Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-alpha-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods.
The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-alpha-mannosidases. The 5' and 3' ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-alpha-mannosidases. Class I 1,2-alpha-mannosidase enzyme activity was also detected in the T27K vaccine using the substrate, Man-alpha-1,2-Man-alpha-OCH(3) in a spectrophotometric assay.
Lunetta
Jennine M
JM
Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA. jmlunetta@ucdavis.edu
Simmons
Keira A
KA
Johnson
Suzanne M
SM
Pappagianis
Demosthenes
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003045:Coccidioides / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005657:Fungal Vaccines / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase / Q000235:genetics* / Q000502:physiology*
D018076:DNA, Complementary; D005657:Fungal Vaccines; D011993:Recombinant Fusion Proteins; D043323:alpha-Mannosidase