Intracellular transport of sucrase-isomaltase in patients with a congenital sucrase-somaltase deficiency

The sucrase-isomaltase enzyme complex (pro-SI) is a type II integral membrane glycoprotein of the intestinal brush border membrane. Its synthesis commences with the isomaltase (IM) subunit and ends with sucrase (SUC). Both domains reveal striking structural similarities, suggesting a pseudo-dimeric assembly of a correctly folded and an enzymatically active pro-SI. The impact of each domain on the folding and function of pro-SI has been analyzed by individual expression and coexpression of the individual subunits. SUC acquires correct folding, enzymatic activity and transport competence and is secreted into the external milieu independent of the presence of IM. By contrast, IM persists as a mannose-rich polypeptide that interacts with the endoplasmic reticulum resident molecular chaperone calnexin. This interaction is disrupted when SUC is coexpressed with IM, indicating that SUC competes with calnexin for binding of IM. The interaction between SUC and the membrane-anchored IM leads to maturation of IM and blocks the secretion of SUC into the external milieu. We conclude that SUC plays a role as an intramolecular chaperone in the context of the pro-SI protein.To our knowledge all intramolecular chaperones so far identified are located at the N-terminal end. SUC is therefore the first C-terminally located intramolecular chaperone in mammalian cells.Membrane and secretory proteins are subject to a complex array of cotranslational and post-translational processes that precede sorting to the organelle in which they exert their biological functions. The most crucial events take place in the ER 1 and include folding, subunit assembly, and in many cases oligomerization (for review see Ref. 1). These steps, individually or altogether, lead ultimately to the acquisition of the protein to a transport-competent conformation that enables its egress from the ER. A number of resident proteins of the ER, such as the molecular chaperones BiP and calnexin and protein disulfide isomerase, play primordial roles in these processes that vary from binding of unfolded proteins to the formation of disulfide bonds between individual subunits or within a single polypeptide chain (2-4). A large number of proteins assemble into homodimers, oligomers, or heterodimers preceding protein exit from the ER (5, 6). Correct protein folding of the individual monomers or subunits is required for optimal completion of these events. Many proteins are known not to go into dimers or heterodimers, such as the brush border proteins sucrase-isomaltase (SI) (7), angiotensin-converting enzyme (8), and maltase-glucoamylase (9). An interesting and common feature of these proteins is their composition of an even number of homologous domains. SI, for example, is an enzyme complex that is made of two subunits that share Ն 35% of amino acid sequence similarity and 41% of conserved sequences (10). Although no three-dimensional structure of this large protein is so far available, algorithmic predictions suggest that the two large domains are folded similarly and that...

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confidence: 98%

Sucrase Is an Intramolecular Chaperone Located at the C-terminal End of the Sucrase-Isomaltase Enzyme Complex

Jacob¹,

Pürschel²,

Naim³

2002

“…Clinically, the disease is manifested as an osmoticfermentative diarrhea upon ingestion of disaccharides and oligosaccharides (2). Analysis of this disorder at the molecular and subcellular levels has unraveled a number of phenotypes of CSID, which are characterized by perturbations in the intracellular transport, polarized sorting, aberrant processing, and defective function of SI (3)(4)(5). A few examples have been also reported, in which the misfolded protein products may escape the quality control system, instead of being either degraded or retained in the ER.…”

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confidence: 99%

“…3 This intestinal autosomal recessive disorder is characterized by the absence of the sucrase and most of the maltase digestive activity within the sucraseisomaltase (SI) enzyme complex. The isomaltase activity varies from absent to normal (1).…”

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confidence: 99%

Altered Folding, Turnover, and Polarized Sorting Act in Concert to Define a Novel Pathomechanism of Congenital Sucrase-Isomaltase Deficiency

Keiser¹,

Alfalah²,

Pröpsting³

et al. 2006

Naturally occurring mutants of membrane and secretory proteins are often associated with the pathogenesis of human diseases. Here, we describe the molecular basis of a novel phenotype of congenital sucrase-isomaltase deficiency (CSID), a disaccharide malabsorption disorder of the human intestine in which several structural features and functional capacities of the brush-border enzyme complex sucrase-isomaltase (SI) are affected. The cDNA encoding SI from a patient with CSID reveals a mutation in the isomaltase subunit of SI that results in the substitution of a cysteine by an arginine at amino acid residue 635 (C635R). When this mutation is introduced into the wild type cDNA of SI a mutant enzyme, SI C635R , is generated that shows a predominant localization in the endoplasmic reticulum. Nevertheless, a definite localization of SI C635R in the Golgi apparatus and at the cell surface could be also observed. Epitope mapping with conformation-specific mAbs protease sensitivity assays, and enzymatic activity measurements demonstrate an altered folding pattern of SI C635R that is responsible for a substantially increased turnover rate and an aberrant sorting profile. Thus, SI C635R becomes distributed also at the basolateral membrane in contrast to wild type SI. Concomitant with the altered sorting pattern, the partial detergent extractability of wild type SI shifts to a complete detergent solubility with Triton X-100. The mutation has therefore affected an epitope responsible for the apical targeting fidelity of SI. Altogether, the combined effects of the C635R mutation on the turnover rate, function, polarized sorting, and detergent solubility of SI constitute a unique and novel pathomechanism of CSID.The utilization of misfolding-related diseases has proven to be invaluable in dissection of the molecular mechanisms of protein transport and has unraveled several intriguing cell biological phenomena, such as in cases of congenital sucrase-isomaltase deficiency (CSID).3 This intestinal autosomal recessive disorder is characterized by the absence of the sucrase and most of the maltase digestive activity within the sucraseisomaltase (SI) enzyme complex. The isomaltase activity varies from absent to normal (1). Clinically, the disease is manifested as an osmoticfermentative diarrhea upon ingestion of disaccharides and oligosaccharides (2). Analysis of this disorder at the molecular and subcellular levels has unraveled a number of phenotypes of CSID, which are characterized by perturbations in the intracellular transport, polarized sorting, aberrant processing, and defective function of SI (3-5). A few examples have been also reported, in which the misfolded protein products may escape the quality control system, instead of being either degraded or retained in the ER. A mutation at the position 620 of SI (L620P), for example, has been identified as one of the possible genetic modifications occurring in the CSID (6). Although this mutant is mainly found to be localized in the ER, it can be at least partially expressed als...

“…Sorting to the correct membrane is essential for the proteins to exhibit their biological functions, whereas missorting often results in pathological conditions (3,4). The recognition event responsible for sorting has been under intense investigation for two decades, and a number of peptide sequences capable of specifying transport to the basolateral surface of epithelial cells (5)(6)(7)(8)(9)(10)(11), or cell body of neurons (12)(13)(14)(15)(16), have been characterized.…”

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confidence: 99%

Hierarchy of Sorting Signals in Chimeras of Intestinal Lactase-Phlorizin Hydrolase and the Influenza Virus Hemagglutinin

Jacob¹,

Preuß

Panzer

et al. 1999

Polarized cells such as neurons and epithelial cells maintain separate plasma membrane domains, each with a distinct protein and lipid composition, through intracellular sorting mechanisms that recognize classes of proteins and deliver them into separate vesicles for transport to the correct surface domain (1, 2). Sorting to the correct membrane is essential for the proteins to exhibit their biological functions, whereas missorting often results in pathological conditions (3, 4). The recognition event responsible for sorting has been under intense investigation for two decades, and a number of peptide sequences capable of specifying transport to the basolateral surface of epithelial cells (5-11), or cell body of neurons (12-16), have been characterized. All of these signals are located in the cytoplasmic domains of transmembrane glycoproteins. In addition to basolateral signals, three types of signals for sorting proteins to the apical surface of epithelial cells, or axon of neurons, are known. Glycolipid anchors direct proteins to the apical surface of several types of epithelial cells (17, 18), apparently by associating in the trans-Golgi network (19, 20) with detergent-insoluble membrane domains enriched in glycosphingolipids and cholesterol (21). Oligosaccharides on some secreted proteins appear to specify apical transport (22), although this mechanism does not apply to all secreted proteins (23-26).For many transmembrane glycoproteins, deletion of cytoplasmic sequences containing a basolateral sorting signal results in efficient transport of the protein to the apical surface, rather than the random transport expected for the deletion of specific sorting information (10,(27)(28)(29)(30). For other proteins, deletion of cytoplasmic sequences caused randomized transport, proving that transport to the apical surface does not occur by default (9, 31). In the reverse approach, introducing basolateral sorting signals into the cytoplasmic domain of the influenza hemagglutinin (HA) 1 was shown to have a dominant effect over apical sorting information (8, 31, 32) that has been recently localized to the transmembrane domain (8). These observations implied that some proteins carry apical sorting information that is recessive to cytoplasmic basolateral sorting signals. Basolateral signals could dominate over apical signals simply by being recognized earlier in the biosynthetic pathway, or sorting could occur in a common compartment where basolateral signals might bind tighter to the sorting machinery than apical signals. To investigate these questions, we attached a series of basolateral sorting signals to the strictly polarized membrane protein of small intestinal epithelial cells, lactasephlorizin hydrolase (LPH, EC 3.2.1.23-3.2.1.62), and determined their effect on the sorting of LPH.LPH, an integral type I membrane glycoprotein, is 1927 amino acids long containing a membrane anchor of 19 contiguous hydrophobic amino acids and a cytoplasmic domain of 26 amino acids. It is synthesized as a precursor with apparent mo...