{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T14:49:19Z","timestamp":1780757359849,"version":"3.54.1"},"reference-count":49,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,5,30]],"date-time":"2020-05-30T00:00:00Z","timestamp":1590796800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A (rh-\u03b1-Gal A) is the standard treatment for Fabry disease (FD). ERT has shown a significant impact on patients; however, there is still morbidity and mortality in FD, resulting in progressive cardiac, renal, and cerebrovascular pathology. The main pathway for delivery of rh-\u03b1-Gal A to lysosome is cation-independent mannose-6-phosphate receptor (CI-M6PR) endocytosis, also known as insulin-like growth factor 2 receptor (IGF2R) endocytosis. This study aims to investigate the mechanisms of uptake of rh-\u03b1-Gal-A in different cell types, with the exploration of clathrin-dependent and caveolin assisted receptor-mediated endocytosis and the dynamics of autophagy-lysosomal functions. rh-\u03b1-Gal-A uptake was evaluated in primary fibroblasts, urine originated kidney epithelial cells, and peripheral blood mononuclear cells derived from Fabry patients and healthy controls, and in cell lines HEK293, HTP1, and HUVEC. Uptake of rh-\u03b1-Gal-A was more efficient in the cells with the lowest endogenous enzyme activity. Chloroquine and monensin significantly blocked the uptake of rh-\u03b1-Gal-A, indicating that the clathrin-mediated endocytosis is involved in recombinant enzyme delivery. Alternative caveolae-mediated endocytosis coexists with clathrin-mediated endocytosis. However, clathrin-dependent endocytosis is a dominant mechanism for enzyme uptake in all cell lines. These results show that the uptake of rh-\u03b1-Gal-A occurs rapidly and activates the autophagy-lysosomal pathway.<\/jats:p>","DOI":"10.3390\/biom10060837","type":"journal-article","created":{"date-parts":[[2020,6,2]],"date-time":"2020-06-02T09:19:27Z","timestamp":1591089567000},"page":"837","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Rapid Clathrin-Mediated Uptake of Recombinant \u03b1-Gal-A to Lysosome Activates Autophagy"],"prefix":"10.3390","volume":"10","author":[{"given":"Margarita M.","family":"Ivanova","sequence":"first","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Dao","sequence":"additional","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neil","family":"Kasaci","sequence":"additional","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Adewale","sequence":"additional","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Fikry","sequence":"additional","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ozlem","family":"Goker-Alpan","sequence":"additional","affiliation":[{"name":"Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA 22030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,5,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1023\/B:BOLI.0000031101.12838.c6","article-title":"Enzyme replacement and enhancement therapies for lysosomal diseases","volume":"27","author":"Desnick","year":"2004","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_2","first-page":"1","article-title":"The effect of enzyme replacement therapy on clinical outcomes in male patients with Fabry disease: A systematic literature review by a European panel of experts","volume":"19","author":"Germain","year":"2019","journal-title":"Mol. Genet. Metab. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"338","DOI":"10.7326\/0003-4819-138-4-200302180-00014","article-title":"Fabry disease, an under-recognized multisystemic disorder: Expert recommendations for diagnosis, management, and enzyme replacement therapy","volume":"138","author":"Desnick","year":"2003","journal-title":"Ann. Intern. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1016\/S0140-6736(08)61589-5","article-title":"Fabry\u2019s disease","volume":"372","author":"Zarate","year":"2008","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S37","DOI":"10.1016\/j.ymgme.2009.10.157","article-title":"140. A validated disease severity scoring system for Fabry disease","volume":"99","author":"Waldek","year":"2010","journal-title":"Mol. Genet. Metab."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1136\/jmg.38.11.769","article-title":"Anderson-Fabry disease: Clinical manifestations and impact of disease in a cohort of 60 obligate carrier females","volume":"38","author":"MacDermot","year":"2001","journal-title":"J. Med. Genet."},{"key":"ref_7","first-page":"2090","article-title":"Renal function predicts long-term outcome on enzyme replacement therapy in patients with Fabry disease","volume":"32","author":"Lenders","year":"2016","journal-title":"Nephrol. Dial. Transplant."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Najafian, B., T\u00f8ndel, C., Svarstad, E., Sokolovkiy, A., Smith, K., and Mauer, M. (2016). One Year of Enzyme Replacement Therapy Reduces Globotriaosylceramide Inclusions in Podocytes in Male Adult Patients with Fabry Disease. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0152812"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1186\/1471-2369-15-72","article-title":"Fabry nephropathy: A review \u2013 how can we optimize the management of Fabry nephropathy?","volume":"15","author":"Waldek","year":"2014","journal-title":"BMC Nephrol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.jocn.2019.03.064","article-title":"Fabry\u2019s disease and stroke: Effectiveness of enzyme replacement therapy (ERT) in stroke prevention, a review with meta-analysis","volume":"65","author":"Sheng","year":"2019","journal-title":"J. Clin. Neurosci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1136\/hrt.2006.104026","article-title":"Effects of enzyme replacement therapy on the cardiomyopathy of Anderson Fabry disease: A randomised, double-blind, placebo-controlled clinical trial of agalsidase alfa","volume":"94","author":"Hughes","year":"2008","journal-title":"Heart"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2945","DOI":"10.2174\/092986707782794005","article-title":"Mannose 6-phosphate receptor targeting and its applications in human diseases","volume":"14","author":"Jeanjean","year":"2007","journal-title":"Curr. Med. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Prabakaran, T., Nielsen, R., Larsen, J.V., S\u00f8rensen, S.S., Rasmussen, U.F.-, Saleem, M.A., Petersen, C.M., Verroust, P.J., and Christensen, E.I. (2011). Receptor-Mediated Endocytosis of \u03b1-Galactosidase A in Human Podocytes in Fabry Disease. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0025065"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Prabakaran, T., Nielsen, R., Satchell, S.C., Mathieson, P.W., Feldt-Rasmussen, U., S\u00f8rensen, S.S., and Christensen, E.I. (2012). Mannose 6-Phosphate Receptor and Sortilin Mediated Endocytosis of \u03b1-Galactosidase A in Kidney Endothelial Cells. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0039975"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10597","DOI":"10.1021\/nn404719c","article-title":"Biological Functionalization of Drug Delivery Carriers To Bypass Size Restrictions of Receptor-Mediated Endocytosis Independently from Receptor Targeting","volume":"7","author":"Ansar","year":"2013","journal-title":"ACS Nano"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2636","DOI":"10.1007\/s12035-016-9849-7","article-title":"Insulin-Like Growth Factor-II\/Cation-Independent Mannose 6-Phosphate Receptor in Neurodegenerative Diseases","volume":"54","author":"Wang","year":"2016","journal-title":"Mol. Neurobiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"12115","DOI":"10.1016\/S0021-9258(18)63825-6","article-title":"Mannose 6-phosphate receptors and lysosomal enzyme targeting","volume":"264","author":"Dahms","year":"1989","journal-title":"J. Boil. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1093\/glycob\/cwg034","article-title":"A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease","volume":"13","author":"Lee","year":"2003","journal-title":"Glycobiology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"S69","DOI":"10.1016\/j.ymgme.2017.12.170","article-title":"Individualized screening for chaperone activity in Gaucher disease using multiple patient derived primary cell lines","volume":"123","author":"Ivanova","year":"2018","journal-title":"Mol. Genet. Metab."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"11042","DOI":"10.1038\/s41598-018-29240-w","article-title":"Urine-derived cells: A promising diagnostic tool in Fabry disease patients","volume":"8","author":"Slaats","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1307","DOI":"10.1016\/S0021-9258(19)69965-5","article-title":"Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms","volume":"256","author":"Bishop","year":"1981","journal-title":"J. Boil. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1042\/BJ20070479","article-title":"Mutant \u03b1-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: Biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin","volume":"406","author":"Ishii","year":"2007","journal-title":"Biochem. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2411","DOI":"10.1074\/jbc.M116.762807","article-title":"Down-regulation of Claudin-2 Expression and Proliferation by Epigenetic Inhibitors in Human Lung Adenocarcinoma A549 Cells*","volume":"292","author":"Hichino","year":"2017","journal-title":"J. Boil. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1186\/bcr2922","article-title":"Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles","volume":"13","author":"Tveito","year":"2011","journal-title":"Breast Cancer Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.3892\/ijo.2014.2512","article-title":"Knockdown of IGF2R suppresses proliferation and induces apoptosis in hemangioma cells in vitro and in vivo","volume":"45","author":"Ou","year":"2014","journal-title":"Int. J. Oncol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1038\/gim.2016.122","article-title":"The validation of pharmacogenetics for the identification of Fabry patients to be treated with migalastat","volume":"19","author":"Benjamin","year":"2016","journal-title":"Genet. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1559","DOI":"10.1161\/01.STR.32.7.1559","article-title":"Enhanced Endothelium-Dependent Vasodilation in Fabry Disease","volume":"32","author":"Altarescu","year":"2001","journal-title":"Stroke"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1093\/glycob\/cwn061","article-title":"Strategies for carbohydrate recognition by the mannose 6-phosphate receptors","volume":"18","author":"Dahms","year":"2008","journal-title":"Glycobiology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"748","DOI":"10.1016\/j.addr.2007.06.008","article-title":"Endocytic mechanisms for targeted drug delivery","volume":"59","author":"Bareford","year":"2007","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1083\/jcb.200711073","article-title":"Mechanisms and functions of endocytosis","volume":"180","author":"Miaczynska","year":"2008","journal-title":"J. Cell Boil."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.semcdb.2014.04.011","article-title":"The role of the cytoskeleton and molecular motors in endosomal dynamics","volume":"31","author":"Granger","year":"2014","journal-title":"Semin. Cell Dev. Boil."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Liebau, M.C., Braun, F., H\u00f6pker, K., Weitbrecht, C., Bartels, V., M\u00fcller, R.-U., Brodesser, S., Saleem, M.A., Benzing, T., and Schermer, B. (2013). Dysregulated Autophagy Contributes to Podocyte Damage in Fabry\u2019s Disease. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0063506"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1186\/2051-5960-2-20","article-title":"Autophagy-lysosome pathway associated neuropathology and axonal degeneration in the brains of alpha-galactosidase A-deficient mice","volume":"2","author":"Nelson","year":"2014","journal-title":"Acta Neuropathol. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ivanova, M.M., Changsila, E., Iaonou, C., and Goker-Alpan, O. (2019). Impaired autophagic and mitochondrial functions are partially restored by ERT in Gaucher and Fabry diseases. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0210617"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1002\/1873-3468.12979","article-title":"Small fluorescent molecules for monitoring autophagic flux","volume":"592","author":"Iwashita","year":"2018","journal-title":"FEBS Lett."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2812","DOI":"10.1073\/pnas.0712309105","article-title":"Elevated globotriaosylsphingosine is a hallmark of Fabry disease","volume":"105","author":"Aerts","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1007\/s10545-011-9395-4","article-title":"Functional analysis of variant lysosomal acid glycosidases of Anderson-Fabry and Pompe disease in a human embryonic kidney epithelial cell line (HEK 293 T)","volume":"35","author":"Ebrahim","year":"2011","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1007\/s10545-012-9472-3","article-title":"Lysosomal delivery of therapeutic enzymes in cell models of Fabry disease","volume":"35","author":"Marchesan","year":"2012","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1111\/j.1600-0854.2007.00541.x","article-title":"Role of an Acidic Cluster\/Dileucine Motif in Cation-Independent Mannose 6-Phosphate Receptor Traffic","volume":"8","author":"Tortorella","year":"2007","journal-title":"Traffic"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.2741\/4552","article-title":"Endocytosis and membrane receptor internalization implication of F-BAR protein Carom","volume":"22","author":"Wang","year":"2017","journal-title":"Front. Biosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1526","DOI":"10.1074\/jbc.M309465200","article-title":"Mannose 6-Phosphate Receptor-mediated Uptake Is Defective in Acid Sphingomyelinase-deficient Macrophages","volume":"279","author":"Dhami","year":"2003","journal-title":"J. Boil. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1366","DOI":"10.1021\/mp5005959","article-title":"Altered Clathrin-Independent Endocytosis in Type A Niemann-Pick Disease Cells and Rescue by ICAM-1-Targeted Enzyme Delivery","volume":"12","author":"Rappaport","year":"2015","journal-title":"Mol. Pharm."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/1755-8417-1-6","article-title":"Abnormal mannose-6-phosphate receptor trafficking impairs recombinant alpha-glucosidase uptake in Pompe disease fibroblasts","volume":"1","author":"Cardone","year":"2008","journal-title":"Pathogenetics"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1021\/acs.molpharmaceut.5b00542","article-title":"A Comparative Study on the Alterations of Endocytic Pathways in Multiple Lysosomal Storage Disorders","volume":"13","author":"Rappaport","year":"2016","journal-title":"Mol. Pharm."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1016\/j.ymgme.2013.12.003","article-title":"Neuroinflammation and \u03b1-synuclein accumulation in response to glucocerebrosidase deficiency are accompanied by synaptic dysfunction","volume":"111","author":"Ginns","year":"2014","journal-title":"Mol. Genet. Metab."},{"key":"ref_46","first-page":"295","article-title":"Secrets of caveolae- and lipid raft-mediated endocytosis revealed by mammalian viruses","volume":"1746","author":"Pelkmans","year":"2005","journal-title":"BBA Bioenerg."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1111\/j.1582-4934.2007.00148.x","article-title":"Clathrin-independent internalization and recycling","volume":"12","author":"Gong","year":"2007","journal-title":"J. Cell. Mol. Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1016\/j.cell.2004.09.003","article-title":"Caveolin-Stabilized Membrane Domains as Multifunctional Transport and Sorting Devices in Endocytic Membrane Traffic","volume":"118","author":"Pelkmans","year":"2004","journal-title":"Cell"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Martins, A.S., Ord\u00f3\u00f1ez, J.L., Amaral, A.T., Prins, F., Floris, G., Debiec-Rychter, M., Hogendoorn, P.C.W., and \u00c1lava, E. (2011). IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-\/Caveolin-Dependent Endocytosis. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0019846"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/6\/837\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:34:02Z","timestamp":1760175242000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/6\/837"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,5,30]]},"references-count":49,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2020,6]]}},"alternative-id":["biom10060837"],"URL":"https:\/\/doi.org\/10.3390\/biom10060837","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,5,30]]}}}