1. Volume 136, Issue 3, Pages 883-892 (March 2009)
Compound Heterozygous Mutations Affect Protein Folding and Function in Patients With Congenital Sucrase-Isomaltase Deficiency 
Marwan Alfalah, Markus Keiser, Tosso Leeb, Klaus–Peter Zimmer, Hassan Y. Naim 
Gastroenterology 
Volume 136, Issue 3, Pages (March 2009)
DOI: /j.gastro
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2. Figure 1
Biosynthetic forms of wild-type and SI mutants in transiently transfected COS-1 cells. (A) COS-1 cells were transfected transiently with cDNAs encoding wild-type SI or the mutant forms SI-V577G, SI-G1073D, SI-C1229Y, and SI-F1745C. The cells were labeled continuously for 4 hours with 35S-methionine followed by solubilization and immunoprecipitation with mAb anti-SI. Complex glycosylation or maturation of the proteins was examined by treatment of one part of each immunoprecipitate with endo H. To examine the presence of secreted forms of the mutants the culture media of the labeled cells also were immunoprecipitated with mAb anti-SI. The immunoprecipitates were subjected to SDS-PAGE on 6% slab gels and the band detection was achieved by a phosphorimaging device. The Figure combines several separately run gels. L, cell lysates; M, culture medium; SIc, complex glycosylated SI; SIh, mannose-rich glycosylated SI. (B) Quantification of the biosynthetic forms of wild-type SI and SI-C1229Y in A. The error bars represent standard deviations.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Processing kinetics of SI and SI-C1229Y. (A) The trafficking kinetics of SI-C1229Y and wild-type SI were investigated by pulse-chase experiments of transiently transfected COS-1 cells. The cells were labeled with 35S-methionine for 1 hour, and chased with Dulbecco's modified Eagle medium for up to 4 hours. Detergent extracts of the labeled cells were immunoprecipitated with mAb anti-SI followed by analysis by SDS-PAGE on 6% slab gels and detection of the band pattern in a phosphorimaging device. SIc, complex glycosylated SI; SIh, mannose-rich glycosylated SI. (B) The labeled bands in A and from another 2 similarly conducted experiments were quantified to compare the proportion of the mannose-rich SI vs its mature complex glycosylated counterpart. The error bars represent standard deviations.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Subcellular localization of wild-type and mutant SI proteins. (A) Transiently transfected COS-1 cells expressing wild-type SI, SI-V577G, SI-G1073D, SI-C1229Y, or SI-F1745C were immunostained with mAb–anti-SI as the primary antibody, and anti-mouse IgG conjugated with Alexa Fluor 488 as the secondary antibody. The images show a predominant localization of all mutants in the ER, whereas the wild-type protein is localized in the Golgi apparatus, in transport vesicles, and at the cell surface. (B and C) Wild-type SI or the SI mutants were coexpressed in COS-1 cells with the (B) ER-marker dsRed-ER or with the (C) Golgi-marker galactosyltransferase coupled to the cyan fluorescent protein. All mutants colocalized with the ER-marker. Mutant SI-C1229Y, and to a lower extent SI-F1745C, additionally were detected in Golgi (colocalizations are shown in yellow). No cell surface staining corresponding to the mutant SI-C1229Y was revealed, indicating that this mutant is blocked in the Golgi apparatus. Blue arrows show transport vesicles of SI; G with white arrowheads, Golgi; ER, endoplasmic reticulum; n, nucleus. Scale bars, 20μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 3
Subcellular localization of wild-type and mutant SI proteins. (A) Transiently transfected COS-1 cells expressing wild-type SI, SI-V577G, SI-G1073D, SI-C1229Y, or SI-F1745C were immunostained with mAb–anti-SI as the primary antibody, and anti-mouse IgG conjugated with Alexa Fluor 488 as the secondary antibody. The images show a predominant localization of all mutants in the ER, whereas the wild-type protein is localized in the Golgi apparatus, in transport vesicles, and at the cell surface. (B and C) Wild-type SI or the SI mutants were coexpressed in COS-1 cells with the (B) ER-marker dsRed-ER or with the (C) Golgi-marker galactosyltransferase coupled to the cyan fluorescent protein. All mutants colocalized with the ER-marker. Mutant SI-C1229Y, and to a lower extent SI-F1745C, additionally were detected in Golgi (colocalizations are shown in yellow). No cell surface staining corresponding to the mutant SI-C1229Y was revealed, indicating that this mutant is blocked in the Golgi apparatus. Blue arrows show transport vesicles of SI; G with white arrowheads, Golgi; ER, endoplasmic reticulum; n, nucleus. Scale bars, 20μm.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 4
Assessment of the trypsin sensitivity of wild-type SI and the mutant forms as a measure of protein folding. (A) Transfected COS-1 cells expressing wild-type and mutant SI were labeled biosynthetically with 35S-methionine for 4 hours. The SI proteins were immunoprecipitated with anti-SI mAb. The immunoprecipitates were divided into similar parts, treated with trypsin, and analyzed by SDS-PAGE. Note the trypsin-resistant digestion form of mutants SI-C1229Y and SI-F17435C. (B) To identify the trypsin-resistant bands in SI-C1229Y and SI-F17435C, transfected cells expressing these 2 protein forms were immunoprecipitated, digested with trypsin, and subjected to immunoblotting using anti-IM (HBB 3/705) or anti-SUC (HBB 2/614). Anti-IM and anti-SUC were compiled from different blots with similar samples applied.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 5
Enzymatic activity of SI and SI mutants. Transfected COS-1 cells expressing wild-type SI or the mutants, SI-V577G, SI-G1073D, SI-C1229Y, or SI-F1745C, were solubilized 48 hours after transfection and immunoprecipitated with anti-SI mAbs. The SUC and IM activities were assessed by determining the concentration of the released glucose by high-pressure liquid chromatography. The enzymatic activities of SUC and IM in the mutant forms of SI were compared with their counterparts in the wild-type protein, which were placed as 100%.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 6
Co-expression of the mutants SI-V577G/SI-G1037D or SI-C1229Y/SI-F1745C and assessment of possible resulting effects on the biosynthesis and enzymatic activities. COS-1 cells were co-transfected with the cDNA clones encoding either the mutant forms SI-V577G and SI-G1073D or SI-C1229Y and SI-F1745C. (A) The cells were biosynthetically labeled continuously for 4 hours with 35S-methionine and further processed as in Figure 1. Note that no substantial change in the biosynthetic forms of the mutant proteins has been detected (compare with Figure 1). (B) The co-transfected cells were processed further as described in the Figure 5 legend. Here again, no change in the specific activities of SUC and IM were obtained in comparison with Figure 5.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 7
Trafficking and localization of wild-type and mutant SI. (A) Wild-type SI is transported from the ER, processed in the Golgi, and sorted to the cell surface via O-glycans in its stalk domain,10,17 which presumably bind to a putative galactose-binding protein (gal-binding protein). (B) The mutant SI-C1229Y is partially transport-competent, but is not sorted to the cell surface, presumably because the altered SUC folding prevents its binding to the gal-binding protein. (C) SI-F1745C is located predominantly in the ER and a minor proportion is transported to the cis-Golgi. (D) SI-G1073D and SI-V577G are located exclusively in the ER. ER chaperones also are indicated that bind unfolded forms of SUC or IM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions