Protein Domains Implicated in Intracellular Transport and Sorting of Lactase-Phlorizin Hydrolase

Panzer, Petra; Preuß, Ute; Joberty, Gérard; Naim, Hassan Y.

doi:10.1074/jbc.273.22.13861

Cited by 17 publications

(20 citation statements)

References 39 publications

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“…This domain harbors the catalytic site of the lactose hydrolytic activity. We could show that deletion of 236 amino acids located in the immediate vicinity of the membrane in homologous region IV had almost no influence on the quaternary structure, transport, and sorting of LPH to the plasma membrane (24). By contrast, a further deletion of 87 amino acids upstream of the 236-amino acid stretch have led to an inhibition of the dimerization event and to a substantial reduction in the transport capacity of the deletion mutant.…”

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confidence: 68%

“…The labeled cells were rinsed two times with phosphate-buffered saline. Cells were solubilized with 1 ml/dish of cold lysis buffer essentially as described before (24). The cell extracts were centrifuged to remove nuclei and debris.…”

Section: P1743smentioning

confidence: 99%

“…Deletion of this NH 2 -terminal domain leads to a drastic reduction in the proportion of correctly folded and transport-competent molecules (21,22). Apart from the LPH␣ domain, the transmembrane and perhaps also the cytoplasmic domains of pro-LPH play a key role in acquisition of a correct dimeric protein structure (23) and transport competence of the enzyme (24). The homologous region IV contains a subdomain with structural characteristics of unfolded, highly O-glycosylated stalk regions found in a variety of membrane proteins such as low density lipoprotein receptor, sucrase isomaltase, and aminopeptidase N (25,26).…”

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confidence: 99%

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

Jacob¹,

Weiner²,

Stadge³

et al. 2000

Journal of Biological Chemistry

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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...

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confidence: 68%

Section: P1743smentioning

confidence: 99%

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confidence: 99%

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

Jacob¹,

Weiner²,

Stadge³

et al. 2000

Journal of Biological Chemistry

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“…Furthermore, O-glycosylation does not correlate with the apical targeting of aminopeptidase N and lactase-phlorizin hydrolase, which are apical membrane proteins with O-glycosylated stalk regions near their transmembrane domains. Deletion of these stalk regions does not affect their apical targeting in MDCK cells (25,26).…”

Section: Discussionmentioning

confidence: 99%

Mucin-like Domain of Enteropeptidase Directs Apical Targeting in Madin-Darby Canine Kidney Cells

Zheng¹,

Sadler²

2002

Journal of Biological Chemistry

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Enteropeptidase, a type II transmembrane protein of the enterocyte brush border, is sorted directly to the apical membrane of Madin-Darby canine kidney II cells. Apical targeting appears to be mediated by an N-terminal segment that contains a 27-amino acid residue Oglycosylated mucin-like domain consisting of two short mucin-like repeats, A and B. Targeting signals within these repeats were characterized by using green fluorescent protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion to a different O-glycosylated motif did not alter the random secretion of GFP, and several constructs with mutations in either repeat A or B were O-glycosylated and secreted randomly. In addition, repeat B appears to contain an apical targeting signal that functions in the absence of glycosylation. Density gradient centrifugation indicated that, unlike several other apically targeted membrane and soluble proteins, apical sorting of mucin-GFP chimeric proteins does not appear to utilize lipid rafts.Enteropeptidase, a serine protease localized to the brush border of duodenal enterocytes (1), is targeted directly to the apical surface of Madin-Darby canine kidney II (MDCK) 1 cells (2). Apical sorting of enteropeptidase may involve at least two distinct signals. One is located in the C-terminal serine protease domain and depends on N-glycosylation; another is proposed to be located in an N-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites (2). The apical targeting signals within the Nterminal region of enteropeptidase have not been characterized structurally.Several distinct classes of apical sorting signals have been identified, suggesting the existence of several apical targeting mechanisms. For example, apical sorting can be mediated by transmembrane domains (3-5), by glycosylphosphatidylinositol anchors (6, 7), by PDZ-interacting domains (8, 9), or by N-linked oligosaccharides (10, 11). In some proteins, such as sucrase-isomaltase (12) and dipeptidyl peptidase IV (13, 14), O-linked oligosaccharides also appear capable of mediating apical sorting. The juxtamembrane segment of the neurotrophin receptor p75 contains clustered O-linked oligosaccharides and is required for apical targeting (15, 16). The O-linked glycan-dependent apical sorting of sucrase-isomaltase also is accompanied by association with glycosphingolipid and cholesterol-rich membrane microdomains or lipid rafts (13,14). Inhibition of terminal ␣2,3-sialylation with benzyl-2-acetamido-2-deoxy-␣-D-galactopyranoside (GalNAc-␣-O-benzyl) blocks the apical transport of several brush border-associated glycoproteins, including dipeptidylpeptidase IV (14) and the mucin MUC1 (17), suggesting that the terminal structures of certain O-linked olig...

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“…This domain, however, plays a central regulatory role in the context of the function and trafficking of LPH. It contains the LAC236 stretch that is required for dimerization of LPH (24,36). The essential function of domain IV within the LPH complex becomes evident when considering its role in the dimerization of LPH.…”

Section: Discussionmentioning

confidence: 99%

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

Behrendt¹,

Polaina

Naim³

2010

Journal of Biological Chemistry

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Protein Domains Implicated in Intracellular Transport and Sorting of Lactase-Phlorizin Hydrolase

Cited by 17 publications

References 39 publications

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

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

Mucin-like Domain of Enteropeptidase Directs Apical Targeting in Madin-Darby Canine Kidney Cells

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

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