Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder Nikolaj

21 Pages·2001·0.15 MB·English

JBC Papers in Press. Published on May 4, 2001 as Manuscript C100219200
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Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder
Nikolaj Spodsberg‡, Ralf Jacob‡, Marwan Alfalah‡, Klaus-Peter Zimmer# and Hassan Y.
Naim‡*
‡Department of Physiological Chemistry, School of Veterinary Medicine Hannover,
Germany, #University Children’s Hospital Münster, Germany
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*Correspondence to: e
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Hassan Y. Naim, Ph.D. m
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Department of Physiological Chemistry, School of Veterinary Medicine Hannover ://w
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Bünteweg 17, D-30559 Hannover, Germany .jb
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Tel.: + 49 511 953 8780; Fax: + 49 511 953 8585 brg/
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E-mail: [email protected] ue
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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
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 (CSID). Molecular characterization of this novel phenotype
reveals a point mutation in the coding region of the 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
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phenotype in epithelial MDCK reveals a randomly targeted SI protein to the apical and wn
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basolateral membranes confirming an exclusive role of the Q117R mutation in generating this d fro
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phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton-X ttp
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100 despite the presence of O-glycans that serves in the wild type protein as an apical sorting w
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signal and are required for the association of SI with detergent-insoluble lipid microdomains. rg
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Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most ue
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likely due to altered folding of the P-domain that ultimately affects the access of the O- Ap
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glycans to a putative sorting element. , 20
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INTRODUCTION
The composition and function of the plasma membrane of polarized cells is 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
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diseases constitutes therefore a powerful means to unravel the molecular mechanisms wn
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underlying intracellular protein transport and sorting. A small intestinal disorder that directly d fro
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associated with a folding mutants and defective intracellular protein transport is congenital ttp
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sucrase isomaltase deficiency1 (CSID). CSID is an autosomal recessive disease that is w.jb
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characterized by an absent sucrase activity within the sucrase-isomaltase (SI) enzyme b/
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complex, while the isomaltase activity can vary from absent to normal. The disease is s
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clinically manifested as an osmotic-fermentative diarrhea upon ingestion of di- and pril 5
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oligosaccharides (4). Several phenotypes of the disease have been characterized on the basis of 1
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cellular mislocalisation 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, the 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 non-covalent ionic interactions (9;10). This
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The Abbreviations used are: CSID, congenital sucrase-isomaltase deficiency; SI, sucrase-isomaltase
(all forms); pro-SI, uncleaved sucrase-isomaltase; ER, endoplasmic reticulum; TGN, trans-Golgi
network; TX-100, Triton X 100; mAb, monoclonal antibody; Endo H, endoglyosidase H; MDCK,
Madin-Darby canine kidney cells; benzyl-GalNAc, benzyl-N-acetyl-a-D-galactosaminide
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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
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rarely observed, perhaps because such defects have lethal consequences during early stages of wn
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development. Abnormalities in protein trafficking in polycsytic kidney disease and cystic d fro
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fibrosis have been shown to be associated with mutations in the coding regions of the ttp
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corresponding genes (3;12). The mutation identified in this report, however, is to our knowledge the w
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first of its kind, in which aberrant polarized sorting of an intestinal protein is disease- rg
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associated. By establishing the functional details of this mutation and its implication in the ue
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polarized sorting of SI, the structure and function of the sorting elements possibly interacting Ap
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within the region harboring the mutation could be elucidated. , 20
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MATERIALS AND METHODS
Processing of biopsy samples- CSID in a four 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 before (13). Messenger RNA was isolated using
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Dynabeads Oligo(dT) (Dynal, Oslo, Norway) and cDNA was synthesized with the First
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Strand cDNA Synthesis Kit (Amersham/Pharmacia, Freiburg, Germany) using random
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hexamer nucleotide primers. For each PCR reaction 1/10 (2µl) of the reaction mixture was
used as a template. PCR reactions were performed with the seven primer pairs published by
Ouwendijk et al. (13). The negative controls underwent the same procedure, only cDNA template
was excluded from each reaction mixture. The products of two independent PCR reactions
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were directly cloned into the pCR 2.1 Vector (Invitrogen, Groningen, The Netherlands) and
sequenced in both orientations with M13 universe and reverse primers. The sequence analysis
revealed a single mutation A/G at position 412. For transfection in MDCK cells the complete
SI cDNA in the plasmid vector pSG8 (phSI) (14) was mutated at position 412 by oligonucleotide
directed mutagenesis with the Quick ChangeTM in vitro Mutagenesis System from Stratagene
(Amsterdam, The Netherlands). The following oligonucleotides were used in this context:
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SIA/Gup: 5´- TGG TTA TAA CGT TCG AGA CAT GAC AAC AAC -3´ nlo
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SIA/Gdo: 5´- GTT GTT GTC ATG TCT CGA ACG TTA TAA CCA -3´ d fro
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Mutation of A to G was confirmed by sequencing and the plasmid obtained was denoted h
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phSIA/G. w
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Transfection and metabolic labeling of MDCK cells- MDCK cells were co-transfected with c
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phSIA/G and the neomycin-resistance vector pcDNA3 (Invitrogen, Groningen, The b/
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Netherlands) using the calcium phosphate procedure as described before (15). Stable cell lines st o
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expressing wild type or mutant SI were selected by using 0.25 mg/ml active G418-containing p
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selection medium (Life Technologies, Inc.). Isolated clones were screened by metabolic , 2
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labeling and immunoprecipitation as described below. In control experiments the MDCK-
SIWT cell line that expresses wild type SI cell line was used (16). MDCK cells were biosynthetically
labeled with 80µCi L-(35S) RedivueTM PRO-MIXTM (Amersham/Pharmacia, Freiburg,
Germany) as described by Naim et al. (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-a-D-galactosaminide) (Sigma, Deisenhofen,
Germany), an inhibitor of O-glycosylation (17;18).
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
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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 ice-old lysis buffer was used for each 100-mm culture dish (~2-4 x
106 cells). The detergent extracts were centrifuged for 1 h at 100,000 x 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 were used. 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
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grown on membrane filters were performed as described previously (11). Here, MDCK cells were nlo
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labeled for 4 h with 100 µCi [35S]methionine (Amersham/Pharmacia, Freiburg, Germany). d fro
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Wild type or mutant SI expressed at the cell surface were immunoprecipitated from intact h
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cells by addition of mAb anti-SI to either the apical or basolateral compartments. The cells w
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were solubilized in the lysis buffer described above and the antigen-antibody complex was c
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captured by protein A-Sepahrose (Amersham/Pharmacia, Freiburg, Germany ). b/
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Association of wild type and mutant SI with lipid microdomains- The association of SI with st o
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sphingolipid/cholesterol-rich microdomains was assessed in detergent extractability assays p
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using TX-100 essentially as described before (11). Here, MDCK cells containing either wild type , 2
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SI or mutant SI were biosynthetically-labeled for 4 h and solubilized for 2 h at 4°C in a lysis
buffer containing 1 % TX-100, 25 mM Tris-HCl, pH 8.0, 50 mM NaCl. The detergent
extracts were centrifuged at 100,000 x 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 10 fold volume
of a buffer containing 1% Triton X-100 was added and these extracts as well as the TX-100-
soluble fraction were immunoprecipitated with the mixture of mAb anti-SI antibodies
mentioned above that recognizes native and denatured forms of SI (19).
Other procedures. Washing, digestion of immunoprecipitates with endo-ß-N-
acetylglucosaminidase H (endo H) and processing for analysis by for SDS-PAGE on 6% slab
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gels (20) was 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
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 protein. In comparison, the normal activity D
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of sucrase and that of isomaltase in the small intestine range from 40 to 60 IU/mg protein. a
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Another disaccharidase in the small intestine, lactase-phlorizin hydrolase, revealed 42 IU/mg m
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protein, which is in the normal range, indicating that the sucrose malabsorption is restricted to ://w
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the sucrase-isomaltase complex. The next step was therefore to analyze the molecular basis .jbc
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and the corresponding mechanisms leading to this defect. Due to the limited amount of the y
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biopsy specimen we were not able to investigate the biosynthesis and processing of the SI t o
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complex in organ culture as has been performed with other cases of CSID (8;22). However, the ril 5
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tissue available was adequate for the isolation of the cDNA encoding the patient’s SI by RT-
PCR following a similar strategy to that described before (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
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and in modulating cell growth (for a review see (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 Madin-Darby canine kidney (MDCK) cells, which does not express
endogenously 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.
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Continuous metabolic labeling of the cells containing the wild type SI protein for 6 h revealed wn
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a 210-kDa mannose-rich polypeptide (SIh) that shifted upon reaction with endo H to a fro
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smaller size and an endo H-resistant 245-kDa complex glycosylated mature protein (SIc) ://w
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(Fig. 2A, denoted WT). A similar band pattern was also observed with the cells containing the .jb
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mutant protein (Fig. 2A, denoted mutant). However, the proportion of the complex by/
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glycosylated protein in the total synthesized and processed SI protein was significantly less t o
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than that in its wild type counterpart suggesting a slower rate of processing of the mannose- ril 5
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rich to the complex form. We therefore determined the transport and processing kinetics of 9
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 half time for the conversion of the mannose-rich mutant SI into a
complex glycosylated form was 4.5 h as compared to approximately 1.5 h of the wild type
protein (Fig. 2C) compatible with delayed transport kinetics of this mutant from the ER to the
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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 I and
S 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
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often implicated in the folding pattern of membrane glycoproteins already during early stages wn
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of posttranslational processing in the ER (29;30). The pattern of O-glycosylation is similar in both d fro
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cases, since treatments of cells with benzyl-GalNAc, an inhibitor of O-glycosylation (11;18), ttp
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revealed reductions in the size of the wild type and the mutant SI forms reminiscent of w
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comparable contents of O-glycans in both protein forms (Fig. 2D). rg
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The next step was to determine whether complex glycosylated mutant SI is correctly targeted ue
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to the apical membrane or the mutation is associated with the generation of a randomly or Ap
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missorted SI phenotype. For this MDCK cells expressing the mutant and wild type proteins , 20
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were grown on membrane filters and their biosynthesis and polarized transport was 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
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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 it to 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-
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glycosylation strongly suggests that a protein structure implicated in the sorting process has wn
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been altered. A Ser/Thr-rich stalk region immediately upstream the transmembrane domain d fro
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of SI is heavily O-glycosylated and is required for the association of SI with lipid rafts (16). ttp
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Deletion of this domain eliminates almost completely O-glycosylation of SI affecting thus its w
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detergent extractability concomitant with loss of its sorting fidelity, while the transport rg
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competence of SI per se remains unchanged. As shown here, the mutation Q117R induces ue
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similar effects with respect to detergent solubility and sorting of SI as the deletion mutants do. Ap
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Unlike the deletion mutants, however, mutant SI is extensively O-glycosylated, but it is , 20
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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 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 hampered thus 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