title: Biotinylation and characterization of Cryptococcus neoformans cell surface proteins.
abstract: OBJECTIVE
  To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation.
  
  
  RESULTS
  Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls.
  
  
  CONCLUSIONS
  A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma.
  
  
  CONCLUSIONS
  A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.
authors.affiliation: Life and Health Sciences, Aston University, Birmingham, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Foster
authors.forename: R A
authors.initials: RA
authors.lastname: Bird
authors.forename: S N
authors.initials: SN
authors.lastname: Smith
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation / Q000379:methods; D003455:Cryptococcus neoformans / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000032:analysis; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007202:Indicators and Reagents; D008024:Ligands; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008828:Microbiological Techniques / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D011485:Protein Binding; D019809:Streptavidin; D013388:Succinimides
chemical_list: D005456:Fluorescent Dyes; D005656:Fungal Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008565:Membrane Proteins; D013388:Succinimides; C065130:sulfo-N-hydroxysuccinimide-biotin; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate