title: Naturally occurring mutations in intestinal sucrase-isomaltase provide evidence for the existence of an intracellular sorting signal in the isomaltase subunit.
abstract: Mutations in the sucrase-isomaltase gene can lead to the synthesis of transport-incompetent or functionally altered enzyme in congenital sucrase-isomaltase deficiency (CSID) (Naim, H. Y., J. Roth, E. Sterchi, M. Lentze, P. Milla, J. Schmitz, and H. P. Hauri. J. Clin. Invest. 82:667-679). In this paper we have characterized two novel mutant phenotypes of CSID at the subcellular and protein levels. The first phenotype revealed a sucrase-isomaltase protein that is synthesized as a single chain, mannose-rich polypeptide precursor (pro-SI) and is electrophoretically indistinguishable from pro-SI in normal controls. By contrast to normal controls, however, pro-SI does not undergo terminal glycosylation in the Golgi apparatus. Subcellular localization of pro-SI by immunoelectron microscopy revealed unusual labeling of the molecule in the basolateral membrane and no labeling in the brush border membrane thus indicating that pro-SI is missorted to the basolateral membrane. Mapping of biosynthetically labeled pro-SI with four epitope- and conformation-specific monoclonal antibodies suggested that conformational and/or structural alterations in the pro-SI protein have prevented posttranslational processing of the carbohydrate chains of the mannose-rich precursor and have lead to its missorting to the basolateral membrane. The second phenotype revealed two variants of pro-SI precursors that differ in their content of mannose-rich oligosaccharides. Conversion of these forms to a complex glycosylated polypeptide occurs at a slow rate and is incomplete. Unlike its counterpart in normal controls, pro-SI in this phenotype is intracellularly cleaved. This cleavage produces an isomaltase-like subunit that is transport competent and is correctly sorted to the brush border membrane since it could be localized in the brush border membrane by anti-isomaltase mAb. The sucrase subunit is not transported to the cell surface and is most likely degraded intracellularly. We conclude that structural features in the isomaltase region of pro-SI are required for transport and sorting of the sucrase-isomaltase complex.
authors.affiliation: Laboratory for Electron Microscopy, University of Leiden, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Fransen
authors.forename: H P
authors.initials: HP
authors.lastname: Hauri
authors.forename: L A
authors.initials: LA
authors.lastname: Ginsel
authors.forename: H Y
authors.initials: HY
authors.lastname: Naim
mesh_terms: D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D009154:Mutation; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D013394:Sucrase-Isomaltase Complex / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*
chemical_list: D000939:Epitopes; D013394:Sucrase-Isomaltase Complex