No abstract

Sucrase-isomaltase (SI) is a highly N- and O-glycosylated intestinal brush border membrane protein. SI is sorted with high fidelity to the apical membrane via O-linked glycans that mediate its association with lipid rafts or detergent-resistant membranes (DRMs). Here, we show that N- and O-glycosylation and DRMs are implicated in the regulation of the function of SI in intestinal Caco-2 cells. The activities of sucrase (SUC) and isomaltase (IM) increase substantially in DRMs when N- and O-glycosylation patterns are intact. Disruption of DRMs by solubilization with Triton X-100 at 37 degrees C substantially reduces the activities of SUC and IM. Furthermore, modulation of O-glycosylation by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside and N-glycosylation by deoxymannojirimycin is linked to a decreased capacity of SI to associate with DRMs, with a subsequent reduction of the enzymatic activities of SUC and IM. This is the first report that reveals a direct role of N- and O-glycans in association with DRMs in regulating the function of a membrane glycoprotein.

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with N-butyldeoxynojirimycin (NB-DNJ) in vitro was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients.

Human aminopeptidase N (APN) is used as a routine marker for myelomonocytic cells in hematopoietic malignant disorders. Its gene and surface expressions are increased in cases of malignant transformation, inflammation, or T cell activation, whereas normal B and resting T cells lack detectable APN protein expression. In this study we elucidated the intracellular distribution, expression pattern, and enzymatic activity of a naturally occurring mutation in the coding region of the APN gene. At physiological temperatures the mutant protein is enzymatically inactive, persists as a mannoserich polypeptide in the endoplasmic reticulum, and is ultimately degraded by an endoplasmic reticulum-associated degradation pathway. It shows in part the distinct behavior of a temperaturesensitive mutant with a permissive temperature of 32°C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cellsurface membrane and without regaining its enzymatic activity. Because the patient bearing this mutation suffered from leukemia, possible links to the pathogenesis of leukemia are discussed.Membrane alanyl aminopeptidase (aminopeptidase N, APN 2 (1), CD13, EC 3.4.11.2) is a 967-amino acid type II transmembrane protein that is expressed on the surface of a broad variety of cell types, most strongly in intestinal mucosa and kidney tissue (1, 2). Although not all aspects of its function are fully understood, it is established that the enzyme preferentially cleaves neutral amino acids from the N terminus of oligopeptides leading to degradation of neuropeptides (3-12), cytokines, immunomodulatory peptides (13-15), and angiotensins (16 -18). Depending on its location, APN is involved in terminal degradation of small peptides in the intestinal brush border (19), inactivation of endorphins and enkephalins in synaptic membranes (2), and angiogenesis (21-23).Furthermore, APN may contribute to extracellular matrix degradation (24, 25) and antigen processing via trimming of major histocompatibility complex class I and II associated peptides (26, 27). There are also several reports of its distinct functions as a receptor for various viruses like the human and murine cytomegalovirus (28 -30) Recently it has been shown that APN is capable of promoting phagocytosis by supporting Fc-␥ receptors on peripheral blood monocytes (41).APN has been implicated in the growth and function of immune cells, including T cells and T cell subsets (42-44). A significant fraction of malignant lymphocytes and corresponding cell lines appear CD13-positive in flow cytometry. In addition, CD13-negative T and B cell lines were shown to contain considerable amounts of APN mRNA (45). Therefore, it is suggested that a dysregulation of APN expression may contribute to or result from malignant transformation of lymphocytes and/or enhanced cellular growth. A previous study reported mutations in the gene coding for APN in 18 and 6% of cases of leukemia and lymphoma, respectively. No such mutations were f...

The molecular basis of gastrointestinal intolerances in a severe case of Niemann-Pick type C disease was analyzed in an intestinal biopsy specimen. The enzyme activities of intestinal sucrase-isomaltase and maltaseglucoamylase are reduced in the patient, while that of lactase is comparable to the control. The association of SI with lipid rafts is reduced in the patient's biopsy as a consequence of altered composition of membrane microdomains. As association with lipid rafts influences the intracellular transport and the enzyme activities of sucraseisomaltase and maltase-glucoamylase, these data explain reduced carbohydrate digestion in the intestinal lumen and delineate the effect of deficient cholesterol and sphingolipid homeostasis in development of gastrointestinal symptoms in NPC patients.