title: Identification, molecular characterization, and expression analysis of a DOMON-like type 9 carbohydrate-binding module domain-containing protein of Coccidioides posadasii.
abstract: Previously, we investigated the effect of N-acetylglucosamine (GlcNAc) on Coccidioides posadasii chitinolytic enzymes during in vitro spherule-endospore (S/E) phase culture. During those studies, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of supernatants from S/E phase cultures grown in Converse medium with or without added GlcNAc revealed a ∼ 28-kDa band (CFP28), whose abundance was increased by GlcNAc in parallel with the chitinolytic enzymes. Mass spectrometry (MS) of the CFP28 band revealed peptides that matched an open reading frame found in the tentative consensus sequence, TC20325, retrieved from the Dana Farber Cancer Institute C. posadasii Gene Index Database. The TC20325 cDNA sequence was used to design internal primers based on MS peptides and a full-length cDNA was isolated using a combination of rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. The deduced amino acid sequence of the full-length cDNA consists of 231 amino acid residues with a 19 aa signal peptide. The mature protein has a calculated molecular mass of ∼ 24.5 kDa, a theoretical pI of 6.09, and consists of a single DOMON-like type 9 carbohydrate-binding module (CBM9-like-3) conserved domain. The protein shares the highest sequence similarity (≥57%) to hypothetical proteins from fungi within the Pezizomycotina subphylum of Ascomycota. Antiserum against a recombinant version of CFP28 recognized native CFP28 in S/E phase cells and culture supernatants. CFP28 mRNA and protein expression were detectable in S/E phase in Converse medium, but were increased in the presence of added GlcNAc. Purified native CFP28 reacted with pooled sera from patients with coccidioidomycosis.
authors.affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California, USA jmlunetta@ucdavis.edu.
authors.forename: Jennine M
authors.initials: JM
authors.lastname: Lunetta
authors.affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California, USA.
authors.forename: Demosthenes
authors.initials: D
authors.lastname: Pappagianis
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003045:Coccidioides / Q000235:genetics*; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins
keywords: Coccidioides; DOMON; N-acetylglucosamine; carbohydrate-binding module; seroreactive