DOCSLIB.ORG

N-Terminal Region Sulfation in a Conserved Extracellular CCR2B Is

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
N-Terminal Region Sulfation in a Conserved Extracellular CCR2B Is Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region This information is current as of September 27, 2021. Alexander A. Preobrazhensky, Sofya Dragan, Tomonori Kawano, Mikhail A. Gavrilin, Irina V. Gulina, Leena Chakravarty and P. E. Kolattukudy J Immunol 2000; 165:5295-5303; ; doi: 10.4049/jimmunol.165.9.5295 Downloaded from http://www.jimmunol.org/content/165/9/5295 References This article cites 68 articles, 39 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/9/5295.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region Alexander A. Preobrazhensky, Sofya Dragan, Tomonori Kawano, Mikhail A. Gavrilin, Irina V. Gulina, Leena Chakravarty, and P. E. Kolattukudy1 Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed muta- tions in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological Downloaded from consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamelli- .podium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2؉ influx into cytoplasm Mutations that prevented adenylate cyclase inhibition and Ca2؉ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of http://www.jimmunol.org/ the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational mod- ifications probably have significant biological functions. The Journal of Immunology, 2000, 165: 5295–5303. hemotactic cytokines (chemokines) recruit leukocytes to fated in proteins (6, 11). Several examples are known where sulfa- sites of injury, infection, and inflammation (1–4). Che- tion of tyrosines was shown to be important either for protein-protein C mokines are classified mainly in terms of the patterns of interaction (12–16) or for protein precursor processing (11). Cys residues, which form structurally important disulfide bonds. Monocyte chemotactic protein-1 (MCP-1)2 receptor (CCR2B), by guest on September 27, 2021 Those with an amino acid residue between the N-terminal pair of which is known to play an important role in a variety of diseases Cys residues, called ␣ or CXC chemokines, chemotax neutrophils involving inflammation and/or injury such as wound healing, ath- and nonhemopoietic cells, whereas those without an intervening erosclerosis, and arthritis (1–4), also contains a consensus sulfa- ␤ amino acid residue, called or CC chemokines, chemotax mono- tion domain at the N-terminal region. We noticed that a hexad of cytes, T cells, eosinophils, and NK cells. These two groups con- amino acid residues in this region is conserved in several chemo- stitute the majority of known chemokines. Chemokines specifi- kines, and therefore we suspected it to be functionally important. cally bind to seven transmembrane G protein-coupled receptors To test whether these residues are involved in the biological func- present on the target cells and cause chemotaxis and signal trans- tion of the receptor, site-directed mutagenesis was used. This mu- duction events that are not well understood. tagenesis involved the consensus sulfation site, including tyrosine, A chemokine receptor, CCR5 was recently reported to require the suspected site of sulfation, and the neighboring amino acid tyrosine sulfation for its function as a coreceptor for HIV-1 (5). residue that should be a part of the consensus required for enzy- Tyrosine sulfation of proteins is a modification that widely occurs matic sulfation by protein tyrosine sulfotransferases (6, 11). The in multicellular eukaryotic organisms (6, 7). Recently, two protein results presented here demonstrate that the hexad is important for tyrosine sulfotransferases, PTST-1 and PTST-2, have been cloned ligand binding, lamellipodium formation, chemotaxis, and signal (8–10). The sulfation of tyrosines occurs post-translationally, in the trans part of the Golgi network (6). The most important feature transduction. Structural alterations in this region of the receptor of tyrosine sulfation consensus sequences is the presence of an caused differential effects on lamellipodium formation and chemo- 2ϩ acidic or neutral amino acid residue directly before a tyrosine to be taxis vs Ca influx and adenylate cyclase inhibition, indicating sulfated, although not all theoretical consensus sequences are sul- that the receptor plays a role beyond the known function of causing ␣ ␤␥ dissociation of the trimeric G protein into Gi and G . Our re- sults also show that tyrosine within the hexad is sulfated and that Neurobiotechnology Center and Departments of Biochemistry and Molecular and the mutation in the consensus residue thought to be required for Cellular Biochemistry, Ohio State University, Columbus, OH 43210 sulfation severely inhibits sulfation and consequently inhibits the Received for publication March 14, 2000. Accepted for publication August 9, 2000. biological function of the receptor. Some chemokine receptors, The costs of publication of this article were defrayed in part by the payment of page such as CXCR4, are known to be glycoproteins, although no CC charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. P. E. Kolattukudy, Neurobio- technology Center, Ohio State University, 206 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. E-mail address: [email protected] 2 Abbreviation used in this paper: MCP-1, monocyte chemotactic protein-1. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 5296 SULFATION AND GLYCOSYLATION OF CCR2B chemokine receptor has been shown to be glycosylated. Our stud- once with HEPES containing binding buffer (1 mM CaCl2, 5 mM MgCl2, ies show that CCR2B is a glycoprotein and is sulfated at 0.5% BSA, and 50 mM HEPES, pH 7.2) (24). The final assay volume (250 ␮ ϫ 6 tyrosine 26. l) contained 5 10 transfected and washed cells, various amounts of MCP-1, and 0.02 pmol of iodinated MCP-1 protein. Various transfected cell lines were incubated with labeled and unlabeled MCP-1. Following Materials and Methods incubation, the cells were passed through a Whatman GF/C filter, and Reagents radioactivity on the filter was measured in a Packard COBRA gamma radiation counter (Downers Grove, IL). Dissociation constant (Kd) values The expression vector, pcDNA3 was obtained from Invitrogen (San Diego, for MCP-1 binding to CCR2B and mutated receptor in various cell lines of CA). Human embryonic kidney (HEK293) cells were obtained from Amer- HEK293 were determined using the LIGAND program (25). ican Type Culture Collection (ATCC CRL 1573; Manassas, VA). Lipo- fectamine, G418, penicillin-streptomycin solution, DMEM/F-12, BAC- ϩ TO-BAC baculovirus expression system, and FBS were purchased from Measurement of Ca2 influx induced by MCP-1 Life Technologies (Gaithersburg, MD). MEM/Eagle’s medium was ob- tained from ICN. Anti-Flag M2 mAb was purchased from Eastman Kodak Calcium influx into the cytoplasm was measured in HEK293 cell lines ␮ (Rochester, NY). Mouse IgG1, ␬ (MOPC-21), FITC-conjugated sheep anti- expressing CCR2B or its mutants after incubation with 2 M fura-2/ace- mouse IgG (whole molecule), phalloidin-FITC, chloramine-T, isobutyl- toxymethyl ester for 30 min at 37°C in a CO2 incubator. Fluorescence was methylxanthine, forskolin, QAE-Sephadex A-25, and Igepal CA-630 were measured after the addition of different concentrations
Load more

Recommended publications
  • ECO-Ssls for Pahs
    Ecological Soil Screening Levels for Polycyclic Aromatic Hydrocarbons (PAHs) Interim Final OSWER Directive 9285.7-78 U.S. Environmental Protection Agency Office of Solid Waste and Emergency Response 1200 Pennsylvania Avenue, N.W. Washington, DC 20460 June 2007 This page intentionally left blank TABLE OF CONTENTS 1.0 INTRODUCTION .......................................................1 2.0 SUMMARY OF ECO-SSLs FOR PAHs......................................1 3.0 ECO-SSL FOR TERRESTRIAL PLANTS....................................4 5.0 ECO-SSL FOR AVIAN WILDLIFE.........................................8 6.0 ECO-SSL FOR MAMMALIAN WILDLIFE..................................8 6.1 Mammalian TRV ...................................................8 6.2 Estimation of Dose and Calculation of the Eco-SSL ........................9 7.0 REFERENCES .........................................................16 7.1 General PAH References ............................................16 7.2 References Used for Derivation of Plant and Soil Invertebrate Eco-SSLs ......17 7.3 References Rejected for Use in Derivation of Plant and Soil Invertebrate Eco-SSLs ...............................................................18 7.4 References Used in Derivation of Wildlife TRVs .........................25 7.5 References Rejected for Use in Derivation of Wildlife TRV ................28 i LIST OF TABLES Table 2.1 PAH Eco-SSLs (mg/kg dry weight in soil) ..............................4 Table 3.1 Plant Toxicity Data - PAHs ..........................................5 Table 4.1
    [Show full text]
  • Biosynthesis and Secretion of the Microbial Sulfated Peptide Raxx and Binding to the Rice XA21 Immune Receptor
    Biosynthesis and secretion of the microbial sulfated peptide RaxX and binding to the rice XA21 immune receptor Dee Dee Luua,b,1, Anna Joea,b,c,1, Yan Chend, Katarzyna Paryse, Ofir Bahara,b,2, Rory Pruitta,b,3, Leanne Jade G. Chand, Christopher J. Petzoldd, Kelsey Longa,b, Clifford Adamchaka,b, Valley Stewartf, Youssef Belkhadire, and Pamela C. Ronalda,b,c,4 aDepartment of Plant Pathology, University of California, Davis, CA 95616; bThe Genome Center, University of California, Davis, CA 95616; cFeedstocks Division, Joint Bioenergy Institute, Emeryville, CA 94608; dTechnology Division, Joint Bioenergy Institute, Emeryville, CA 94608; eGregor Mendel Institute, Austrian Academy of Sciences, 1030 Vienna, Austria; and fDepartment of Microbiology & Molecular Genetics, University of California, Davis, CA 95616 Edited by Jonathan D. G. Jones, The Sainsbury Laboratory, Norwich, United Kingdom, and approved March 7, 2019 (received for review October 24, 2018) The rice immune receptor XA21 is activated by the sulfated micro- to Xoo strains lacking RaxX (7–10). Xoo strains lacking RaxX are bial peptide required for activation of XA21-mediated immunity X also compromised in virulence of rice plants lacking XA21 (10). (RaxX) produced by Xanthomonas oryzae pv. oryzae (Xoo). Muta- These results suggest a role for RaxX in bacterial virulence. tional studies and targeted proteomics revealed that the RaxX pre- Tyrosine sulfated RaxX16, a 16-residue synthetic peptide de- cursor peptide (proRaxX) is processed and secreted by the protease/ rived from residues 40–55 of the 60-residue RaxX precursor transporter RaxB, the function of which can be partially fulfilled by peptide (proRaxX), is the shortest characterized immunogenic a noncognate peptidase-containing transporter component B derivative of RaxX (10).
    [Show full text]
  • NDA/BLA Multi-Disciplinary Review and Evaluation
    NDA/BLA Multi-disciplinary Review and Evaluation NDA 214154 Nextstellis (drospirenone and estetrol tablets) NDA/BLA Multi-Disciplinary Review and Evaluation Application Type NDA Application Number(s) NDA 214154 (IND 110682) Priority or Standard Standard Submit Date(s) April 15, 2020 Received Date(s) April 15, 2020 PDUFA Goal Date April 15, 2021 Division/Office Division of Urology, Obstetrics, and Gynecology (DUOG) / Office of Rare Diseases, Pediatrics, Urologic and Reproductive Medicine (ORPURM) Review Completion Date April 15, 2021 Established/Proper Name drospirenone and estetrol tablets (Proposed) Trade Name Nextstellis Pharmacologic Class Combination hormonal contraceptive Applicant Mayne Pharma LLC Dosage form Tablet Applicant proposed Dosing x Take one tablet by mouth at the same time every day. Regimen x Take tablets in the order directed on the blister pack. Applicant Proposed For use by females of reproductive potential to prevent Indication(s)/Population(s) pregnancy Recommendation on Approval Regulatory Action Recommended For use by females of reproductive potential to prevent Indication(s)/Population(s) pregnancy (if applicable) Recommended Dosing x Take one pink tablet (drospirenone 3 mg, estetrol Regimen anhydrous 14.2 mg) by mouth at the same time every day for 24 days x Take one white inert tablet (placebo) by mouth at the same time every day for 4 days following the pink tablets x Take tablets in the order directed on the blister pack 1 Reference ID: 4778993 NDA/BLA Multi-disciplinary Review and Evaluation NDA 214154 Nextstellis (drospirenone and estetrol tablets) Table of Contents Table of Tables .................................................................................................................... 5 Table of Figures ................................................................................................................... 7 Reviewers of Multi-Disciplinary Review and Evaluation ...................................................
    [Show full text]
  • Biotransformation: Basic Concepts (1)
    Chapter 5 Absorption, Distribution, Metabolism, and Elimination of Toxics Biotransformation: Basic Concepts (1) • Renal excretion of chemicals Biotransformation: Basic Concepts (2) • Biological basis for xenobiotic metabolism: – To convert lipid-soluble, non-polar, non-excretable forms of chemicals to water-soluble, polar forms that are excretable in bile and urine. – The transformation process may take place as a result of the interaction of the toxic substance with enzymes found primarily in the cell endoplasmic reticulum, cytoplasm, and mitochondria. – The liver is the primary organ where biotransformation occurs. Biotransformation: Basic Concepts (3) Biotransformation: Basic Concepts (4) • Interaction with these enzymes may change the toxicant to either a less or a more toxic form. • Generally, biotransformation occurs in two phases. – Phase I involves catabolic reactions that break down the toxicant into various components. • Catabolic reactions include oxidation, reduction, and hydrolysis. – Oxidation occurs when a molecule combines with oxygen, loses hydrogen, or loses one or more electrons. – Reduction occurs when a molecule combines with hydrogen, loses oxygen, or gains one or more electrons. – Hydrolysis is the process in which a chemical compound is split into smaller molecules by reacting with water. • In most cases these reactions make the chemical less toxic, more water soluble, and easier to excrete. Biotransformation: Basic Concepts (5) – Phase II reactions involves the binding of molecules to either the original toxic molecule or the toxic molecule metabolite derived from the Phase I reactions. The final product is usually water soluble and, therefore, easier to excrete from the body. • Phase II reactions include glucuronidation, sulfation, acetylation, methylation, conjugation with glutathione, and conjugation with amino acids (such as glycine, taurine, and glutamic acid).
    [Show full text]
  • A Novel Method for Site-Determination of Tyrosine O- Sulfation in Peptides and Proteins
    A novel method for site-determination of tyrosine O- sulfation in peptides and proteins Yonghao Yu Genome Center, Depts of Chemistry and Molecular Cell Biology, University of California, Davis, CA, 95616 and Dept of Chemistry, University of California, Berkeley, CA, 94720, current address: Dept of Cell Biology, Harvard Medical School, Boston, MA 02115 Adam J. Hoffhines Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 Kevin L. Moore Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Julie A. Leary Genome Center, Departments of Chemistry and Molecular Cell Biology, University of California, Davis, CA, 95616 Method Article Keywords: tyrosine sulfation, mass spectrometry, post-translational modication, protein-protein interaction, extracelluar matrix, proteomics Posted Date: September 20th, 2007 DOI: https://doi.org/10.1038/nprot.2007.388 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/12 Abstract Introduction Tyrosine O-sulfation is one of many posttranslational modications described in nature that can impart critical functional properties to proteins that are independent of the genes encoding them1, 2. It plays a key role in regulating protein-protein interactions in the extracellular space3 and is catalyzed by two closely related Golgi enzymes called tyrosylprotein sulfotransferases \(TPST-1 and TPST–2)4, 5. In this protocol, we describe a novel subtractive strategy to determine the sites of tyrosine sulfation in peptides and proteins6. Hydroxyl groups on unsulfated tyrosines are stoichiometrically acetylated by a one-step reaction using sulfosuccinimidyl acetate in the presence of imidazole at pH 7.0.
    [Show full text]
  • Tyrosine Sulfation: a Modulator of Extracellular Protein–Protein Interactions Provided by Elsevier - Publisher Connector John W Kehoe1 and Carolyn R Bertozzi1,2
    cm7309.qxd 03/10/2000 11:46 Page R57 CORE Minireview R57 brought to you by Elsevier - Publisher Connector Tyrosine sulfation: a modulator of extracellular protein–protein interactions provided by John W Kehoe1 and Carolyn R Bertozzi1,2 Tyrosine sulfation is a post-translational modification of P-selectin on activated endothelial cells with the many secreted and membrane-bound proteins. Its P-selectin glycoprotein ligand (PSGL)-1 on cognate biological roles have been unclear. Recent work has leukocytes [3]. PSGL-1 was identified at the molecular implicated tyrosine sulfate as a determinant of level in 1993, using expression cloning [4]. Glycosylation protein–protein interactions involved in leukocyte of PSGL-1 was known to be a major determinant of adhesion, hemostasis and chemokine signaling. P-selectin binding, but was not sufficient to explain the binding avidities observed in vivo. Addresses: Departments of 1Molecular and Cell Biology and 2 Chemistry, University of California, Berkeley, CA 94720, USA. Further investigations revealed that tyrosine sulfation near Correspondence: Carolyn R Bertozzi the amino terminus of PSGL-1 plays an essential role in E-mail: [email protected] P-selectin binding (shown schematically in Figure 1b). Three papers published within one month of each other in Chemistry & Biology 2000, 7:R57–R61 1995 provided the first evidence of this phenomenon 1074-5521/00/$ – see front matter [5–7]. Progressive deletions of the PSGL-1 amino termi- © 2000 Elsevier Science Ltd. All rights reserved. nus by Pouyani and Seed [5] revealed that a 20-residue peptide was required for high-affinity binding. Further- The study of living systems has been transformed by an more, mutagenesis within this sequence showed that the explosion of genetic information.
    [Show full text]
  • NST110: Advanced Toxicology Lecture 4: Phase I Metabolism
    Absorption, Distribution, Metabolism and Excretion (ADME): NST110: Advanced Toxicology Lecture 4: Phase I Metabolism NST110, Toxicology Department of Nutritional Sciences and Toxicology University of California, Berkeley Biotransformation The elimination of xenobiotics often depends on their conversion to water-soluble chemicals through biotransformation, catalyzed by multiple enzymes primarily in the liver with contributions from other tissues. Biotransformation changes the properties of a xenobiotic usually from a lipophilic form (that favors absorption) to a hydrophilic form (favoring excretion in the urine or bile). The main evolutionary goal of biotransformation is to increase the rate of excretion of xenobiotics or drugs. Biotransformation can detoxify or bioactivate xenobiotics to more toxic forms that can cause tumorigenicity or other toxicity. Phase I and Phase II Biotransformation Reactions catalyzed by xenobiotic biotransforming enzymes are generally divided into two groups: Phase I and phase II. 1. Phase I reactions involve hydrolysis, reduction and oxidation, exposing or introducing a functional group (-OH, -NH2, -SH or –COOH) to increase reactivity and slightly increase hydrophilicity. O R1 - O S O sulfation O R2 OH Phase II Phase I R1 R2 R1 R2 - hydroxylation COO R1 O O glucuronidation OH R2 HO excretion OH O COO- HN H -NH2 R R1 R1 S N 1 Phase I Phase II COO O O oxidation glutathione R2 OH R2 R2 conjugation 2. Phase II reactions include glucuronidation, sulfation, acetylation, methylation, conjugation with glutathione, and conjugation with amino acids (glycine, taurine and glutamic acid) that strongly increase hydrophilicity. Phase I and II Biotransformation • With the exception of lipid storage sites and the MDR transporter system, organisms have little anatomical defense against lipid soluble toxins.
    [Show full text]
  • 6. References
    332 IARC MONOGRAPHS VOLUME 91 6. References Abramov, Y., Borik, S., Yahalom, C., Fatum, M., Avgil, G., Brzezinski, A. & Banin, E. (2004) The effect of hormone therapy on the risk for age-related maculopathy in postmenopausal women. Menopause, 11, 62–68 Adams, M.R., Register, T.C., Golden, D.L., Wagner, JD. & Williams, J.K. (1997) Medroxyproges- terone acetate antagonizes inhibitory effects of conjugated equine estrogens on coronary artery atherosclerosis. Arterioscler. Thromb. vasc. Biol., 17, 217–221 Adjei, A.A. & Weinshilboum, R.M. (2002) Catecholestrogen sulfation: Possible role in carcino- genesis. Biochem. biophys. Res. Commun., 292, 402–408 Adjei, A.A., Thomae, B.A., Prondzinski, J.L., Eckloff, B.W., Wieben, E.D. & Weinshilboum, R.M. (2003) Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: Gene resequencing and functional genomics. Br. J. Pharmacol., 139, 1373–1382 Ahmad, M.E., Shadab, G.G.H.A., Hoda, A. & Afzal, M. (2000) Genotoxic effects of estradiol-17β on human lymphocyte chromosomes. Mutat. Res., 466, 109–115 COMBINED ESTROGEN−PROTESTOGEN MENOPAUSAL THERAPY 333 Ahmad, M.E., Shadab, G.G.H.A., Azfer, M.A. & Afzal, M. (2001) Evaluation of genotoxic poten- tial of synthetic progestins-norethindrone and norgestrel in human lymphocytes in vitro. Mutat. Res., 494, 13–20 Aitken, J.M., Hart, D.M. & Lindsay, R. (1973) Oestrogen replacement therapy for prevention of osteoporosis after oophorectomy. Br. med. J., 3, 515–518 Al-Azzawi, F., Wahab, M., Thompson, J., Whitehead, M. & Thompson, W. (1999) Acceptability and patterns of uterine bleeding in sequential trimegestone-based hormone replacement therapy: A dose-ranging study. Hum. Reprod., 14, 636–641 Albert, C., Vallée, M., Beaudry, G., Belanger, A.
    [Show full text]
  • Serves As High-Affinity Substrate for Tyrosylprotein Sulfotransferase
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 6143-6147, September 1985 Cell Biology (Glu62,Ala30,Tyr8). serves as high-affinity substrate for tyrosylprotein sulfotransferase: A Golgi enzyme (tyrosine sulfation/protein sorting/chromaffin granules/tyrosine phosphorylation/tubulin) RAYMOND W. H. LEE AND WIELAND B. HUTTNER* Department of Neurochemistry, Max-Planck-Institute for Psychiatry, 8033 Martinsried, Federal Republic of Germany Communicated by Fritz Lipmann, May 10, 1985 ABSTRACT Tyrosylprotein sulfotransferase, the enzyme as substrate are as follows: (i) the only sulfatable hydroxyl catalyzing the sulfation of proteins on tyrosine residues, was group in Glu,Ala,Tyr is on tyrosine, in contrast to physio- characterized by using the acidic polymer containing tyrosine logical protein substrates, which may also contain sulfatable (Glu62,Ala3',Tyr8). (referred to as Glu,Ala,Tyr) as exogenous carbohydrate, serine, and threonine residues; (ii) "protein" substrate. After subcellular fractionation ofa bovine Glu,Ala,Tyr is available in completely unsulfated form in adrenal medulla homogenate, tyrosylprotein sulfotransferase sufficient quantities for enzymological studies, whereas activity was found to be highest in fractions enriched in Golgi physiologically sulfated proteins purified from bi6logical membrane vesicles. Tyrosylprotein sulfotransferase required sources need to be desulfated before being used as sub- the presence of a nonionic detergent for sulfation of exogenous strates; (iii) Glu,Ala,Tyr is likely to contain tyrosine residues Glu,Ala,Tyr, indicating an orientation of the catalytic site of in the vicinity ofacidic amino acid residues. A comparison of the enzyme toward the Golgi lumen. Tyrosylprotein the amino acid sequences surrounding the sulfated tyrosine sulfotransferase was solubilized by Triton X-100, suggesting residues in gastrin (10), cholecystokinin (11), fibrinopeptide that the enzyme was tightly associated with the Golgi mem- B (12), and hirudin (13) shows that these sequences always brane, possibly as an integral membrane protein.
    [Show full text]
  • Estrogen Metabolism: Are We Assessing It Properly? Filomena Trindade, MD, MPH
    Estrogen Metabolism: Are We Assessing It Properly? Filomena Trindade, MD, MPH The views and opinions expressed herein are solely those of the presenter and do not necessarily represent those of Genova Diagnostics. Thus, Genova Diagnostics does not accept liability for consequences of any actions taken on the basis of the information provided. Michael Chapman, ND Medical Education Specialist - Asheville Filomena Trindade, MD, MPH Technical Issues & Clinical Questions Please type any technical issue or clinical question into either the “Chat” or “Questions” boxes, making sure to send them to “Organizer” at any time during the webinar. We will be compiling your clinical questions and answering as many as we can the final 15 minutes of the webinar. DISCLAIMER: Please note that any and all emails provided may be used for follow up correspondence and/or for further communication. Estrogen Metabolism: Are We Assessing It Properly? Filomena Trindade, MD, MPH The views and opinions expressed herein are solely those of the presenter and do not necessarily represent those of Genova Diagnostics. Thus, Genova Diagnostics does not accept liability for consequences of any actions taken on the basis of the information provided. Objectives • Understand the importance of estrogen metabolism with respect to cancer risk • Be able to devise a treatment plan for a pt with an unfavorable estrogen metabolism profile • Gain a basic understanding of the importance of methylation in estrogen metabolism, health promotion and cancer prevention • Learn to apply these
    [Show full text]
  • Intratumoral Estrogen Sulfotransferase Induction Contributes to the Anti-Breast Cancer Effects of the Dithiocarbamate Derivative TM208
    npg Acta Pharmacologica Sinica (2015) 36: 1246–1255 © 2015 CPS and SIMM All rights reserved 1671-4083/15 www.nature.com/aps Original Article Intratumoral estrogen sulfotransferase induction contributes to the anti-breast cancer effects of the dithiocarbamate derivative TM208 Xi-wei JI 1, 2, Guang-ping CHEN4, Yan SONG5, Ming HUA1, Li-jie WANG1, Liang LI1, Yin YUAN1, Si-yuan WANG1, Tian-yan ZHOU1, 3, *, Wei LU1, 3 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; 2Institute of Clinical Pharmacology, Peking University First Hospital, Beijing 100191, China; 3State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China; 4Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA; 5Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Aim: Sulfotransferase-catalyzed sulfation is the most important pathway for inactivating estrogens. Thus, activation of estrogen sulfotransferase (EST) may be an alternative approach for the treatment of estrogen-dependent breast cancer. In this study we investigated the involvement of EST in anti-breast cancer effects of the dithiocarbamate derivative TM208 in vitro and in vivo. Methods: The viability of human breast cancer MCF-7 cells was determined using a SBB assay. Nude mice bearing MCF-7 cells were orally administered TM208 (50 and 150 mg·kg-1·d-1) for 18 days. The xenograft tumors and uteri were collected. The mRNA expression of EST was examined with real-time PCR. EST protein was detected with Western blot, ELISA or immunohistochemical staining assays. A radioactive assay was used to measure the EST activity.
    [Show full text]
  • Nutritional Flavonoids Impact on Nuclear and Extranuclear Estrogen Receptor Activities
    Genes & Nutrition Vol. 1, No. 3/4, pp. 161-176, 2006 ISSN 1555-8932 print, Copyright © 2006 by New Century Health Publishers, LLC www.newcenturyhealthpublishers.com All rights of reproduction in any form reserved NUTRITIONAL FLAVONOIDS IMPACT ON NUCLEAR AND EXTRANUCLEAR ESTROGEN RECEPTOR ACTIVITIES Paola Galluzzo and Maria Marino Department of Biology, University “Roma Tre”, Viale G. Marconi 446, I-00146 Roma, Italy [Received March 14, 2006; Accepted April 30, 2006] ABSTRAABSTRAABSTRACTCTCT: Flavonoids are a large group of non-nutrient important are the plant secondary metabolites flavonoids. They compounds naturally produced from plants as part of their are present in all terrestrial vascular plants whereas, in mammals, defence mechanisms against stresses of different origins. They they occur only through dietary intake (Birt et al., 2001; Dixon, emerged from being considered an agricultural oddity only after 2004). More than 4,000 different flavonoids have been it was observed that these compounds possess a potential protective described and categorized into 6 subclasses as a function of function against several human degenerative diseases. This has the type of heterocycle involved: flavonols, flavones, flavanols, led to recommending the consumption of food containing high flavanonols, flavanones, and isoflavones (Table 1) (Birt et al., concentrations of flavonoids, which at present, especially as soy 2001; Manach et al., 2004). isoflavones, are even available as over-the-counter nutraceuticals. The increased use of flavonoids has occurred even though their mechanisms are not completely understood, in particular those TABLE 11:1TABLE Chemical structures of certain commonly occurring plant involving the flavonoid impact on estrogen signals. In fact, most flavonoid aglycones of the human health protective effects of flavonoids are described either as estrogen-mimetic, or as anti-estrogenic, while others do not involve estrogen signaling at all.
    [Show full text]