DOCSLIB.ORG

Type I Collagen During Lung Fibrosis Fibrocytes Are Not an Essential

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Type I Collagen During Lung Fibrosis Fibrocytes Are Not an Essential Fibrocytes Are Not an Essential Source of Type I Collagen during Lung Fibrosis Kathryn R. Kleaveland, Miranda Velikoff, Jibing Yang, Manisha Agarwal, Richard A. Rippe, Bethany B. Moore and This information is current as Kevin K. Kim of September 27, 2021. J Immunol 2014; 193:5229-5239; Prepublished online 3 October 2014; doi: 10.4049/jimmunol.1400753 http://www.jimmunol.org/content/193/10/5229 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/10/02/jimmunol.140075 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 59 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/193/10/5229.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Fibrocytes Are Not an Essential Source of Type I Collagen during Lung Fibrosis Kathryn R. Kleaveland,* Miranda Velikoff,* Jibing Yang,* Manisha Agarwal,* Richard A. Rippe,† Bethany B. Moore,* and Kevin K. Kim* Progressive fibrosis involves accumulation of activated collagen-producing mesenchymal cells. Fibrocytes are hematopoietic- derived cells with mesenchymal features that potentially have a unique and critical function during fibrosis. Fibrocytes have been proposed as an important direct contributor of type I collagen deposition during fibrosis based largely on fate-mapping studies. To determine the functional contribution of hematopoietic cell-derived type I collagen to fibrogenesis, we use a double-transgenic sys- tem to specifically delete the type I collagen gene across a broad population of hematopoietic cells. These mice develop a robust fibrotic response similar to littermate genotype control mice injured with bleomycin indicating that fibrocytes are not a necessary source of type I collagen. Using collagen–promoter GFP mice, we find that fibrocytes express type I collagen. However, fibrocytes Downloaded from with confirmed deletion of the type I collagen gene have readily detectable intracellular type I collagen indicating that uptake of collagen from neighboring cells account for much of the fibrocyte collagen. Collectively, these results clarify several seemingly conflicting reports regarding the direct contribution of fibrocytes to collagen deposition. The Journal of Immunology, 2014, 193: 5229–5239. ibrosis is a common feature of many systemic inflamma- matrix proteins and eventual replacement with fibrillar collagens http://www.jimmunol.org/ tory conditions and can also occur as a primary progres- derived from activated fibrogenic cells. Initiation of these events F sive disease. Fibrosis can lead to organ dysfunction and can occur through recruitment of circulating cells with fibrogenic significant morbidity. Collectively, fibrosis is the leading cause of potential into the microenvironment, proliferation and activation death in developed countries. Idiopathic pulmonary fibrosis is the of quiescent fibroblasts, and transdifferentiation of structural cells most common primary fibrotic lung disorder, affecting .5 million into fibroblast-like cells. The original paradigm assumed that the people worldwide. The median survival of patients with this con- accumulated activated fibroblasts were derived from proliferation dition is 3–5 y from the time of diagnosis and current medical and activation of quiescent resident tissue fibroblasts. However, therapy is largely ineffective (1–5). Unfortunately, despite intense recently other possibilities for the origin of collagen producing by guest on September 27, 2021 investigation, we still have a poor understanding of the pathogenesis cells have been proposed including epithelial cells, endothelial of tissue fibrosis and fibrotic diseases are difficult to treat. Recent cells, pericytes, mesenchymal stem cells, and fibrocytes (7–14). evidence suggests that fibrogenesis is more complex than originally Differentiating among these possibilities is important because thought and involves activation of and coordination of many cells pathways leading to their activation may be distinct. There is sig- types that contribute directly and indirectly to fibrogenesis. A more nificant overlap in mechanisms of activation of fibroblasts, epi- thorough delineation of the processes that underlie fibrogenesis thelial cells, endothelial cells, and pericytes. For example, TGF-b requires the investigation of novel pathways and cell types. is the most well established cytokine leading to fibroblast to Fibrosis is characterized by accumulation of activated fibroblasts myofibroblast transition and epithelial to mesenchymal transition and excessive deposition of fibrotic extracellular matrix proteins, (15, 16). Most, if not all, factors promoting mesenchymal transition especially type I collagen. Extensive research has focused on of epithelial and endothelial cells have also been studied in models mechanisms of type I collagen synthesis by fibroblasts. However, of fibroblast activation. Fibrocyte recruitment may be unique in the origin of type I collagen secreting cells remains unclear and their ability to respond to a number of cytokines and chemokines controversial (6). Injury leads to sequential remodeling of the typically associated with activation of inflammatory cells (17). extracellular matrix with rapid replacement by plasma derived Fibrocytes are hematopoietic bone marrow–derived cells that express both fibroblast and leukocyte markers. They circulate in *Division of Pulmonary and Critical Care Medicine, Department of Internal Medi- peripheral blood and can be isolated from many tissues including cine, University of Michigan, Ann Arbor, MI 48109; and †National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892 the lung. Cultured fibrocytes have been shown to express a number of fibrotic extracellular matrix proteins including collagen I, col- ORCID: 0000-0003-3051-745X (B.B.M.). lagen III, and fibronectin (17–21). In addition, fibrocytes maintain Received for publication March 24, 2014. Accepted for publication September 8, 2014. expression of common leukocyte markers including CD45, CD13, This work was supported by National Institutes of Health Grants R01 HL108904 (to and CD34. Fibrocytes have been shown to secrete a number of K.K.K.) and R01 HL115618 (to B.B.M.). profibrotic cytokines that could potentially help orchestrate fibro- Address correspondence and reprint requests to Dr. Kevin K. Kim, 109 Zina Pitcher genesis. Importantly, fibrocytes express a number of chemokine Place, BSRB 4061, Ann Arbor, MI 48109. E-mail address: [email protected] receptors, including CXCR4, CCR7, and CCR2, which likely The online version of this article contains supplemental material. mediate recruitment and activation of fibrocytes to areas of tissue Abbreviations used in this article: Col-GFP, collagen-GFP; MSC, mesenchymal damage (22–24). Thus, recruitment of fibrocytes to sites of injury stem/stromal cell. suggests an important transition from inflammation to fibrogenesis Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 with the ability of fibrocytes to respond to inflammatory cytokines, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400753 5230 FIBROCYTE-DERIVED COLLAGEN I DOES NOT PROMOTE LUNG FIBROSIS produce fibrotic extracellular matrix proteins, and foster further plemented with 10% FBS in a 37˚C, 5% CO2 incubator allowing primary activation of fibroblasts and other cell types (17). Although several lung mesenchymal cell outgrowth over 2–4 wk. Primary mesenchymal reports indicate that fibrocytes express type I collagen, others have cells were analyzed between passages 2-4 and within 4 wk of isolation. In some experiments, primary lung mesenchymal cells from col1a1fl/fl mice suggested that uptake of secreted type I collagen by hematopoietic were treated with adenovirus encoding Cre (50 PFU/cell) (37). cells accounts for the fibrocyte population (25, 26). Thus, whether fibrocytes contribute to matrix accumulation via direct or indirect Isolation of whole lung single cell suspension mechanisms remains unclear. Whole-lung single-cell suspensions were prepared as described previously Prior studies on the origin of fibrogenic effector cells have relied (22, 40, 41). In brief, mice were euthanized at the time points indicated. primarily on generation of fate-mapping chimeric mice by either Lungs were perfused with PBS, excised, and minced as above. Minced lungs were incubated in digestion solution containing collagenase (1 mg/ml) transplanting bone marrow from a transgenic reporter mouse into and DNase (25 U/ml) in RPMI 1640 medium at 37˚C for 30 min. Samples a wild-type mice or by crossing cell type–specific Cre mice with were dispersed by passing up and down 20 times with a 10-ml syringe. transgenic lox-stop-lox
Load more

Recommended publications
  • Single-Cell RNA Sequencing Demonstrates the Molecular and Cellular Reprogramming of Metastatic Lung Adenocarcinoma
    ARTICLE https://doi.org/10.1038/s41467-020-16164-1 OPEN Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma Nayoung Kim 1,2,3,13, Hong Kwan Kim4,13, Kyungjong Lee 5,13, Yourae Hong 1,6, Jong Ho Cho4, Jung Won Choi7, Jung-Il Lee7, Yeon-Lim Suh8,BoMiKu9, Hye Hyeon Eum 1,2,3, Soyean Choi 1, Yoon-La Choi6,10,11, Je-Gun Joung1, Woong-Yang Park 1,2,6, Hyun Ae Jung12, Jong-Mu Sun12, Se-Hoon Lee12, ✉ ✉ Jin Seok Ahn12, Keunchil Park12, Myung-Ju Ahn 12 & Hae-Ock Lee 1,2,3,6 1234567890():,; Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell tran- scriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions. 1 Samsung Genome Institute, Samsung Medical Center, Seoul 06351, Korea.
    [Show full text]
  • Cell Communication and Signaling Biomed Central
    Cell Communication and Signaling BioMed Central Review Open Access Extravasation of leukocytes in comparison to tumor cells Carina Strell and Frank Entschladen* Address: Institute of Immunology, Witten/Herdecke University, Stockumer Str. 10, 58448 Witten, Germany Email: Carina Strell - [email protected]; Frank Entschladen* - [email protected] * Corresponding author Published: 4 December 2008 Received: 18 November 2008 Accepted: 4 December 2008 Cell Communication and Signaling 2008, 6:10 doi:10.1186/1478-811X-6-10 This article is available from: http://www.biosignaling.com/content/6/1/10 © 2008 Strell and Entschladen; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The multi-step process of the emigration of cells from the blood stream through the vascular endothelium into the tissue has been termed extravasation. The extravasation of leukocytes is fairly well characterized down to the molecular level, and has been reviewed in several aspects. Comparatively little is known about the extravasation of tumor cells, which is part of the hematogenic metastasis formation. Although the steps of the process are basically the same in leukocytes and tumor cells, i.e. rolling, adhesion, transmigration (diapedesis), the molecules that are involved are different. A further important difference is that leukocyte interaction with the endothelium changes the endothelial integrity only temporarily, whereas tumor cell interaction leads to an irreversible damage of the endothelial architecture. Moreover, tumor cells utilize leukocytes for their extravasation as linkers to the endothelium.
    [Show full text]
  • Lysosomal Membrane Glycoproteins Bind Cholesterol and Contribute to Lysosomal Cholesterol Export
    1 Lysosomal membrane glycoproteins bind cholesterol and 2 contribute to lysosomal cholesterol export 3 4 Jian Li and Suzanne R. Pfeffer* 5 Department of Biochemistry, Stanford University School of Medicine 6 Stanford, CA USA 94305-5307 7 8 *Correspondence to: [email protected]. 9 10 Abstract: LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins 11 that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. 12 LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 13 is needed for chaperone-mediated autophagy, and its expression improves tissue function in 14 models of aging. We show here that LAMP1 and LAMP2 bind cholesterol in a manner that 15 buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that 16 export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels 17 suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome 18 limiting membrane, and may signal cholesterol availability. Functional rescue experiments show 19 that the ability of LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to 20 bind cholesterol directly. 21 22 23 Introduction 24 Eukaryotic lysosomes are acidic, membrane-bound organelles that contain proteases, lipases and 25 nucleases and degrade cellular components to regenerate catabolic precursors for cellular use (1- 26 3). Lysosomes are crucial for the degradation of substrates from the cytoplasm, as well as 27 membrane bound compartments derived from the secretory, endocytic, autophagic and 28 phagocytic pathways. The limiting membrane of lysosomes is lined with so-called lysosomal 29 membrane glycoproteins (LAMPs) that are comprised of a short cytoplasmic domain, a single 30 transmembrane span, and a highly, N- and O-glycosylated lumenal domain (4-6).
    [Show full text]
  • Sensitization to the Lysosomal Cell Death Pathway by Oncogene- Induced Down-Regulation of Lysosome-Associated Membrane Proteins 1 and 2
    Research Article Sensitization to the Lysosomal Cell Death Pathway by Oncogene- Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2 Nicole Fehrenbacher,1 Lone Bastholm,2 Thomas Kirkegaard-Sørensen,1 Bo Rafn,1 Trine Bøttzauw,1 Christina Nielsen,1 Ekkehard Weber,3 Senji Shirasawa,4 Tuula Kallunki,1 and Marja Ja¨a¨ttela¨1 1Apoptosis Department and Centre for Genotoxic Stress Response, Institute for Cancer Biology, Danish Cancer Society; 2Institute of Molecular Pathology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 3Institute of Physiological Chemistry, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Halle, Germany; and 4Department of Cell Biology, School of Medicine, Fukuoka University, Fukuoka, Japan Abstract molecules of the cell to breakdown products available for Expression and activity of lysosomal cysteine cathepsins metabolic reuse (3, 4). Cathepsin proteases are among the best- correlate with the metastatic capacity and aggressiveness of studied lysosomal hydrolases. They are maximally active at the tumors. Here, we show that transformation of murine acidic pH of lysosomes (pH 4–5). However, many of them can be Y527F embryonic fibroblasts with v-H-ras or c-src changes the active at the neutral pH outside lysosomes, albeit with a decreased distribution, density, and ultrastructure of the lysosomes, efficacy and/or altered specificity (5). For example, transformation and tumor environment enhance the expression of lysosomal decreases the levels of lysosome-associated membrane pro- teins (LAMP-1 and LAMP-2) in an extracellular signal- cysteine cathepsins and increase their secretion into the extracel- regulated kinase (ERK)- and cathepsin-dependent manner, lular space (6). Once outside the tumor cells, cathepsins stimulate and sensitizes the cells to lysosomal cell death pathways angiogenesis, tumor growth, and invasion in murine cancer induced by various anticancer drugs (i.e., cisplatin, etoposide, models, thereby enhancing cancer progression (7, 8).
    [Show full text]
  • Dipeptidase Aminodipeptidase, Quiescent Cell Proline of a Novel
    Vesicular Localization and Characterization of a Novel Post-Proline-Cleaving Aminodipeptidase, Quiescent Cell Proline Dipeptidase This information is current as of September 26, 2021. Murali Chiravuri, Fernando Agarraberes, Suzanne L. Mathieu, Henry Lee and Brigitte T. Huber J Immunol 2000; 165:5695-5702; ; doi: 10.4049/jimmunol.165.10.5695 http://www.jimmunol.org/content/165/10/5695 Downloaded from References This article cites 28 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/165/10/5695.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Vesicular Localization and Characterization of a Novel Post-Proline-Cleaving Aminodipeptidase, Quiescent Cell Proline Dipeptidase1 Murali Chiravuri,* Fernando Agarraberes,† Suzanne L. Mathieu,* Henry Lee,* and Brigitte T. Huber2* A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus.
    [Show full text]
  • Fluid Biomarkers in Frontotemporal Dementia: Past, Present and Future
    Neurodegeneration J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2020-323520 on 13 November 2020. Downloaded from Review Fluid biomarkers in frontotemporal dementia: past, present and future Imogen Joanna Swift ,1 Aitana Sogorb- Esteve,1,2 Carolin Heller ,1 Matthis Synofzik,3,4 Markus Otto ,5 Caroline Graff,6,7 Daniela Galimberti ,8,9 Emily Todd,2 Amanda J Heslegrave,1 Emma Louise van der Ende ,10 John Cornelis Van Swieten ,10 Henrik Zetterberg,1,11 Jonathan Daniel Rohrer 2 ► Additional material is ABSTRACT into a behavioural form (behavioural variant fron- published online only. To view, The frontotemporal dementia (FTD) spectrum of totemporal dementia (bvFTD)), a language variant please visit the journal online (primary progressive aphasia (PPA)) and a motor (http:// dx. doi. org/ 10. 1136/ neurodegenerative disorders includes a heterogeneous jnnp- 2020- 323520). group of conditions. However, following on from a series presentation (either FTD with amyotrophic lateral of important molecular studies in the early 2000s, major sclerosis (FTD- ALS) or an atypical parkinsonian For numbered affiliations see advances have now been made in the understanding disorder). Neuroanatomically, the FTD spectrum end of article. of the pathological and genetic underpinnings of the is characteristically associated with dysfunction and disease. In turn, alongside the development of novel neuronal loss in the frontal and temporal lobes, but Correspondence to more widespread cortical, subcortical, cerebellar Dr Jonathan Daniel Rohrer, methodologies for measuring proteins and other Dementia Research molecules in biological fluids, the last 10 years have and brainstem involvement is now recognised. Centre, Department of seen a huge increase in biomarker studies within FTD.
    [Show full text]
  • An Underappreciated Yet Critical Hurdle for Successful Cancer Immunotherapy Robert Sackstein1,2,3,4, Tobias Schatton1,3,5,6 and Steven R Barthel1,3,5
    Laboratory Investigation (2017) 97, 669–697 © 2017 USCAP, Inc All rights reserved 0023-6837/17 $32.00 PATHOBIOLOGY IN FOCUS T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy Robert Sackstein1,2,3,4, Tobias Schatton1,3,5,6 and Steven R Barthel1,3,5 Advances in cancer immunotherapy have offered new hope for patients with metastatic disease. This unfolding success story has been exemplified by a growing arsenal of novel immunotherapeutics, including blocking antibodies targeting immune checkpoint pathways, cancer vaccines, and adoptive cell therapy (ACT). Nonetheless, clinical benefit remains highly variable and patient-specific, in part, because all immunotherapeutic regimens vitally hinge on the capacity of endogenous and/or adoptively transferred T-effector (Teff) cells, including chimeric antigen receptor (CAR) T cells, to home efficiently into tumor target tissue. Thus, defects intrinsic to the multi-step T-cell homing cascade have become an obvious, though significantly underappreciated contributor to immunotherapy resistance. Conspicuous have been low intralesional frequencies of tumor-infiltrating T-lymphocytes (TILs) below clinically beneficial threshold levels, and peripheral rather than deep lesional TIL infiltration. Therefore, a Teff cell ‘homing deficit’ may arguably represent a dominant factor responsible for ineffective immunotherapeutic outcomes, as tumors resistant to immune-targeted killing thrive in such permissive, immune-vacuous microenvironments. Fortunately, emerging
    [Show full text]
  • Protein Composition of the Hepatitis a Virus Quasi-Envelope
    Protein composition of the hepatitis A virus quasi-envelope Kevin L. McKnighta,b,c, Ling Xiec,d, Olga González-Lópeza,b,c, Efraín E. Rivera-Serranoa,b,c, Xian Chenc,d, and Stanley M. Lemona,b,c,1 aDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292; bDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292; cLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295; and dDepartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260 Edited by Mary K. Estes, Baylor College of Medicine, Houston, TX, and approved April 12, 2017 (received for review November 27, 2016) The Picornaviridae are a diverse family of RNA viruses including many within extracellular vesicles before cell lysis, often with many pathogens of medical and veterinary importance. Classically consid- capsids packaged within a single vesicle (4–6). The cellular egress ered “nonenveloped,” recent studies show that some picornaviruses, of these classically nonenveloped viruses in membrane-bound notably hepatitis A virus (HAV; genus Hepatovirus) and some mem- vesicles has blurred the distinction between enveloped and bers of the Enterovirus genus, are released from cells nonlytically in nonenveloped viruses and has important implications for path- membranous vesicles. To better understand the biogenesis of quasi- ogenesis (2). enveloped HAV (eHAV) virions, we conducted a quantitative proteo- Several lines of evidence indicate that the biogenesis of quasi- mics analysis of eHAV purified from cell-culture supernatant fluids by enveloped eHAV virions is dependent upon components of the isopycnic ultracentrifugation.
    [Show full text]
  • Glycosylation Inhibition Reduces Cholesterol Accumulation in NPC1 Protein-Deficient Cells
    Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells Jian Li1, Maika S. Deffieu1, Peter L. Lee1, Piyali Saha, and Suzanne R. Pfeffer2 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved October 28, 2015 (received for review October 16, 2015) Lysosomes are lined with a glycocalyx that protects the limiting In this paper, we have used two approaches to modify the gly- membrane from the action of degradative enzymes. We tested the cocalyx. Alteration of glycosylation decreases lysosomal cholesterol hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer levels in NPC1-deficient Chinese hamster ovary (CHO) and human of low density lipoprotein-derived cholesterol across this glycocalyx. A cells. These experiments support a model in which NPC1 protein prediction of this model is that cells will be less dependent upon NPC1 functions to help cholesterol traverse the glycocalyx that lines the if their glycocalyx is decreased in density. Lysosome cholesterol content interior of lysosome limiting membranes. was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyrano- Results and Discussion side, an inhibitor of O-linked glycosylation. Direct biochemical LAMP1 and LAMP2 proteins represent the major glycoproteins measurement of cholesterol showed that lysosomes purified from of the lysosome membrane (12). These proteins are glycosylated NPC1-deficient fibroblasts contained at least 30% less cholesterol on both asparagine residues (N linked) and serine or threonine when O-linked glycosylation was blocked.
    [Show full text]
  • Mai Muuttunut Hiu Li Huwuluhan
    MAIMUUTTUNUT US009809653B2 HIU LI HUWULUHAN (12 ) United States Patent ( 10 ) Patent No. : US 9 ,809 ,653 B2 Baudat et al. (45 ) Date of Patent: Nov . 7 , 2017 (54 ) ANTI- LAMP1 ANTIBODIES AND ANTIBODY ( 58) Field of Classification Search DRUG CONJUGATES , AND USES THEREOF None See application file for complete search history . ( 71 ) Applicant: SANOFI, Paris (FR ) (72 ) Inventors: Yves Baudat, Paris (FR ) ; Francis ( 56 ) References Cited Blanche, Paris ( FR ) ; Béatrice Cameron , Paris (FR ) ; Tarik Dabdoubi, U . S . PATENT DOCUMENTS Le Coudray Montceaux (FR ) ; 4 , 179 , 337 A 12 / 1979 Davis et al. Anne -Marie LeFebvre , Le Plessis Pâté 4 ,256 ,746 A 3 / 1981 Miyashita et al . ( FR ) ; Magali Mathieu , Choisy le Roi 4 , 294 , 757 A 10 / 1981 Asai 4 , 301 , 144 A 11/ 1981 Iwashita et al. (FR ) ; Ana Merino - Trigo , Paris (FR ) ; 4 , 307 ,016 A 12 / 1981 Asai et al. Manoel Nunes, Paris (FR ) 4 ,313 ,946 A 2 / 1982 Powell et al . 4 , 315 , 929 A 2 / 1982 Freedman et al. 4 ,322 , 348 A 3 / 1982 Asai et al. ( 73 ) Assignee : SANOFI, Paris ( FR ) 4 ,331 , 598 A 5 / 1982 Hasegawa et al. 4 , 361, 650 A 11/ 1982 Asai et al . ( * ) Notice : Subject to any disclaimer, the term of this 4 , 362 ,663 A 12 / 1982 Kida et al. patent is extended or adjusted under 35 4 , 364 , 866 A 12 / 1982 Asai et al. U . S . C . 154 ( b ) by 0 days . 4 ,371 , 533 A 2 / 1983 Akimoto et al . 4 ,424 ,219 A 1 / 1984 Hashimoto et al. 4 ,450 , 254 A 5 / 1984 Isley et al.
    [Show full text]
  • Supplemental Materials 1 SUPPLEMENTAL METHODS CSC
    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Identification of MM immunotherapy targets by MS – Supplemental materials 1 SUPPLEMENTAL METHODS 2 CSC-technology 3 Approximately 100 million cells from each biological replicate (n=3-6) were taken 4 through the CSC-Technology workflow as previously described in detail.(1-3) Cells were 5 washed with PBS and oxidized by treatment with 1 mM sodium meta-periodate (Pierce, 6 Rockford, IL) in PBS pH 7.6 for 15 min at 4°C followed by 2.5 mg/ml biocytin hydrazide 7 (Biotium, Hayward, CA) in PBS pH 6.5 for 1 hour at 4°C. Cells were then collected and 8 homogenized in 10mM Tris pH 7.5, 0.5 mM MgCl2 and the resulting cell lysate was centrifuged 9 at 800 x g for 10 min at 4°C. The supernatant was centrifuged at 210,000 x g for 16 hours at 4°C 10 to collect the membranes. The supernatant was removed and the membrane protein pellet was 11 washed with 25 mM Na2CO3 to disrupt peripheral protein interactions. To the resulting 12 membrane pellet, 300µl 100 mM NH4HCO3, 5 mM Tris(2-carboxyethyl) phosphine (Sigma, St. 13 Louis, MO), and 0.1% (v/v) Rapigest (Waters, Milford, MA) were added and placed on a 14 Thermomixer (750 rpm) to continuously vortex. Proteins were allowed to reduce for 10 min at 15 25°C followed by alklylation with 10 mM iodoacetamide for 30 min.
    [Show full text]
  • Lamp2s and Chaperone-Mediated Autophagy 4443 of Trichloroacetic Acid to a final Concentration of 10%
    Journal of Cell Science 113, 4441-4450 (2000) 4441 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1804 Unique properties of lamp2a compared to other lamp2 isoforms A. M. Cuervo* and J. F. Dice Department of Physiology, Tufts University School of Medicine, Boston, MA, USA *Author for correspondence (e-mail: [email protected]) Accepted 28 September; published on WWW 16 November 2000 SUMMARY Lamp2a acts as a receptor in the lysosomal membrane for other lamp2s. Four positively-charged amino acids substrate proteins of chaperone-mediated autophagy. uniquely present in the cytosolic tail of lamp2a are required Using antibodies specific for the cytosolic tail of lamp2a and for the binding of substrate proteins. Lamp2a also others recognizing all lamp2 isoforms, we found that in rat distributes to an unique subpopulation of perinuclear liver lamp2a represents 25% of lamp2s in the lysosome. We lysosomes in cultured fibroblasts in response to serum show that lamp2a levels in the lysosomal membrane in withdrawal, and lamp2a, more than other lamp2s, tends to rat liver and fibroblasts in culture directly correlate with multimerize. These characteristics may be important for rates of chaperone-mediated autophagy in a variety lamp2a to act as a receptor for chaperone-mediated of physiological and pathological conditions. The autophagy. concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Key words: Lysosome, Protein degradation, Membrane receptor, Furthermore, substrate proteins bind to lamp2a but not to Chaperone, Rat liver INTRODUCTION Part of this matrix form of lamp2 reversibly aggregates with other lysosomal enzymes in a pH-dependent manner (Jadot et Lysosome-associated membrane proteins (lamps) are a al., 1997).
    [Show full text]