Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity.

The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes "especially thermophilic fungi" could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0-8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.

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

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.