (22, 23). The correctness
of the cDNA was corroborated by sequencing in both directions 5'-3'
and vice versa (24). A single point mutation could be identified in the
patient's cDNA replacing an adenine by a guanine at nucleotide 412 (Fig. 1). This mutation results in an
amino acid substitution of glutamine to arginine at residue 117 of the
isomaltase subunit. Limited algorithmic analysis of this region
revealed a trefoil motif or a P-domain also found in
maltase-glucoamylase (25), a brush border protein with striking
homologies to sucrase-isomaltase. These domains have been shown to be
implicated in protein-protein interaction or lectin-like interactions,
in the pathology of mucous epithelia and in modulating cell growth (for
a review, see Ref. 26). To analyze the detailed function and the
significance of this mutation in this CSID phenotype, we expressed the
mutant SI cDNA in the epithelial cell line MDCK cells, which do not
express endogenous SI. This cell line is particularly convenient for
these analyses borne out by its successful application in the
heterologous expression of several intestinal proteins, including SI,
which revealed structural and functional features as well polarized sorting behavior similar to their endogenously expressed wild type
counterparts in intestinal epithelial cells (16). Biosynthesis and
processing of wild type and mutant SI in MDCK are shown in Fig.
2. Continuous metabolic labeling of the
cells containing the wild type SI protein for 6 h revealed a
210-kDa mannose-rich polypeptide (SIh) that shifted upon
reaction with endo H to a smaller size and an endo H-resistant 245-kDa
complex glycosylated mature protein (SIc) (Fig.
2A, denoted WT). A similar band pattern was also
observed with the cells containing the mutant protein (Fig.
2A, denoted mutant). However, the proportion of
the complex glycosylated protein in the total synthesized and processed
SI protein was significantly less than that in its wild type
counterpart, suggesting a slower rate of processing of the mannose-rich
to the complex form. We therefore determined the transport and
processing kinetics of both wild type and mutant SI employing a
pulse-chase protocol. At early chase time points wild type SI appears
as a 210-kDa mannose-rich SIh polypeptide that is gradually
converted with increasing chase periods to the complex glycosylated
SIc species (Fig. 2B, denoted WT).
Qualitatively a similar biosynthetic pattern could be observed for the
mutant SI protein exemplified by the mannose-rich and complex
glycosylated polypeptides (Fig. 2B, denoted
mutant). However, the t1/2 time for the
conversion of the mannose-rich mutant SI into a complex glycosylated
form was 4.5 h as compared with ~1.5 h of the wild type protein
(Fig. 2C) compatible with delayed transport kinetics of this
mutant from the ER to the Golgi apparatus. Clearly the effects on the
intracellular transport of the mutant SI are due to the Q117R
substitution, which has most likely altered some of the structural
features of SI, perhaps in the domain containing the mutation itself,
and may result in an increased degradation of mutant SI. This could
explain the substantial reduction in the activity of isomaltase
and sucrase in the biopsy specimen as well as in transfected
MDCK cells. The Q117R substitution itself does not generate a novel
sequon for N-linked glycosylation (27) and at the same time
does not conform with a putative O-glycosylation motif (28).
This strongly suggests that potential alterations in mutant SI are not
due to differences in the N-glycosylation pattern, which is
often implicated in the folding pattern of membrane glycoproteins
already during early stages of posttranslational processing in the ER
(29, 30). The pattern of O-glycosylation is similar in both
cases, since treatments of cells with benzyl-GalNAc, an inhibitor of
O-glycosylation (11, 18), revealed reductions in the size of
the wild type and the mutant SI forms reminiscent of comparable
contents of O-glycans in both protein forms (Fig. 2D).
The next step was to determine whether complex glycosylated mutant SI
is correctly targeted to the apical membrane or whether the mutation is
associated with the generation of a randomly or missorted SI phenotype.
For this MDCK cells expressing the mutant and wild type proteins were
grown on membrane filters, and their biosynthesis and polarized
transport were followed by isolating the cell surface antigens from the
apical or basolateral compartments with a mAb anti-SI. Fig.
3 shows that the wild type SI protein was
predominantly found at the apical membrane with almost 90% sorting
fidelity, confirming previous data (16). By contrast, the mutant
protein followed a random pattern of transport to the cell surface,
since it appeared almost equally at both sides of the membrane. The
results demonstrate therefore that the mutation Q117R is responsible
for the generation of a mistargeted SI phenotype. This finding was at
first glance surprising, since mutant SI similar to the wild type
protein is O-glycosylated, and apical sorting of SI is known
to take place through O-linked carbohydrates as a sorting
signal and association of the protein with Triton X-100-insoluble cholesterol- and sphingolipids-rich membrane microdomains (11). The
sorting mechanism of mutant SI was therefore examined and compared with
that of the wild type protein. As shown in Fig. 4 a proportion of wild type SI was
recovered in the Triton X-100-insoluble pellet, while the mutant SI
protein was exclusively recovered in the detergent-soluble phase.
Mutant SI therefore does not associate with lipid microdomains despite
the presence of O-glycans that are required for this
association and for apical sorting. The fact that the mutation is not
directly implicated in O-glycosylation strongly suggests
that a protein structure implicated in the sorting process has been
altered. A Ser/Thr-rich stalk region immediately upstream of the
transmembrane domain of SI is heavily O-glycosylated and is
required for the association of SI with lipid rafts (16). Deletion of
this domain eliminates almost completely O-glycosylation of
SI, thus affecting its detergent extractability concomitant with loss
of its sorting fidelity, while the transport competence of SI per
se remains unchanged. As shown here, the mutation Q117R induces
similar effects with respect to detergent solubility and sorting of SI
as the deletion mutants do. Unlike the deletion mutants, however,
mutant SI is extensively O-glycosylated, but it is possible
that these chains are not adequately recognized by a putative sorting
element, a lectin-like protein for example. A recognition and
subsequent binding would trigger the sequence of events leading to
association of SI with lipid microdomains and its delivery to the
apical membrane. The Q117R mutation is located in the P-domain in the
immediate vicinity of the O-glycosylated stalk domain (24).
It is likely that structural alterations in this domain have partially
or completely masked the O-linked carbohydrates and thus
hampered their recognition. Along this concept a correctly folded
P-domain would be implicated in the sorting event by providing ample
spatial requirements required for an adequate recognition of the
O-glycosylated sorting signal by a specific sorting protein.
The existence, identity, and structure of components of the apical
sorting machinery, in particular that of a putative cellular
lectin-like protein specific for binding residues comprising
O-linked chains, such as galactose or
N-acetylgalactoseamine, are far from being unraveled. It has
been proposed that lectin-like proteins in trans-Golgi network and
post-Golgi compartments may recognize N-linked chains that
function as apical sorting signals on some proteins and thus mediate
their incorporation into apical carrier vesicles (31). VIP 36 is one of
these candidates that binds specifically galactose and
N-acetylgalactoseamine residues (32) and can therefore
function as a receptor for both N- and O-linked
glycans. Its detection, however, in basolateral carriers has raised
questions as to its specific role in apical sorting. Nevertheless, some apical proteins are targeted to the apical membrane
by transcytosis through the basolateral membrane and would also be
transiently found in basolateral vesicles (33).
Molecular Basis of Aberrant Apical Protein Transport in an
Intestinal Enzyme Disorder*
,,,¶
Department of Physiological Chemistry,
School of Veterinary Medicine, Bünteweg 17, D-30559 Hannover,
Germany and the § University Children's Hospital, D-41489
münster, Münster, Germany
ABSTRACT
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MATERIALS AND METHODS
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-D-galactosaminide) (Sigma,
Deisenhofen, Germany), an inhibitor of O-glycosylation (17,
18).
-N-acetylglucosaminidase H (endo H), and
processing for analysis by for SDS-PAGE on 6% slab gels (20) were
according to Naim et al. (10). Following electrophoresis the
radioactive bands were visualized using a phosphorimager (Bio-Rad,
Munich, Germany) or autoradiography. Isomaltase, sucrase, and lactase
activities were measured according to Dahlqvist (21).
RESULTS AND DISCUSSION
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MATERIALS AND METHODS
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View larger version (20K):
[in a new window]
Fig. 1.
Location of the Q117R mutation in SI.
Structural features of pro-SI were deduced from biosynthetic studies
(8) and cDNA cloning (34). As a type II membrane glycoprotein
(Nin/Cout) SI is synthesized with an
uncleavable signal sequence, which also serves as a membrane anchoring
domain (34). The first 12 amino acid residues represent the cytoplasmic
tail, followed by a membrane anchor of 20 amino acids containing the
signal sequence for ER translocation and a Ser/Thr-rich stalk domain of
28 amino acids that is considered to be part of the isomaltase subunit.
Isomaltase ends with amino acid residue Arg1007, and
sucrase starts immediately thereafter with Ile1008. The
Arg/Ile peptide sequence between isomaltase and sucrase is a trypsin
site where the mature large precursor pro-SI is cleaved in the
intestinal lumen by pancreatic trypsin (19). The location of the Q117R
mutation in the CSID patient is indicated. Part of the DNA sequence
flanking the mutation is shown. The mutation A to G at nucleotide 412 is highlighted. The corresponding amino acid substitution
glutamine to arginine at residue 117 is shown in bold. Amino
acid residues around the mutation are indicated in italic
numbers.
View larger version (21K):
[in a new window]
Fig. 2.
Expression of mutant SI cDNA in MDCK
cells. A, stable MDCK cells expressing wild type SI
(indicated WT) or mutant SI (indicated mutant)
were biosynthetically labeled with [35S]methionine for
6 h. The cell lysates were immunoprecipitated with mAb anti-SI,
and the immunoprecipitates were treated or not treated with endo H. The
samples were finally analyzed by SDS-PAGE on 6% slab gels and
autoradiography. SIh is the mannose-rich endo H-sensitive
form, and SIc is the complex glycosylated endo H-resistant
form. B, stable MDCK cells containing either wild type SI
(WT) or mutant SI (mutant) were pulse-labeled for
30 min with [35S]methionine followed by a chase with cold
methionine for the indicated time points. The cell lysates were
immunoprecipitated with mAb anti-SI and subjected to SDS-PAGE on 6%
slab gels. SIn, mannose-rich SI;
SIc, complex glycosylated SI. C,
Quantification of the gels shown in B. D, stable
MDCK cells were biosynthetically labeled for 4 h (WT)
or 8 h (mutant) with [35S]methionine in
the presence or absence of the 4 mM benzyl-GalNAc, an
inhibitor of O-glycosylation. Detergent extracts of the
cells were immunoprecipitated with mAb anti-SI and analyzed by SDS-PAGE
on 6% slab gels.
View larger version (15K):
[in a new window]
Fig. 3.
Polarized expression of mutant SI in MDCK
cells. A, MDCK cells expressing wild type
(WT) and mutant SI were grown on transmembrane filters,
labeled with [35S]methionine for 4 h followed by
cell-surface immunoprecipitation of SI from the apical (panel
a) or basolateral (panel b) membranes. The
immunoprecipitates were subjected to SDS-PAGE. B,
quantification of the proportions of complex glycosylated SI shown in
A.
View larger version (16K):
[in a new window]
Fig. 4.
Mutant SI is not associated with lipid
microdomains. A, MDCK cells expressing wild type
(WT) and mutant (mutant) SI were biosynthetically
labeled with [35S]methionine for 4 h. Triton X-100
extracts were centrifuged at 100,000 × g for 1 h
at 4 °C. The supernatants (S) as well as the pellets
after lysis with 0.1% SDS (P) were immunoprecipitated with
a mixture of mAb anti-SI antibodies that recognizes native as well as
denatured forms of the protein. The immunoprecipitates were thereafter
analyzed by SDS-PAGE. B, quantification of the proportions
of SI detected in the supernatants and pellets shown in
A.
In summary, the first identification of a naturally occurring mutation
responsible for random transport of an otherwise highly polarized
protein of the brush border membrane provides a strong indication for
the existence of a receptor protein that is required to recognize a
sorting signal. The fact that the available O-linked glycans
in mutant SI are not recognized by such a putative receptor suggests
that its conformation should properly fit in the context of three
structures of the SI protein, the P-domain, the
O-glycosylated stalk region, and the transmembrane domain,
which has been assigned a decisive role in the sorting pathway of SI
(16). Strategies could therefore be designed to discover this putative
protein by utilizing the SI protein and its mutant phenotype as
promising models.
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ACKNOWLEDGEMENTS |
|---|
We thank Dr. Hans-Peter Hauri, Biozentrum, University of Basel and Dr. Erwin Sterchi, Institute of Biochemistry and Molecular Biology, University of Bern for generous gifts of monoclonal anti-SI antibodies.
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FOOTNOTES |
|---|
* This work was supported by Grant Na 331/1-2 form the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Sonderforschungsbereich 280 (both 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 correspondence should be addressed: Dept. of Physiological Chemistry, School of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany. Tel.: 49-511-953-8780; Fax: 49-511-953-8585; E-mail: hassan.naim@tiho-hannover.de.
Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.C100219200
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ABBREVIATIONS |
|---|
The abbreviations used are:
CSID, congenital
sucrase-isomaltase deficiency;
SI, sucrase-isomaltase (all forms);
pro-SI, uncleaved sucrase-isomaltase;
ER, endoplasmic reticulum;
mAb, monoclonal antibody;
endo H, endoglyosidase H;
MDCK, Madin-Darby canine
kidney cells;
benzyl-GalNAc, benzyl-N-acetyl--D-galactosaminide;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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ABSTRACT
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MATERIALS AND METHODS
RESULTS AND DISCUSSION
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