title: Tracing myelin protein zero (P0) in vivo by construction of P0-GFP fusion proteins.
abstract: BACKGROUND
  Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein.
  
  
  RESULTS
  In contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed.
  
  
  CONCLUSIONS
  The present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation.
authors.affiliation: Institute of Human Genetics, Friedrich-Alexander-University, Erlangen, Germany. aekici@humgenet.uni-erlangen.de
authors.forename: Arif B
authors.initials: AB
authors.lastname: Ekici
authors.forename: Sevinc
authors.initials: S
authors.lastname: Oezbey
authors.forename: Christina
authors.initials: C
authors.lastname: Fuchs
authors.forename: Eva
authors.initials: E
authors.lastname: Nelis
authors.forename: Christine
authors.initials: C
authors.lastname: Van Broeckhoven
authors.forename: Melitta
authors.initials: M
authors.lastname: Schachner
authors.forename: Bernd
authors.initials: B
authors.lastname: Rautenstrauss
mesh_terms: D000344:Afferent Pathways / Q000473:pathology / Q000503:physiopathology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002675:Child, Preschool; D003593:Cytoplasm / Q000737:chemistry; D049452:Green Fluorescent Proteins; D015417:Hereditary Sensory and Motor Neuropathy / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D007202:Indicators and Reagents; D007313:Insecta; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional / Q000235:genetics; D018993:Myelin P0 Protein / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D009431:Neural Conduction / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D013497:Sural Nerve / Q000737:chemistry / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D014162:Transfection
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018993:Myelin P0 Protein; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins