Gastroenterology

Gastroenterology

Volume 136, Issue 3, March 2009, Pages 883-892
Gastroenterology

Basic—Alimentary Tract
Compound Heterozygous Mutations Affect Protein Folding and Function in Patients With Congenital Sucrase-Isomaltase Deficiency

https://doi.org/10.1053/j.gastro.2008.11.038Get rights and content

Background & Aims

Congenital sucrase-isomaltase (SI) deficiency is an autosomal-recessive intestinal disorder characterized by a drastic reduction or absence of sucrase and isomaltase activities. Previous studies have indicated that single mutations underlie individual phenotypes of the disease. We investigated whether compound heterozygous mutations, observed in some patients, have a role in disease pathogenesis.

Methods

We introduced mutations into the SI complementary DNA that resulted in the amino acid substitutions V577G and G1073D (heterozygous mutations found in one group of patients) or C1229Y and F1745C (heterozygous mutations found in another group). The mutant genes were expressed transiently, alone or in combination, in COS cells and the effects were assessed at the protein, structural, and subcellular levels.

Results

The mutants SI-V577G, SI-G1073D, and SI-F1745C were misfolded and could not exit the endoplasmic reticulum, whereas SI-C1229Y was transported only to the Golgi apparatus. Co-expression of mutants found on each SI allele in patients did not alter the protein's biosynthetic features or improve its enzymatic activity. Importantly, the mutations C1229Y and F1745C, which lie in the sucrase domains of SI, prevented its targeting to the cell's apical membrane but did not affect protein folding or isomaltase activity.

Conclusions

Compound heterozygosity is a novel pathogenic mechanism of congenital SI deficiency. The effects of mutations in the sucrase domain of SIC1229Y and SIF1745C indicate the importance of a direct interaction between isomaltase and sucrose and the role of sucrose as an intermolecular chaperone in the intracellular transport of SI.

Section snippets

Mutagenesis of a cDNA Encoding SI

The mutations identified previously18 were introduced by site-directed mutagenesis into the wild-type SI cloned in the vector pSG8-SI.7 The generated SI mutants are denoted hereafter as SI-V577G, SI-G1073D, SI-C1229Y, and SI-F1745C. The mutagenesis was performed by polymerase chain reaction using Isis-DNA Polymerase (MP Biomedicals, Eschwege, Germany) and the following oligonucleotides as primers: (1) SI-V577G: 5′-GAG CAA GCA GTA CAA AAA GGT TTT CCT AAT AAG AGA AGC-3′ (forward) and 5′-GCT TCT

General Description of Novel Mutations Associated With CSID

We recently identified the mutations V577G, G1073D, C1229Y, and F1745C in the SI protein by isolating and sequencing the genomic DNA of patients with symptoms typical of CSID.18 All the patients were of Hungarian origin. The mutations are compiled in Table 1, which also compares sequences in the immediate regions of affected residues and highlights the conserved nature of these residues among different species.

The mutation C1229Y is located in SUC and was found in 2 individuals who are full

Discussion

During the past decade several mutations have been characterized in the SI gene that are associated with the onset of disaccharide malabsorption in CSID. These mutations elicit alterations in the folding pattern, apical sorting fidelity, intracellular localization of SI, or function of SI. The diversity of these phenotypes has been instrumental in identifying sorting signals or proposing novel cell biological mechanisms such as secondary quality control. Until now each CSID phenotype has been

Acknowledgements

The authors thank Hans–Peter Hauri and Erwin Sterchi (Biozentrum Basel and University of Bern, respectively, both Switzerland) for the generous gifts of monoclonal anti–sucrase-isomaltase antibodies. The authors thank Dr Irma Korponay–Szabo, Children's Hospital, Budapest, Hungary, and Dr Petra Sander, University of Veterinary Medicine, Hannover, Germany, for participation in the initial phases of this project.

References (30)

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M.A. and M.K. contributed equally to this work.

Conflict of interest The authors disclose no conflicts.

Funding Dr Naim was supported by the German Research Foundation (SFB 621, C8 project).

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