Costa M¹., Parente J.A.¹, Castro N. S.¹, Felipe M.S.S.², Pereira M¹.and Soares C.M.A¹.

1 Laboratório de Biologia Molecular, ICB II, Universidade Federal de Goiás, Goiânia, Goiás, Brazil; 2 Laboratório de Biologia Molecular, IB, Universidade de Brasília, Distrito Federal, Brazil.

Key words: Paracoccidioides brasiliensis, proteinases, aspartyl.

Paracoccidioides brasiliensis causes Paracoccidioidomycosis the most prevalent systemic mycosis in Latin America. It is a dimorphic fungus that undergoes a complex transformation in vivo. The mycelia produce conidia, which upon inhalation into the lungs transform to the yeast form (Brummer et al., 1993 and Franco et al., 1993). Proteinases have been described as virulence factors in microorganisms, allowing them to evade the host defenses by degrading immunoglobulins and complement proteins and also by promoting adherence and degradation of host barriers during invasion (Schaller et al., 2000)

Aspartyl proteinases are synthesized as zymogens that are self-actived by proteolytic removal of a peptide segment under acidic conditions, first in the endoplasmic reticulum and after in the vacuole to generate the active, mature form of the protein. (von Heijine, 1985). In protein the region preceding the cleavage site it is found a single lysine identified as important for the processing. This lysine residue is present in P. brasiliensis proteinase (PbAP) in a conserved sequence of five amino acids (LGQKY). Conceivably, the region containing these amino acids could interact with conserved regions in the mature protein to determine the site of cleavage. So it can be suggested that PbAP can be synthesized as a propeptide.

In fungi, aspartyl proteinases activate other zymogens such as alkaline phosphatase, chitin synthase and other proteinases (Inoue et al., 1998). In Candida albicans, secreted aspartyl proteinases are described as kexin-processed substrates and play role in the microorganism adhesion to epithelial cells (Newport et al., 2003).

In this work we present the characterization of a 1.3Kb cDNA (Pbap), encoding a protein homologous to related sequences of aspartyl proteinases of several organisms. The deduced amino acid sequence (PbAP) with 43.8 kDa and p.I 5.7 shows identity to sequences of aspartyl proteinases of several fungi. The PbAP presents two highly conserved motifs DTGSSNLWVPS and DTGTSLLALPS, described as essential for the catalytic activity. A putative signal peptide is present between amino acids 1 to 19, with predicted cleavage site at amino acids 19 and 20. The complete cDNA and the deduced protein were submitted to GenBank (AY278218).

The NCBI BLAST program (http://www.ncbi.nlm.nih.gov) was used for search for nucleotide and protein sequences similarity to the PbcatP. Protein sequence analysis was performed with the PROSITE (http://us.expasy.org/prosite) and Pfam databases (http://www.sanger.ac.uk/softwer/pfam/index.shtml). The alignment was generated with Clustal X software (Thompson et al., 1997).

PCR amplification were designed for cloning of the cDNA into expression vector. The PCR of Pbap was performed for obtain the cDNA with insertion of sites EcoR I and Xho I using oligonucleotides constructed on basis of sequences in 5’(Pbap-sense) and 3’(Pbap-atsense).

Pbap was used as a template for PCR amplification of fragment encoding the aspartyl. Oligonucleotides primers were designed based on the nucleotide sequences 5’(Pbap-sense) and 3’(Pbap-atsense) of Pbap with insertion of sites EcoR I and Xho I

The cloned cDNA was 1361 nucleotides in length and contained a 63 base at the 5’UTR (untranslated region). The cDNA had a single ORF. The deduced amino acid sequence indicated a protein of 400 amino acid residues. The ATG codon at base 64 encoded the presumed initiating methionine. The cDNA included 95 bp in the 3’UTR, exclusive of the poly-A tail. The stop codon TGA was located at position1264.

The deduced amino acid sequence of PbAP was alignment with several species. The sequences aligned are: Paracoccidioides brasiliensis (GenBank AY278218), Coccidioides immitis (GenBank AF162132), Aspergillus fumigatus (GenBank AFY15744) and Neurospora crassa (GenBank AABX01000355).

PCR amplification were designed for cloning of the cDNA into expression vector. The PCR of Pbap was performed for obtain the cDNA with insertion of sites EcoR I and Xho I using oligonucleotides constructed on basis of sequences in 5’(Pbap-sense) and 3’(Pbap-atsense).

• A 1,3Kb cDNA encoding an aspartyl proteinase of Paracoccidiodes brasiliensis was cloned and sequenced.
• The deduced amino acid sequence presents molecular mass of 43,8kDa and pI 5,7. Sequence analysis revealed three probable N-glycosylation sites and 20 amino acids at N-terminus, characteristic of a signal peptide. The signal peptide is evidenced by the hydrophobic profile of PbAP sequence in amino terminal region.
• The PbAP presents two highly conserved motifs DTGSSNLWVPS and DTGTSLLALPS, described as essential for the catalytic activity.
• The complete cDNA and the deduced protein were deposited at GenBank (AY278218).
• The characterization of the cDNA of aspartyl proteinase from Pb open the possibility of analyzing the role of proteinases as virulence factor in this fungus.
• The cloning of the aspartyl cDNA and its heterologous expression will provide the tools for the understanding of the physiological role of this protein in Paracoccidiodes brasiliensis.

Brummer, E., Castaneda, E., Restrepo, A. 1993. Paracoccidiodomycosis: an update. Clin Microbiol Rev. 6:89-117.

Franco, M., Peracoli, M.T., Soares, A., Montenegro, R., Mendes, R.P., Meira, D. A. 1993. Host-parasite relationship in a paracoccidiodomycosis.Curr Top Med Mycol. 5: 115-149.

Inoue, H., Huan, X.P., Hayashi, T., Athauda, S.B.P., Yamagata, H., Udaka, S., Takahashi, K., 1998. The roles of the basic residues in the prosegment of the aspergillopepsinogen I. In: James, M.N.G. (Ed.), Aspartic Proteinases: Retroviral and Cellular Enzymes. Plenum Press, New York, pp239-244.

Newport, G., Kuo, A., Flattery, A., Gill, C., .Blake, J.J., Kurtz, M.B., Abruzzo, G.K., Agabian, N. 2003 Inactivation of Kex2p diminishes the virulence of Candida albicans. The Journal of Biological Chemistry. 17:1713-1720.

Schaller M., Schackert, C., Korting, H.C., Januschke E., Hube, B. 2000. Invasion of Candida albicans correlates with expression of secreted aspartic proteinase during experimental infection of human epidermitis. J Invest. 114: 712-717.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. 1997. The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876-4882.

von Heijine, G. 1985. Signal sequences: the limits of variation. J. Mol. Biol. 184, 99-105.

CAPES, CNPq and FUNAPE-UFG