32 results on '"Alfalah M"'
Search Results
2. PP, PYY and NPY: Synthesis, Storage, Release and Degradation
- Author
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von Hörsten, S., primary, Hoffmann, T., additional, Alfalah, M., additional, Wrann, C. D., additional, Karl, T., additional, Pabst, R., additional, and Bedoui, S., additional
- Published
- 2004
- Full Text
- View/download PDF
3. Prostate-specific membrane antigen (PSMA) associates early in the endoplasmic reticulum with lipid microdomains
- Author
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Castelletti, D., Giulio Fracasso, Alfalah, M., Colombatti, M., and Naim, H. Y.
- Subjects
prostate ,trafficking ,PSMA - Published
- 2005
4. Congenital sucrase-isomaltase deficiency because of an accumulation of the mutant enzyme in the endoplasmic reticulum
- Author
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Ritz, V., Alfalah, M., Zimmer, K.p., Schmitz, J., Jacob, R., and Naim, H.Y.
- Abstract
Background & Aims: Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive human disorder characterized by reduced activities of the brush border enzyme sucrase-isomaltase (SI). Here, we elucidate the pathogenesis of a new variant of CSID at the cellular and molecular level. Methods: Assessment of the CSID phenotype was achieved by enzymatic activity measurements, biosynthetic labeling of intestinal biopsy specimens, immunoprecipitation of SI, and immunoelectronmicroscopy. The putative mutation was identified by sequencing of the SI cDNA isolated by RT-PCR from intestinal biopsy samples. The function of the mutation was verified by immunoprecipitation and confocal microscopy of transiently transfected cells. Results: Biosynthetic labeling and immunoelectron microscopy reveal a predominant localization of SI in the endoplasmic reticulum (ER) similar to phenotype I of CSID. Unlike phenotype I, however, a partial conversion of SI to a complex glycosylated mature form takes place. The SI cDNA in this phenotype revealed 3 mutations, 2 of which, Val to Phe at residue 15 and Ala to Thr at residue 231, had no effect on the structure or function of SI. By contrast, the third mutation resulted in an exchange of leucine by proline at position 620 (L620P) and revealed in transfected COS cells structural features and subcellular localization similar to the phenotype identified in the patient's enterocytes. Conclusions: This is the first identification at the molecular and subcellular levels of a novel variant of CSID in which SI accumulates predominantly in the ER, and a minor proportion is further processed and transported to the apical membrane of enterocytes.
- Published
- 2003
- Full Text
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5. Structural determinants required for apical sorting of an intestinal brush-border membrane protein.
- Author
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Jacob, R, Alfalah, M, Grünberg, J, Obendorf, M, and Naim, H Y
- Abstract
The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain.
- Published
- 2000
6. Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin.
- Author
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Jacob, R, Preuss, U, Panzer, P, Alfalah, M, Quack, S, Roth, M G, Naim, H, and Naim, H Y
- Abstract
Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.
- Published
- 1999
7. Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV.
- Author
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Naim, H Y, Joberty, G, Alfalah, M, and Jacob, R
- Abstract
The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing of the mannose-rich N-linked glycans is blocked by deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the presence of swainsonine, an inhibitor of Golgi mannosidase II. The results indicate that removal of the outermost mannose residues by mannosidase I from the mannose-rich N-linked glycans is required before O-glycosylation can ensue. On the other hand, subsequent mannose residues in the core chain impose no sterical constraints on the progression of O-glycosylation. Reduction or modification of N- and O-glycosylation do not affect the transport of pro-SI, ApN, or DPPIV to the cell surface per se. However, the polarized sorting of two of these proteins, pro-SI and DPPIV, to the apical membrane is substantially altered when O-glycans are not completely processed, while the sorting of ApN is not affected. The processing of N-linked glycans, on the other hand, has no influence on sorting of all three proteins. The results indicate that O-linked carbohydrates are at least a part of the sorting mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms.
- Published
- 1999
8. Time to pars plana vitrectomy in adults with retained lens fragments: a systematic review and meta-analysis.
- Author
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Lim M, Sverdlichenko I, Wei EY, Mihalache A, Popovic MM, Alfalah M, Pimentel MC, Muni RH, and Kertes PJ
- Abstract
Introduction: This review and meta-analysis compare visual outcomes and complication risk based on the timing of pars plana vitrectomy (PPV) following cataract surgery with retained lens fragments., Methods: MEDLINE (Ovid), EMBASE, and Cochrane Library were searched between 2000 to February 2022 for studies comparing visual outcomes and complications based on time to PPV. Discrete outcomes were analyzed using a random-effects meta-analysis model on Review Manager (RevMan 5.4). The certainty of evidence of outcomes was evaluated using the Grading of Recommendations, Assessment, Development and Evaluation approach., Results: Ten studies and 1,693 eyes were included. The incidence of patients achieving a final best-corrected visual acuity (BCVA) of >6/12 Snellen may be similar among patients receiving PPV within 1 week or after 1 week of cataract surgery (RR = 1.06, 95% CI = [0.96, 1.17], p = 0.25), and patients receiving PPV within 1 month or after 1 month of cataract surgery (RR = 1.12, 95% CI = [0.95, 1.32]; p = 0.18). Incidence of glaucoma or elevated intraocular pressure for patients may be similar among patients receiving PPV within 1 week or after 1 week of cataract surgery (RR = 1.08, 95% CI = [0.62, 1.87]; p = 0.79), and patients receiving PPV within 1 month or after 1 month of cataract surgery (RR = 0.33, 95% CI = [0.09, 1.23]; p = 0.10)., Conclusion: Incidence of patients achieving a final BCVA of >6/12 Snellen or postoperative adverse effects was similar between patients who underwent early and late PPV following cataract surgery. However, all studies had an overall serious risk of bias, primarily because of confounding and reporting bias., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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9. Assessment of Zonular Integrity in Phakic Eyes Following Pars Plana Vitrectomy Using Ultrasound Biomicroscopy: A Prospective Paired Eye Comparative Study.
- Author
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Alfalah M, Eng KT, Felfeli T, Chew HF, Birt C, Maniyali F, and Kertes PJ
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- Humans, Prospective Studies, Female, Male, Middle Aged, Double-Blind Method, Adult, Lens, Crystalline diagnostic imaging, Aged, Visual Acuity physiology, Vitrectomy, Microscopy, Acoustic
- Abstract
Purpose: To assess zonular integrity in phakic patients post vitrectomy using ultrasound biomicroscopy (UBM)., Design: Prospective, comparative, nonrandomized, double-masked, paired eye study., Methods: We used ultrasound biomicroscopy (UBM) to evaluate phakic patients with a history of unilateral pars-plana vitrectomy., Inclusion Criteria: (1) phakic patients with history of pars plana vitrectomy in one eye as the only procedure; (2) normal unoperated fellow eye; and (3) complete gas or air resolution from the vitreous cavity at the time of UBM assessment., Exclusion Criteria: (1) monocular patients; (2) history of intraoperative lenticular trauma; (3) the use of silicone oil tamponade; (4) history of trauma or pseudoexfoliation in either eye; (5) history of other ocular conditions that can affect the integrity of zonules, such as uveitis or ectopia lentis; (6) eyes with extreme myopia or long axial length (> -8.00 D or >30.0 mm); (7) history of intravitreal injection in either eye; (8) age <18 years., Technique: A high-frequency (50 MHz) UBM device was used by a masked technician to obtain radial section images from zonular bundles at 8 different clock positions. Image quality was assessed in real time, captured, and saved. Two experienced masked observers (H.C. and C.B.) then assessed the quality of the images and graded the zonular findings. Only patients with adequate studies have been included. A unique grading system that was specifically devised for this study was used as the following: (0) clear, well-defined zonule(s); (1) uneven, disrupted zonules or stretched zonules; and (2) extensive loss of zonules. Each clock hour was graded according to this system and the total score was then calculated for each eye. In the primary outcome, 2 main groups were analyzed: vitrectomized eyes and healthy contralateral nonvitrectomized eyes. The mean total UBM score (TUS) from each group was compared and analyzed., Results: Thirty-five patients were recruited into this study. Eleven patients were male and 24 were female. The mean age was 66.3 years. Thirty patients had vitrectomy for vitreomacular interface disorders (either macular hole or epiretinal membrane), 1 patient had vitreous hemorrhage and the remaining 4 patients had rhegmatogenous retinal detachments. With regard to tamponade agents, SF6 was used in 21 (60%) patients, air in 9 (26%) patients, and C3F8 in 5 (14%) patients. The mean TUS in the vitrectomized eyes was 2.28 (SD 1.83) vs 2.24 (SD 1.77) in the nonvitrectomized eyes (P = .9531). Overall, in the comparative analysis of mean scores based on 2 graders' assessments for each clock position in vitrectomized and nonvitrectomized eyes, there were no significant differences noted between the groups., Conclusion: This study found no evidence for a difference in the mean total UBM score in eyes following vitrectomy when compared to their contralateral healthy, nonvitrectomized eyes. This likely indicates that vitrectomy may not affect the integrity of zonules in phakic patients, at least for patients with vitreomacular interface disorders undergoing uncomplicated surgery., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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10. Multifocal congenital pyogenic granuloma successfully treated with oral propranolol.
- Author
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Alharbi M, Eber AE, Perper M, ALFalah M, Al-Khenaizan S, Alomair IA, Alfuraih A, Nouri K, and Cho-Vega JH
- Subjects
- Administration, Oral, Granuloma, Pyogenic congenital, Humans, Infant, Male, Skin pathology, Adrenergic beta-Antagonists therapeutic use, Granuloma, Pyogenic drug therapy, Propranolol therapeutic use
- Abstract
Disseminated congenital pyogenic granuloma (DCPG) is an uncommon condition. Individual lesions of DCPG share clinical and histologic similarities with infantile hemangioma (IH); endothelial glucose transporter 1 (GLUT-1), which is highly expressed in IH but generally not in pyogenic granulomas (PG), is an important diagnostic tool. Treatment for DCPG remains difficult. We describe a case of DCPG effectively treated with propranolol., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2019
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11. Alkyl Glucosides.
- Author
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Alfalah M, Loranger C, and Sasseville D
- Subjects
- Humans, Patch Tests, Allergens adverse effects, Cosmetics adverse effects, Dermatitis, Allergic Contact etiology, Glucosides adverse effects, Surface-Active Agents adverse effects
- Abstract
Alkyl glucosides are surfactants synthesized through the condensation of long-chain fatty alcohols and glucose, extracted from vegetal, renewable sources. Although available for more than 4 decades, they have been rediscovered in recent years because of their eco-friendly character. They are used in various leave-on and rinse-off cosmetics and are considered of low irritancy and allergenicity. However, since the early 2000s, cases of allergic contact dermatitis to this family of molecules have been repeatedly reported. Decyl glucoside was found to be a "hidden" allergen in the sunscreen ingredient Tinosorb M and is likely responsible for most allergic contact dermatitis reported to this compound. Members of the North American Contact Dermatitis Group have seen a steady increase of the rate of sensitization to decyl glucoside. Cross-reactions with other glucosides are common but not automatic; thus, patch testing multiple compounds is recommended.
- Published
- 2017
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12. Alkyl Glucosides in Contact Dermatitis.
- Author
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Loranger C, Alfalah M, Ferrier Le Bouedec MC, and Sasseville D
- Subjects
- Humans, Patch Tests, Allergens adverse effects, Cosmetics adverse effects, Dermatitis, Allergic Contact etiology, Glucosides adverse effects, Sunscreening Agents adverse effects
- Abstract
Ecologically sound because they are synthesized from natural and renewable sources, the mild surfactants alkyl glucosides are being rediscovered by the cosmetic industry. They are currently found in rinse-off products such as shampoos, liquid cleansers, and shower gels, but also in leave-on products that include moisturizers, deodorants, and sunscreens. During the past 15 years, numerous cases of allergic contact dermatitis have been published, mostly to lauryl and decyl glucosides, and these compounds are considered emergent allergens. Interestingly, the sunscreen Tinosorb M contains decyl glucoside as a hidden allergen, and most cases of allergic contact dermatitis reported to this sunscreen ingredient are probably due to sensitization to decyl glucoside. This article will review the chemistry of alkyl glucosides, their sources of exposure, as well as their cutaneous adverse effects reported in the literature and encountered in various patch testing centers.
- Published
- 2017
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13. Contact Allergy to Polymyxin B Among Patients Referred for Patch Testing.
- Author
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Alfalah M, Zargham H, Moreau L, Stanciu M, and Sasseville D
- Subjects
- Adult, Aged, Canada epidemiology, Cohort Studies, Dermatitis, Allergic Contact diagnosis, Dermatitis, Allergic Contact epidemiology, Female, Humans, Male, Middle Aged, Patch Tests, Prevalence, Retrospective Studies, Anti-Bacterial Agents adverse effects, Dermatitis, Allergic Contact etiology, Polymyxin B adverse effects
- Abstract
Backgound: Polymyxin B is not included in most standard contact allergen series. The aim of this study was to determine the prevalence of contact sensitization to polymyxin B in a population of patients referred for patch testing., Methods: A retrospective cohort study design was used to collect data on 795 patients referred to the contact dermatitis clinic of the McGill University Health Centre, as well as to the office of one of the authors (L.M.), between March 2014 and November 2015. Patients were patch tested to the North American Contact Dermatitis Group baseline series and polymyxin B sulfate 3% in petrolatum., Results: Out of 795 tested individuals, 18 were allergic to polymyxin B, for a prevalence of 2.3%. The eruptions affected almost all body parts, but mostly the face. The degree of reaction ranged from 1+ to 2+. Isolated reactions to polymyxin B occurred in 9 (50%) patients, whereas reactions to bacitracin and polymyxin B were seen in the other 9. Only 1 patient reacted to bacitracin, polymyxin B, and neomycin (11.1%). Most reactions (12/18) were from past exposure to polymyxin B., Conclusions: Allergic reactions to polymyxin B are not rare, and this antibiotic warrants inclusion in the standard patch testing series.
- Published
- 2016
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14. "Parabenoia" Debunked, or "Who's Afraid of Parabens?".
- Author
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Sasseville D, Alfalah M, and Lacroix JP
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- Androgen Antagonists, Androgens, Animals, Breast Neoplasms chemically induced, Consumer Product Safety, Female, Humans, Preservatives, Pharmaceutical metabolism, Skin Absorption, Breast Neoplasms epidemiology, Cosmetics adverse effects, Estrogens metabolism, Parabens adverse effects, Parabens metabolism, Preservatives, Pharmaceutical adverse effects
- Abstract
Parabens have been used as preservatives in foods, injectables, and topical preparations for nearly 10 decades. Present in nature, rapidly metabolized by skin and liver enzymes, they have an excellent safety record. However, in the past 15 years, they have been under scrutiny for their alleged estrogenic and antiandrogenic effects, as well as their putative role in promoting cancerogenesis through endocrine disruption. Scientific articles supporting these assertions have led the European Community to ban or restrict the use of some parabens. Despite that methylparaben and ethylparaben have negligible endocrine disruption activity, the food, pharmaceutical, and cosmetic industries are under pressure from scare campaigns in the media and are responding by replacing parabens with other biocides that cause multiple cases, and even worldwide epidemics, of allergic contact sensitization. In the present review, we present a balanced account of the published literature about the metabolism and potential toxicology of parabens.
- Published
- 2015
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15. Protocadherin of the liver, kidney, and colon associates with detergent-resistant membranes during cellular differentiation.
- Author
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Krahn MP, Rizk S, Alfalah M, Behrendt M, and Naim HY
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- Adherens Junctions genetics, Animals, Cadherins genetics, Cell Line, Detergents pharmacology, Dogs, Glycosylation, Membrane Proteins genetics, Membrane Proteins metabolism, Occludin, Phosphoproteins genetics, Phosphoproteins metabolism, Protein Transport physiology, RNA, Messenger genetics, RNA, Messenger metabolism, Tight Junctions genetics, Tight Junctions metabolism, Zonula Occludens-1 Protein, Adherens Junctions metabolism, Cadherins metabolism, Calcium metabolism, Cell Differentiation physiology
- Abstract
Protocadherin LKC (PLKC) is a member of the heterogeneous subgroup of protocadherins that was identified and described as a potential tumor-suppressor gene involved in contact inhibition (Okazaki, N., Takahashi, N., Kojima, S., Masuho, Y., and Koga, H. (2002) Carcinogenesis 23, 1139-1148 and Ose, R., Yanagawa, T., Ikeda, S., Ohara, O., and Koga, H. (2009) Mol. Oncol. 3, 54-66). Several aspects of the structure, posttranslational processing, targeting, and function of this new protocadherin are still not known. Here, we demonstrate that the expression of PLKC at the apical membrane domain and its concentration at regions of cell-cell contacts occur concomitantly with significant elevation of PLKC-mRNA levels. Furthermore, it can be found within the adherens junctions, but it does not colocalize with tight junctions proteins ZO-1 and occludin, respectively. Additionally, unlike E-cadherin, PLKC is not redistributed upon Ca(2+) removal. Biosynthetic labeling revealed N- and O-glycosylation as posttranslational modifications as well as a fast transport to the cell surface and a low turnover rate. During differentiation, PLKC associates with detergent-resistant membranes that trigger its redistribution from intracellular membranes to the cell surface. This association occurs concomitant with alterations in the glycosylation pattern. We propose a role for PLKC in the establishment of a proper epithelial cell polarity that requires O-linked glycosylation and association of the protein with detergent-resistant membranes.
- Published
- 2010
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16. Compound heterozygous mutations affect protein folding and function in patients with congenital sucrase-isomaltase deficiency.
- Author
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Alfalah M, Keiser M, Leeb T, Zimmer KP, and Naim HY
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Chlorocebus aethiops, Enzyme Activation genetics, Gene Expression Regulation, Enzymologic, Genetic Heterogeneity, Humans, Mutagenesis, Site-Directed, Protein Folding, Protein Structure, Tertiary, Protein Transport genetics, Transfection, Carbohydrate Metabolism, Inborn Errors genetics, Carbohydrate Metabolism, Inborn Errors metabolism, Point Mutation, Sucrase-Isomaltase Complex chemistry, Sucrase-Isomaltase Complex deficiency, Sucrase-Isomaltase Complex genetics, Sucrose metabolism
- Abstract
Background & Aims: Congenital sucrase-isomaltase (SI) deficiency is an autosomal-recessive intestinal disorder characterized by a drastic reduction or absence of sucrase and isomaltase activities. Previous studies have indicated that single mutations underlie individual phenotypes of the disease. We investigated whether compound heterozygous mutations, observed in some patients, have a role in disease pathogenesis., Methods: We introduced mutations into the SI complementary DNA that resulted in the amino acid substitutions V577G and G1073D (heterozygous mutations found in one group of patients) or C1229Y and F1745C (heterozygous mutations found in another group). The mutant genes were expressed transiently, alone or in combination, in COS cells and the effects were assessed at the protein, structural, and subcellular levels., Results: The mutants SI-V577G, SI-G1073D, and SI-F1745C were misfolded and could not exit the endoplasmic reticulum, whereas SI-C1229Y was transported only to the Golgi apparatus. Co-expression of mutants found on each SI allele in patients did not alter the protein's biosynthetic features or improve its enzymatic activity. Importantly, the mutations C1229Y and F1745C, which lie in the sucrase domains of SI, prevented its targeting to the cell's apical membrane but did not affect protein folding or isomaltase activity., Conclusions: Compound heterozygosity is a novel pathogenic mechanism of congenital SI deficiency. The effects of mutations in the sucrase domain of SIC1229Y and SIF1745C indicate the importance of a direct interaction between isomaltase and sucrose and the role of sucrose as an intermolecular chaperone in the intracellular transport of SI.
- Published
- 2009
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17. The influence of linoleic and linolenic acid on the activity and intracellular localisation of phospholipase D in COS-1 cells.
- Author
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Gemeinhardt A, Alfalah M, Gück T, Naim HY, and Fuhrmann H
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- Animals, COS Cells, Chlorocebus aethiops, Linoleic Acid pharmacology, Phospholipase D metabolism, alpha-Linolenic Acid pharmacology
- Abstract
Phospholipase D (PLD) is a receptor-regulated signalling enzyme involved in biological functions, such as exocytosis, phagocytosis, actin dynamics, membrane trafficking, and is considered to be essential for stimulated degranulation of cells. The purpose of our investigation was to examine how the fatty acid pattern of cellular membranes influences the activities and cellular distribution of the PLD1 and PLD2 isoforms. Expression of GFP-tagged PLD1 and PLD2 in COS-1 cells that were stimulated with mastoparan after cultivation in 20 micromol linoleic (C18:2n6) or linolenic (C18:3n3) acid for 4 d demonstrated that PLD1 dramatically alters its cellular distribution and is redistributed from intracellular vesicles to the cell surface. PLD2, on the other hand, maintains its localisation at the plasma membrane. The activity of PLD, which corresponds to PLD1 and PLD2, significantly increased two- to three-fold in the presence of the fatty acids. We conclude that linoleic acid and linolenic acid supplementation affect the intracellular trafficking of the PLD1 isoform and the activity of PLD most likely due to alterations in the membrane lipid environment conferred by the fatty acids.
- Published
- 2009
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18. Different glycoforms of prostate-specific membrane antigen are intracellularly transported through their association with distinct detergent-resistant membranes.
- Author
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Castelletti D, Alfalah M, Heine M, Hein Z, Schmitte R, Fracasso G, Colombatti M, and Naim HY
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- Antigens chemistry, Antigens, Surface metabolism, Cell Line, Tumor, Cell Membrane drug effects, Glutamate Carboxypeptidase II metabolism, Humans, Lipids chemistry, Male, Membrane Microdomains, Octoxynol pharmacology, Polyethylene Glycols pharmacology, Polysorbates pharmacology, Protein Isoforms, Protein Structure, Quaternary, Antigens, Surface chemistry, Cell Membrane metabolism, Detergents pharmacology, Glutamate Carboxypeptidase II chemistry, Glycoproteins chemistry, Membranes chemistry
- Abstract
Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-transmembrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant membranes) at different stages of the protein maturation pathway in human prostate carcinoma LNCaP cells. At least three PSMA glycoforms were biochemically identified based on their extractability behaviour in different non-ionic detergents. In particular, one precursor glycoform of PSMA is associated with Tween 20-insoluble DRMs, whereas the complex glycosylated protein segregates into membrane structures that are insoluble in Lubrol WX and display a different lipid composition. Association of PSMA with these membranes occurs in the Golgi compartment together with the acquisition of a native conformation. PSMA homodimers reach the plasma membrane of LNCaP cells in Lubrol WX-insoluble lipid/protein complexes. At the steady state, the majority of PSMA remains within these membrane microdomains at the cell surface. We conclude that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.
- Published
- 2008
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19. Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4.
- Author
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Frerker N, Wagner L, Wolf R, Heiser U, Hoffmann T, Rahfeld JU, Schade J, Karl T, Naim HY, Alfalah M, Demuth HU, and von Hörsten S
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- Animals, Base Sequence, COS Cells, Chlorocebus aethiops, DNA Primers, Hydrolysis, Microscopy, Confocal, Microscopy, Fluorescence, Rats, Rats, Inbred F344, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Dipeptidyl Peptidase 4 metabolism, Neuropeptide Y metabolism
- Abstract
N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.
- Published
- 2007
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20. Both cleavage products of the mCLCA3 protein are secreted soluble proteins.
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Mundhenk L, Alfalah M, Elble RC, Pauli BU, Naim HY, and Gruber AD
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- Animals, COS Cells, Cell Line, Chlorocebus aethiops, Humans, Hydrolysis, Hydrophobic and Hydrophilic Interactions, Mice, Predictive Value of Tests, Protein Processing, Post-Translational, Protein Transport, Solubility, Subcellular Fractions chemistry, Subcellular Fractions metabolism, Chloride Channels chemistry, Chloride Channels metabolism, Mucoproteins chemistry, Mucoproteins metabolism
- Abstract
Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein. Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.
- Published
- 2006
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21. Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucrase-isomaltase deficiency.
- Author
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Keiser M, Alfalah M, Pröpsting MJ, Castelletti D, and Naim HY
- Subjects
- Adult, Amino Acid Sequence, Animals, Detergents chemistry, Detergents pharmacology, Endoplasmic Reticulum metabolism, Epitope Mapping, Humans, Intestines, Male, Molecular Sequence Data, Octoxynol pharmacology, Protein Folding, Sequence Homology, Amino Acid, Carbohydrate Metabolism, Inborn Errors genetics, Sucrase-Isomaltase Complex deficiency
- Abstract
Naturally occurring mutants of membrane and secretory proteins are often associated with the pathogenesis of human diseases. Here, we describe the molecular basis of a novel phenotype of congenital sucrase-isomaltase deficiency (CSID), a disaccharide malabsorption disorder of the human intestine in which several structural features and functional capacities of the brush-border enzyme complex sucrase-isomaltase (SI) are affected. The cDNA encoding SI from a patient with CSID reveals a mutation in the isomaltase subunit of SI that results in the substitution of a cysteine by an arginine at amino acid residue 635 (C635R). When this mutation is introduced into the wild type cDNA of SI a mutant enzyme, SI(C635R), is generated that shows a predominant localization in the endoplasmic reticulum. Nevertheless, a definite localization of SI(C635R) in the Golgi apparatus and at the cell surface could be also observed. Epitope mapping with conformation-specific mAbs protease sensitivity assays, and enzymatic activity measurements demonstrate an altered folding pattern of SI(C635R) that is responsible for a substantially increased turnover rate and an aberrant sorting profile. Thus, SI(C635R) becomes distributed also at the basolateral membrane in contrast to wild type SI. Concomitant with the altered sorting pattern, the partial detergent extractability of wild type SI shifts to a complete detergent solubility with Triton X-100. The mutation has therefore affected an epitope responsible for the apical targeting fidelity of SI. Altogether, the combined effects of the C635R mutation on the turnover rate, function, polarized sorting, and detergent solubility of SI constitute a unique and novel pathomechanism of CSID.
- Published
- 2006
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22. A mutation in aminopeptidase N (CD13) isolated from a patient suffering from leukemia leads to an arrest in the endoplasmic reticulum.
- Author
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Alfalah M, Krahn MP, Wetzel G, von Hörsten S, Wolke C, Hooper N, Kalinski T, Krueger S, Naim HY, and Lendeckel U
- Subjects
- Aged, Animals, Bone Neoplasms genetics, Bone Neoplasms metabolism, Bone Neoplasms secondary, CD13 Antigens metabolism, COS Cells metabolism, Carcinoma, Renal Cell genetics, Carcinoma, Renal Cell metabolism, Carcinoma, Renal Cell pathology, Chlorocebus aethiops, Glycosylation, Golgi Apparatus metabolism, Humans, Kidney Neoplasms genetics, Kidney Neoplasms metabolism, Kidney Neoplasms pathology, Lymphoma, Non-Hodgkin metabolism, Lymphoma, Non-Hodgkin pathology, Male, Tumor Cells, Cultured, CD13 Antigens genetics, Endoplasmic Reticulum metabolism, Lymphoma, Non-Hodgkin genetics, Mutation genetics
- Abstract
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 mannose-rich 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 temperature-sensitive mutant with a permissive temperature of 32 degrees C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cell-surface 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.
- Published
- 2006
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23. Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing.
- Author
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Castelletti D, Fracasso G, Alfalah M, Cingarlini S, Colombatti M, and Naim HY
- Subjects
- Amino Acid Motifs, Animals, Biological Transport, Biotinylation, Blotting, Western, COS Cells, Calnexin chemistry, Cell Line, Cell Line, Tumor, Cell Membrane metabolism, Chlorocebus aethiops, Cloning, Molecular, DNA, Complementary metabolism, Dogs, Endoplasmic Reticulum metabolism, Fluorescent Antibody Technique, Indirect, Glycosylation, Golgi Apparatus metabolism, Humans, Immunoprecipitation, Kinetics, Male, Mannose chemistry, Neoplasm Metastasis, Prostatic Neoplasms metabolism, Protein Binding, Protein Folding, Protein Isoforms, Transfection, Trypsin chemistry, Antigens, Surface biosynthesis, Antigens, Surface chemistry, Glutamate Carboxypeptidase II biosynthesis, Glutamate Carboxypeptidase II chemistry, Polysaccharides chemistry
- Abstract
Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.
- Published
- 2006
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24. Novel mutations in the human sucrase-isomaltase gene (SI) that cause congenital carbohydrate malabsorption.
- Author
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Sander P, Alfalah M, Keiser M, Korponay-Szabo I, Kovács JB, Leeb T, and Naim HY
- Subjects
- Biopsy, Genotype, Humans, Malabsorption Syndromes enzymology, Polymorphism, Genetic genetics, Protein Processing, Post-Translational, Carbohydrate Metabolism, Inborn Errors enzymology, Carbohydrate Metabolism, Inborn Errors genetics, Malabsorption Syndromes congenital, Malabsorption Syndromes genetics, Mutation genetics, Sucrase-Isomaltase Complex genetics
- Abstract
Disaccharide intolerance I or congenital sucrase-isomaltase deficiency (CSID) is a disorder leading to maldigestion of disaccharides, which is autosomal recessively inherited. Here we analyzed the sucrase-isomaltase (SI) gene from 11 patients of Hungarian origin with congenital sucrase-isomaltase deficiency. Variants in the SI gene had previously been described in CSID patients, which cause amino acid exchanges that affect the transport, the processing, or the function of the SI protein. None of our patients had known mutations for CSID. Our analyses revealed 43 SI variants in total, 15 within exons and one at a splice site. Eight of the exonic mutations lead to amino acid exchanges, causing hypomorph or null alleles. One new variation affects a splice site, which is also predicted to result in a null allele. All potential pathological alterations were present on one allele only. In six out of the 11 patients the phenotype of CSID could be explained by compound heterozygosity., (2005 Wiley-Liss, Inc.)
- Published
- 2006
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25. A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells.
- Author
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Alfalah M, Wetzel G, Fischer I, Busche R, Sterchi EE, Zimmer KP, Sallmann HP, and Naim HY
- Subjects
- Adenosine Deaminase chemistry, Animals, CD13 Antigens pharmacology, COS Cells, Caco-2 Cells, Cell Line, Chlorocebus aethiops, Cholesterol metabolism, Dipeptidyl Peptidase 4 chemistry, Dogs, Dose-Response Relationship, Drug, Drug Resistance, Endoplasmic Reticulum metabolism, Epithelial Cells pathology, Glycoproteins chemistry, Golgi Apparatus metabolism, Humans, Hybridomas metabolism, Immunoprecipitation, Kinetics, Lactase chemistry, Lactase-Phlorizin Hydrolase chemistry, Lipids chemistry, Mannose chemistry, Membrane Cofactor Protein biosynthesis, Membrane Microdomains chemistry, Mice, Octoxynol pharmacology, Oligo-1,6-Glucosidase chemistry, Phosphatidylglycerols chemistry, Phosphatidylinositols chemistry, Polysorbates pharmacology, Protein Transport, Sphingolipids metabolism, Sucrase chemistry, Sucrose pharmacology, Transfection, trans-Golgi Network metabolism, Cell Membrane metabolism, Detergents pharmacology, Epithelial Cells cytology
- Abstract
One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.
- Published
- 2005
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26. Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease.
- Author
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Schmitz M, Alfalah M, Aerts JM, Naim HY, and Zimmer KP
- Subjects
- Animals, Cricetinae, Endoplasmic Reticulum Chaperone BiP, Fibroblasts cytology, Fibroblasts metabolism, Gaucher Disease genetics, Gaucher Disease metabolism, Glucosylceramidase genetics, Heat-Shock Proteins metabolism, Humans, Immunohistochemistry, Lysosomes metabolism, Mice, Molecular Chaperones metabolism, NIH 3T3 Cells, Phenotype, Point Mutation, Protein Transport, Gaucher Disease enzymology, Glucosylceramidase metabolism, Lysosomes enzymology
- Abstract
Gaucher's disease is the most inherited lysosomal storage disorder. Except for a few cases, the broad phenotypic heterogeneity of Gaucher's disease can be neither predicted from defined mutations nor from differences in residual enzyme activity. Here, we analyse the intracellular trafficking of glucocerebrosidase as an underlying mechanism for the expression of the clinical phenotype. Biosynthetic labeling studies combined with immunofluorescence analyses with fibroblasts from patients with the defined mutations N370S, L444P, D409H and G202R unequivocally demonstrate a retarded transport of glucocerebrosidase carrying the mutation N370S and a transport block in the ER of the enzyme with the mutations G202R, L444P and D409H. We asked whether cellular components in the patients' fibroblasts other than glucocerebrosidase are implicated in the onset of the disease. For this, mutant cDNA's corresponding to the phenotypes N370S, G202R and L444P were expressed in the mouse fibroblasts NIH3T3. Essentially similar biochemical and cellular features were revealed as compared to the patients' fibroblasts strongly suggesting that these mutations are exclusively responsible for the characterized phenotypes. Interestingly, the immunoglobulin binding protein (BiP) binds wild type and the mutant N370S but not the G202R and L444P variants suggesting a discriminatory role played by this chaperone associated with the severity of the disease.
- Published
- 2005
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27. Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells.
- Author
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Jacob R, Heine M, Alfalah M, and Naim HY
- Subjects
- Actin Cytoskeleton physiology, Humans, Lactase-Phlorizin Hydrolase pharmacokinetics, Microscopy, Fluorescence, Myosins physiology, Precipitin Tests, Sucrase-Isomaltase Complex pharmacokinetics, trans-Golgi Network physiology, Cell Membrane physiology, Cytoplasmic Vesicles physiology, Cytoskeleton physiology, Epithelial Cells physiology
- Abstract
A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.
- Published
- 2003
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- View/download PDF
28. Intestinal dipeptidyl peptidase IV is efficiently sorted to the apical membrane through the concerted action of N- and O-glycans as well as association with lipid microdomains.
- Author
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Alfalah M, Jacob R, and Naim HY
- Subjects
- Acetylgalactosamine pharmacology, Benzyl Compounds pharmacology, Caco-2 Cells, Cell Line, Cell Membrane metabolism, Cholesterol metabolism, Cyclodextrins metabolism, Detergents pharmacology, Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors pharmacology, Glycoside Hydrolases metabolism, Glycosylation, Humans, Mannosidases pharmacology, Membrane Microdomains chemistry, Octoxynol pharmacology, Protein Binding, Protein Structure, Tertiary, Serine Endopeptidases metabolism, Swainsonine pharmacology, Trypsin pharmacology, Acetylgalactosamine analogs & derivatives, Dipeptidyl Peptidase 4 chemistry, Dipeptidyl Peptidase 4 metabolism, Intestines enzymology, Membrane Microdomains metabolism, Polysaccharides chemistry
- Abstract
The apical sorting of human intestinal dipeptidyl peptidase IV (DPPIV) occurs through complex N-linked and O-linked carbohydrates. Inhibition of O-linked glycosylation by benzyl-N-acetyl-alpha-d-galactosaminide affects significantly the sorting behavior of DPPIV in intestinal Caco-2 and HT-29 cells. However, random delivery to the apical and basolateral membranes and hence a more drastic effect on the sorting of DPPIV in both cell types is only observed when, in addition to O-glycans, the processing of N-glycans is affected by swainsonine, an inhibitor of mannosidase II. Together the data indicate that both types of glycosylation are critical components of the apical sorting signal of DPPIV. The sorting mechanism of DPPIV implicates its association with detergent-insoluble membrane microdomains containing cholesterol and sphingolipids, whereas an efficient association largely depends on the presence of a fully complex N- and O-linked glycosylated DPPIV. Interestingly, cholesterol is a more critical component in this context than sphingolipids, because cholesterol depletion by beta-cyclodextrin affects the detergent solubility and the sorting behavior of DPPIV more strongly than fumonisin, an inhibitor of sphingolipid synthesis.
- Published
- 2002
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29. Characteristics and structural requirements of apical sorting of the rat growth hormone through the O-glycosylated stalk region of intestinal sucrase-isomaltase.
- Author
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Spodsberg N, Alfalah M, and Naim HY
- Subjects
- Animals, Base Sequence, Cell Line, Cell Membrane metabolism, DNA Primers, Dogs, Glycosylation, Growth Hormone biosynthesis, Rats, Growth Hormone metabolism, Intestine, Small enzymology, Protein Transport, Sucrase-Isomaltase Complex metabolism
- Abstract
The apical sorting of the small intestinal membrane glycoprotein sucrase-isomaltase (SI) depends on the presence of O-linked glycans and the transmembrane domain. Here, we investigate the role of O-glycans carried by the Ser/Thr-rich stalk region of SI as an apical sorting signal and evaluate the spatial requirements for an efficient recognition of this signal. Several hybrid proteins are generated comprising the unsorted and unglycosylated protein, the rat growth hormone (rGH), fused to either the transmembrane domain of SI (GH-SI(TM)), or the transmembrane and the stalk domains (GH-SI(SR/TM)). Both constructs are randomly distributed over the apical and basolateral membranes of MDCK cells indicating that neither the transmembrane domain nor the O-glycans are sufficient per se for an apical delivery. Only when a polyglycine spacer is inserted between the stalk region of SI and the luminal part of rGH in the GH-SI(Gly/SR/TM) fusion protein does efficient apical sorting of an O-glycosylated protein as well as a time-dependent association with detergent-insoluble lipid microdomains occur. Obviously, the polyglycine spacer facilitates the accessibility of the O-glycans in GH-SI(Gly/SR/TM) to a putative sorting receptor, whereas these glycans are inadequately recognized in GH-SI(SR/TM). We conclude that the O-glycans in the stalk region of SI act as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial requirements provided, for example by a polyglycine spacer in the context of rGH or the P-domain within the SI enzyme complex.
- Published
- 2001
- Full Text
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30. Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder.
- Author
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Spodsberg N, Jacob R, Alfalah M, Zimmer KP, and Naim HY
- Subjects
- Animals, Cell Line, Cell Membrane metabolism, Child, Preschool, Dogs, Humans, Membrane Microdomains metabolism, Phenotype, Point Mutation, Protein Transport, Sucrase-Isomaltase Complex metabolism, Carbohydrate Metabolism, Inborn Errors enzymology, Carbohydrate Metabolism, Inborn Errors genetics, Cell Polarity, Sucrase-Isomaltase Complex deficiency, Sucrase-Isomaltase Complex genetics
- Abstract
The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element.
- Published
- 2001
- Full Text
- View/download PDF
31. A point mutation in the juxtamembrane stalk of human angiotensin I-converting enzyme invokes the action of a distinct secretase.
- Author
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Alfalah M, Parkin ET, Jacob R, Sturrock ED, Mentele R, Turner AJ, Hooper NM, and Naim HY
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Amyloid Precursor Protein Secretases, Aspartic Acid Endopeptidases, Cell Line, Cell Membrane enzymology, Endopeptidases chemistry, Humans, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Neuroblastoma, Neurons, Peptidyl-Dipeptidase A genetics, Phenylalanine pharmacology, Protease Inhibitors pharmacology, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Thiophenes pharmacology, Transfection, Endopeptidases metabolism, Peptidyl-Dipeptidase A chemistry, Peptidyl-Dipeptidase A metabolism, Phenylalanine analogs & derivatives, Point Mutation
- Abstract
Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
- Published
- 2001
- Full Text
- View/download PDF
32. O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.
- Author
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Alfalah M, Jacob R, Preuss U, Zimmer KP, Naim H, and Naim HY
- Subjects
- Acetylgalactosamine analogs & derivatives, Acetylgalactosamine pharmacology, Animals, Benzyl Compounds pharmacology, Caco-2 Cells, Cell Line, Dogs, Glycosylation, Humans, Intestines enzymology, Enzyme Precursors metabolism, Lipid Metabolism, Polysaccharides metabolism, Sucrase-Isomaltase Complex metabolism
- Abstract
The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.
- Published
- 1999
- Full Text
- View/download PDF
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