Volume 125, Issue 6, Pages 1678-1685 (December 2003) Congenital sucrase-isomaltase deficiency because of an accumulation of the mutant enzyme in the endoplasmic reticulum  Valentina Ritz, Marwan Alfalah, Klaus-peter Zimmer, Jacques Schmitz, Ralf Jacob, Hassan Y Naim  Gastroenterology  Volume 125, Issue 6, Pages 1678-1685 (December 2003) DOI: 10.1053/j.gastro.2003.09.022

Figure 1 Comparison of SI forms from a CSID patient with the normal control. (A ) Biosynthetically labeled biopsy specimens from a patient with CSID and a person analyzed for other purposes than CSID were homogenized, solubilized, and immunoprecipitated with mAb anti-SI. One part of the immunoprecipitates was treated with endo H, and the other was not treated. Finally, the samples were subjected to SDS-PAGE on 6% slab gels. Gels were analyzed by phosphorimaging. Following immunoprecipitations with mAb anti-SI, the supernatants were depleted from SI and sequentially immunoprecipitated with (B) mAb anti-LPH (C ) or mAb anti-DPPIV. The immunoprecipitates were analyzed on SDS-PAGE on 6% slab gels. Mannose rich (SIh, LPHh, DPPIVh) and complex glycosylated mature forms (SIc, LPHc, DPPIVc) are indicated. Gastroenterology 2003 125, 1678-1685DOI: (10.1053/j.gastro.2003.09.022)

Figure 2 Ultrastructural localization of sucrase-isomaltase on thin frozen sections of small bowel biopsy specimens of the patient and the control. Frozen sections were labeled by pAb anti SI and 12-nm large goat anti-rabbit immunogold particles (SI12). The gold particles are indicated by arrows. In the patient’s enterocytes, most labeling was detected in the ER (b), followed by weaker labeling of the Golgi stacks (c) and some distinct SI molecules on the apical membranes (a). The control person shows strong apical labeling of apical membranes (d) and ER or Golgi-specific staining with pAb anti SI (e). AM, apical membrane; ER, endoplasmic reticulum; G, Golgi apparatus; Lu, lumen; M, mitochondria; Mi, microvilli. Scale bars, 0.5 μm. Gastroenterology 2003 125, 1678-1685DOI: (10.1053/j.gastro.2003.09.022)

Figure 3 Expression of mutant pro-SIV15F, pro-SIA231T, and pro-SIL620P in COS-1 cells. COS-1 cells were transfected with pSG8-hSI (SIWT), pSG8-hSIV15F, pSG8-hSIA231T, and pSG8-hSIL620P. The cells were biosynthetically labeled with 35S-methionine (A ) for 6 hours or (B) 30 minutes. In B, cells were chased in methionine-free medium in the presence of unlabeled methionine for the indicated time points prior to cell lysis. Following immunoprecipitation with mAb anti-SI, the immunoprecipitates were treated or not treated with endo H and analyzed after 6% SDS-PAGE by a phosphorimaging device. Arrowheads indicate the faint band of endo H resistant SIL620P. Gastroenterology 2003 125, 1678-1685DOI: (10.1053/j.gastro.2003.09.022)

Figure 4 Subcellular localization of the pro-SIL620P in transiently transfected COS-1 cells. COS-1 cells were transiently cotransfected with pLPHα-DsRed, pGolgi-CFP, and pSI-YFP or pSIL620P-YFP. Fixed cells were analyzed by confocal microscopy. Expression of SIL620P-YFP showed predominantly ER-specific staining and a minor colocalization with Golgi-CFP, whereas SI-YFP could be detected in the ER, the Golgi apparatus, and at the plasma membrane. The arrows point to plasma membrane staining of SI-YFP. Scale bars, 8 μm. Gastroenterology 2003 125, 1678-1685DOI: (10.1053/j.gastro.2003.09.022)

Figure 5 Trypsin sensitivity of the pro-SIL620P molecule. Transiently transfected COS-1 cells were biosynthetically labeled with 35S-methionine for 5 hours. Detergent extracts were treated with 0, 10, 50, and 200 mg/mL trypsin for 30 minutes at 37°C prior to cell lysis and immunoprecipitation with mAb anti-SI. The precipitates were analyzed by SDS-PAGE on 6% slab gels by phosphorimaging. Gastroenterology 2003 125, 1678-1685DOI: (10.1053/j.gastro.2003.09.022)