Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods

Jennine M. Lunetta, Keira A. Simmons, Suzanne M. Johnson, Demosthenes Pappagianis

Research output: Chapter in Book/Report/Conference proceedingConference contribution

6 Citations (Scopus)

Abstract

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-α-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-α- mannosidases. Class I 1,2-α-mannosidase enzyme activitywas also detected in the T27K vaccine using the substrate, Man-α-1,2-Man-α-OCH 3 in a spectrophotometric assay.

Original languageEnglish (US)
Title of host publicationAnnals of the New York Academy of Sciences
Pages164-180
Number of pages17
Volume1111
DOIs
StatePublished - Sep 2007

Publication series

NameAnnals of the New York Academy of Sciences
Volume1111
ISSN (Print)00778923
ISSN (Electronic)17496632

Fingerprint

Mannosidases
Coccidioides
Cloning
Molecular Cloning
Vaccines
Complementary DNA
Polymerase chain reaction
Molecular Weight
Molecular weight
Coccidioidomycosis
Polymerase Chain Reaction
Consensus Sequence
Electrophoresis, Gel, Two-Dimensional
Transcription
Tandem Mass Spectrometry
Electrophoresis
Recombinant Proteins
Reverse Transcription
Mass spectrometry
Amplification

Keywords

  • Coccidioides
  • Fungus
  • Immunoblot
  • Mannosidase
  • Mass spectrometry
  • Rapid amplification of cDNA ends
  • Reverse-transcription polymerase chain reaction
  • Seroreactive
  • Two-dimensional gel electrophoresis
  • Vaccine

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Lunetta, J. M., Simmons, K. A., Johnson, S. M., & Pappagianis, D. (2007). Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. In Annals of the New York Academy of Sciences (Vol. 1111, pp. 164-180). (Annals of the New York Academy of Sciences; Vol. 1111). https://doi.org/10.1196/annals.1406.015

Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. / Lunetta, Jennine M.; Simmons, Keira A.; Johnson, Suzanne M.; Pappagianis, Demosthenes.

Annals of the New York Academy of Sciences. Vol. 1111 2007. p. 164-180 (Annals of the New York Academy of Sciences; Vol. 1111).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Lunetta, JM, Simmons, KA, Johnson, SM & Pappagianis, D 2007, Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. in Annals of the New York Academy of Sciences. vol. 1111, Annals of the New York Academy of Sciences, vol. 1111, pp. 164-180. https://doi.org/10.1196/annals.1406.015
Lunetta JM, Simmons KA, Johnson SM, Pappagianis D. Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. In Annals of the New York Academy of Sciences. Vol. 1111. 2007. p. 164-180. (Annals of the New York Academy of Sciences). https://doi.org/10.1196/annals.1406.015
Lunetta, Jennine M. ; Simmons, Keira A. ; Johnson, Suzanne M. ; Pappagianis, Demosthenes. / Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. Annals of the New York Academy of Sciences. Vol. 1111 2007. pp. 164-180 (Annals of the New York Academy of Sciences).
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abstract = "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-α-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-α- mannosidases. Class I 1,2-α-mannosidase enzyme activitywas also detected in the T27K vaccine using the substrate, Man-α-1,2-Man-α-OCH 3 in a spectrophotometric assay.",
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