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Identification of ATP8B1 as a Blood–Brain Barrier-Enriched Protein

Cellular and Molecular Neurobiology volume 34, pages 473–478 (2014)Cite this article

Abstract

In order to define the molecular anatomy of the blood–brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.

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References

  • Bessey OA, Lowry OH, Brock MJ (1946) A method for the rapid determination of alkaline phosphatase with five cubic milliliters of serum. J Biol Chem 164:321–329

    CAS  PubMed  Google Scholar 

  • Birnbaum MJ, Haspel HC, Rosen OM (1986) Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc Natl Acad Sci USA 83:5784–5788

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  • Bryde S, Hennrich H, Verhulst PM, Devaux PF, Lenoir G, Holthuis JCM (2010) CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery. J Biol Chem 285:40562–40572. doi:10.1074/jbc.M110.139543

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  • Bull LN, Van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, Liao M, Klomp LW, Lomri N, Berger R, Scharschmidt BF, Knisely AS, Houwen RH, Freimer NB (1998) A gene encoding P-type ATPase mutated in two forms hereditary cholestasis. Nat Genet 18:219–224

    CAS  Article  PubMed  Google Scholar 

  • Chehade JM, Haas MJ, Mooradian AD (2002) Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression. Neurochem Res 27:249–252

    CAS  Article  PubMed  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    CAS  Article  PubMed  Google Scholar 

  • Demarest TG, Murugesan N, Shrestha B, Pachter JS (2012) Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan® low density array. J Neurosci Methods 206:200–204

    Article  PubMed Central  PubMed  Google Scholar 

  • Devraj K, Klinger ME, Myers RL, Mokashi A, Hawkins RA, Simpson IA (2011) GLUT-1 glucose transporters in the blood–brain barrier: differential phosphorylation. J Neurosci Res 89:1913–1925

    CAS  Article  PubMed  Google Scholar 

  • Enerson BE, Drewes LR (2006) The rat blood–brain barrier transcriptome. J Cerebral Blood Flow Metabol 26:959–973

    CAS  Article  Google Scholar 

  • Engelhardt B, Sorokin L (2009) The blood–brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin Immunopathol 31:497–511. doi:10.1007/s00281-009-0177-0

    Article  PubMed  Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific-activity. Anal Biochem 132:6–13

    CAS  Article  PubMed  Google Scholar 

  • Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor

    Google Scholar 

  • Harris MJ, Arias IM (2003) FIC1, a P-type ATPase lined to cholestatic liver disease, has homologues (ATP8B2 and ATP8B3) expressed throughout the body. Biochim Biophys Acta 1633:127–133

    CAS  Article  PubMed  Google Scholar 

  • Kong W, Mou X, Liu Q, Chen Z, Vanderburg CR, Rogers JT, Huang X (2009) Independent component analysis of Alzheimer’s DNA microarray gene expression data. Mol Neurodegen 4:5. doi:10.1186/1750-1326-4-5

    Article  CAS  Google Scholar 

  • Laemlli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685

    Article  Google Scholar 

  • Lange MLB, Cenini G, Piroddi M, Abdul HF, Sultana R, Galli F, Memo M, Butterfield DA (2008) Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease. Neurobiol Dis 29:456–464

    Article  CAS  Google Scholar 

  • Liang L, Pardee AB (1992) Differential display of eukaryotic mRNA by means of the polymerase chain reaction. Science 257:967–971

    CAS  Article  PubMed  Google Scholar 

  • Liang P, Averboukh L, Pardee AB (1993) Distribution and cloning of eukaryotic mRNA by means of differential display: refinements and optimizations. Nucleic Acids Res 14:3269–3275

    Article  Google Scholar 

  • Liu WY, Wang ZB, Zhang LC, Wei X, Li L (2012) Tight junction in blood–brain barrier: an overview of structure, regulation, and regulator substances. CNS Neurosci Ther 18(8):609–615. doi:10.1111/j.1755-5949.2012.00340.x

    CAS  Article  PubMed  Google Scholar 

  • Lowry OH, Rosebrough JN, Farr AL, Randal RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:262–275

    Google Scholar 

  • Lu Q, Murugesan N, Macdonald JA, Wu SL, Pachter JS, Hancock WS (2008) Analysis of mouse brain microvascular endothelium using immuno-laser capture microdissection coupled to a hybrid linear ion trap with Fourier transform-mass spectrophotometry proteomics platform. Electrophoresis 29:2689–2695

    CAS  Article  PubMed  Google Scholar 

  • Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke J, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, Bryant SH (2009) CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 37:D205–D210. doi:10.1093/nar/gkn845

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  • Mooradian AD (1997) Central nervous system complications of diabetes mellitus: a perspective from the blood–brain barrier. Brain Res Brain Res Rev 23:210–218

    CAS  Article  PubMed  Google Scholar 

  • Mooradian AD, Bastani B (1993) Identification of proton-translocating adenosine triphosphatases in rat cerebral microvessels. Brain Res 629:128–132

    CAS  Article  PubMed  Google Scholar 

  • Mooradian AD, Grabau G, Uko-eninn A (1993) In situ quantitative estimates of the age-related and diabetes-related changes in cerebral endothelial barrier antigen. Brain Res 609:41–44

    CAS  Article  PubMed  Google Scholar 

  • Mooradian AD, Grabau G, Bastani B (1994a) Adenosine triphosphatases of rat cerebral microvessels. Effect of age and diabetes mellitus. Life Sci 55:1261–1265

    CAS  Article  PubMed  Google Scholar 

  • Mooradian AD, Pinnas JL, Lung CC, Yahya MD, Meredith K (1994b) Diabetes-related changes in the protein composition of rat cerebral microvessels. Neurochem Res 19:123–128

    CAS  Article  PubMed  Google Scholar 

  • Muruganandam A, Tanha J, Narang S, Stanimirovic D (2002) Selection of phage-displayed llama single-domain antibodies that transmigrate across the human blood–brain barrier endothelium. FASEB J 16:240–242

    CAS  PubMed  Google Scholar 

  • Orlowski M, Meister A (1965) Isolation of gamma-glutamyl transpeptidase from hog kidney. J Biol Chem 240:338–347

    CAS  PubMed  Google Scholar 

  • Partridge WM (2005) Molecular biology of the blood–brain barrier. Mol Biotechnol 30:57–69

    Article  Google Scholar 

  • Sanger F, Nichlen S, Coulson AR (1997) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  Google Scholar 

  • Shah GN, Li J, Mooradian AD (1998) Novel translational repressor (NAT-1) expression is modified by thyroid state and age in brain and liver. Eur J Endocrinol 139:649–653

    CAS  Article  PubMed  Google Scholar 

  • Tang X, Halleck MS, Schlegel RA, Williamson P (1996) A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 272:1495–1497

    CAS  Article  PubMed  Google Scholar 

  • Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci USA 76:4350–4354

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  • Van der Velden LW, Wichers CGK, van Breevoort AED, Coleman JA, Molday RS, Berger R, Klomp LWJ, van de Graaf SFJ (2010) Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases. J Biol Chem 285:40088–40096. doi:10.1074/jbc.M110.139006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Williamson P, Schlegel RA (2002) Transbilayer phospholipid movement and the clearance of apoptotic cells. Biochim Biophys Acta 1585:53–63

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

We would like to sincerely thank Dr. Leonard Petrucelli (Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA) for providing mouse cerebral histological sections. We would also like to sincerely thank Dr. Sania Shuja (Department of Pathology and Laboratory Medicine, University of Florida Jacksonville College of Medicine, Jacksonville, FL, USA) for taking the photomicrographs. GNS and LMOH carried out the experiments described in the manuscript. All of the authors participated in writing and reviewing the manuscript.

Conflict of interest

The authors have no conflicts of interest to declare.

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Affiliations

  1. Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Jacksonville College of Medicine, University of Florida, 653-1 West Eighth Street, L14, Jacksonville, FL, 32209, USA

    Michael J. Haas, Luisa M. Onstead-Haas & Arshag D. Mooradian

  2. Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Saint Louis University, Saint Louis, MO, USA

    Gul N. Shah

Authors
  1. Michael J. Haas
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  2. Gul N. Shah
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  3. Luisa M. Onstead-Haas
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  4. Arshag D. Mooradian
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Correspondence to Michael J. Haas.

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Haas, M.J., Shah, G.N., Onstead-Haas, L.M. et al. Identification of ATP8B1 as a Blood–Brain Barrier-Enriched Protein. Cell Mol Neurobiol 34, 473–478 (2014). https://doi.org/10.1007/s10571-014-0045-x

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  • DOI: https://doi.org/10.1007/s10571-014-0045-x

Keywords

  • Blood–brain barrier
  • ATP8B1
  • Gene expression
  • FIC1