Additional N-Glycosylation and Its Impact on the Folding of Intestinal Lactase-phlorizin Hydrolase

Abstract

Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra-and extracellular proteolytic steps to generate the final brush-border membrane form LPH␤ final . Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861-13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimers per se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most likely by nucleating the association of the ectodomains of the enzyme.The pathways by which membrane and secretory proteins attain their three-dimensional structure, in particular the implication of glycosylation in these events has been the target of extensive investigation in the past few years. N-Glycosylation is necessary for efficient folding in the ER 1 (1-5), interaction with calnexin and calreticulin (6), receptor-ligand binding (7), transport to lysosomes (8), polarized sorting to the apical plasma membrane (9), and also for optimal expression of some proteins (10). The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-XaaSer/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum (ER). However, the presence of the sequon does not ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent (11). N-Glycosyl sugar chains are core-glycosylated in the ER and become complex glycosylated by passing through the Golgi apparatus. Although N-glycosylation in the ER constitutes the critical step in the initial folding of proteins, the complex glycosylated chains in some glycoproteins, acquired in the Golgi, are also required for the acquisition of a correct conform...

Altered Folding, Turnover, and Polarized Sorting Act in Concert to Define a Novel Pathomechanism of Congenital Sucrase-Isomaltase Deficiency

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Apical Transport and Folding of Prostate-specific Membrane Antigen Occurs Independent of Glycan Processing

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Structural Hierarchy of Regulatory Elements in the Folding and Transport of an Intestinal Multidomain Protein

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