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* 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.
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 ofO-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 theO-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.
congenital sucrase-isomaltase deficiency
sucrase-isomaltase (all forms)
Madin-Darby canine kidney cells
polyacrylamide gel electrophoresis
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 (
). 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 (
). 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 (
). 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 (
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 (
). 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.
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 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 (
). 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 (
), 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.
). 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 (
). 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, denotedmutant). However, the
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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 theO-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 comprisingO-linked chains, such as galactose orN-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 (
) 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 (
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, theO-glycosylated stalk region, and the transmembrane domain, which has been assigned a decisive role in the sorting pathway of SI (
). Strategies could therefore be designed to discover this putative protein by utilizing the SI protein and its mutant phenotype as promising models.
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.