Biochemical characterization of peptidylarginine deiminase-like orthologs from thermotolerant Emericella dentata and Aspergillus nidulans.

Affiliation

Enzymology and Fungal Biotechnology Lab (EFBL), Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt. Electronic address: [Email]

Abstract

Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37-40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0-8.0, with putative pI 5.0-5.5, higher structural denaturation at pH 4.0-5.5 and 9.5-12 as revealed from absorbance at λ280nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower Kr value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen.

Keywords

Aspergillus nidulans,Biochemical properties,Emericella dentata,Kinetics,Peptidylarginine deiminase,

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