Molecular cloning, characterization and expression analysis of two β-N-acetylhexosaminidase homologs of Coccidioides posadasii

Abstract

Two full-length cDNAs were isolated from Coccidioides posadasii that encode two deduced proteins (CpHEX1 and CpHEX2) with homology to the glycosyl hydro-lase 20 family of β-N-acetylhexosaminidases. CpHEX1 consists of 595 amino acids, has a predicted molecular mass of 68 kDa and shares the highest identity with the N-acetylhexosaminidase (NAGA) of Aspergillus nidulans, while CpHEX2 consists of 603 amino acids, has a predicted molecular mass of 68.5 kDa and shares the highest identity with NAG1 from Paracoccidioides brasiliensis. CpHEX1 and CpHEX2 share only 23% identity and have dissimilar homologies showing more identity with other fungal β-N-acetylhexosaminidases than with each other. Phylogenetic analysis of selected β-N-acetylhexosaminidases placed CpHEX1 in a cluster with the orthologs from A. nidulans, Aspergillus oryzae, Penicillium chrysogenum and Candida albicans, while CpHEX2 grouped with the orthologs from P. brasiliensis and the Trichoderma spp. β-N-acetylhexosaminidase activity and transcripts encoding CpHEX1 and CpHEX2 were detected in vitro during the spherule-endospore (SE) phase. Expression of the Cphex1 transcript exhibited a temporal increase that correlated with β-N-acetylhexosaminidase activity, while the Cphex2 transcript remained relatively constant. The addition of N-acetylglucosamine to the cultures increased β-N-acetylhexosaminidase activity and the expression of the Cphex1 transcript. A native β-N-acetylhexosaminidase enzyme was purified from in vitro SE phase and identified as CpHEX1 by mass spectrometric analysis. Both the CpHEX1 and CpHEX2 cDNAs were expressed as recombinant fusion proteins and purified under denaturing conditions to apparent homogeneity but they lacked enzymatic activity.

Keywords:

• Coccidioides
• fungi
• β-N-acetylhexosaminidase
• β-N-acetylglucosaminidase
• chitobiase
• rapid amplification of cDNA ends
• reverse-transcription polymerase chain reaction
• mRNA expression
• purification

Acknowledgements

The technical assistance of Mark Oamek, Corey Quacken-bush, Cheryl Miller and Jason Sanchez is greatly appreciated. This work was supported, in part, by a grant from the California HealthCare Foundation, the Department of Health Services of the State of California and California State University, Bakersfield.

Declaration of interest: None

This paper was first published Online on Early Online on 27 January 2010.