1. IDENTIFICATION, PURIFICATION AND CHARACTERIZATION OF PROTEINS FROM THE PROTECTIVE FRACTION (F0) OF Paracoccidioides brasiliensis Fernandes, V. C. 1 ; Reis, B. S. 2 ; Goes, A. M. 3 - 1,2,3 UFMG - Bioquímica e Imunologia Financial support: CNPq and FAPEMIG Results %NaCl% 66- 45- 29- 14- kDa FIIIF0FIFIIFIVFVFVIPbAg MW 66- 45- 29- 14- kDa FIIIF0FIFIIFIVFVFVIPbAg Picos do refracionamento 01 Optical Density (280nm) 20 40 60 80 100 NaCl Gradient [M] 0.20 0.40 0.80 1.00 0.60 0 20 40 60 80 100 0.20 0.40 0.80 1.00 0.60 0 0203 66- 29- 24- 18,4- kDa 45- F0 Peak 1Peak 2 Peak 3 66- 29- 24- 18,4- kDa 45- MW Figura 1: Elution profile of PbAg in Q-Sepharose anion-exchange column connected to FPLC system. Figura 2: SDS-PAGE analysis of PbAg fractions from FPLC. Aliquots of 20 μg of each antigenic preparation were separated on 10 % polyacrylamide gels, under reducing conditions, followed by Coomassie-blue staining. MW= Molecular weight; F0,FI, FII, FIII, FIV, FV, FVI= PbAg fractions; PbAg= P. brasiliensis yeast cells lysate Figura 3: Elution profile of F0 in Mono-S anion-exchange column connected to FPLC system. Figura 4: SDS-PAGE analysis of F0 peaks from FPLC. Aliquots of 2 and 4 μg of each antigenic preparation were separated on 10 % polyacrylamide gels, under reducing conditions, followed by silver staining. MW= Molecular weight; F0= fraction 0 from PbAg; Peaks 1, 2, 3= F0 peaks. Figura 5: Western-blot analysis of F0, Peak 1 and Peak 2 using sera from PCM patients. The arrows show the proteins that were sequenced. MW= Molecular weight F0 Pico 1Pico 2 PM 38,5 18,1 kDa 201 125 81 F 0Peak 1Peak 2MW F0 Pico 1Pico 2 PM 38,5 18,1 kDa 201 125 81 6,9 F0 Pico 1Pico 2 PM 31,3 18,1 kDa 201 125 81 27 kDa antigen [Paracoccidioides brasiliensis] aralssdelktvvsvlaqkldslnidyaimggaatcllsgdpnrrtedvdlvihvdhrkitadnlttqllksfpsdfegvsqfghtipay klrrpggtvqlvvelevfdyqswpqrpqydlqtatrttlningqkvklfspewilrekilsqyqrqgsrkegtdirdiismiplavpgkp elnfnqsqelqtalanlvqkrpdlssalkakikcsavfhn Homology a AnimalLengthSimilarityIdentityScore 27 kDa antigenParacoccidioides brasiliensis220 aa7/7 (100%)7/7 (100%)24.0 a: N-terminal sequence of PB29: A P L S I Y E L K T V V S This peptide was compared to protein sequences deposited in non-redundant databases, using the Basic Local Alignment Search Tool program, BLASTp Immunodominant antigen Gp43 [Paracoccidioides brasiliensis] mnfsslnlalascvlawvclasasshvashivprqagsaiygvniggwlllepwispsvfeaggsssvdeytlsknlgrdakrhl skhwdtfiteddfkniaaaglnhvripigywavnpiegepyvqgqldyldkalvwaknsnlrvvidlhgvpgsqnfdnsghrga inwqkgdtirqtliaihtlairyanrtdvvdsielvnkpsipggvqvsllkeyyedgyhivrdidstvgvsisdaslpprtwngflapkt yknvyidtyhnqvfddifrtftidqhvklacslphgrlrgadkplivkewsgamtdcamylngrgigsrfdgsfpsgkpsgacgar skgssselsaqqkkdtllyieaqldafevgagwyfwtwktegapgwdmqdllnqklfpqpiwarkyggcr Homology a AnimalLengthSimilarityIdentityScore Immunodominant antigen Gp43 Paracoccidioides brasiliensis416 aa12/12 (100%),12/12 (100%)38.4 a: N-terminal sequence of PB43: AGSAIYGVNIGGAGSAIYGVNIGG This peptide was compared to protein sequences deposited in non-redundant databases, using the Basic Local Alignment Search Tool program, BLASTp. 81 kDa antigen [Paracoccidioides brasiliensis] No significative homology was found with any other protein present in the data bank. a: N-terminal sequence of PB81: EPPPPPPPPPPPPPPPPPPP This peptide was compared to protein sequences deposited in non-redundant databases, using the Basic Local Alignment Search Tool program, BLASTp Introduction Paracoccidioidomycosis (PCM) is a human systemic granulomatous disease, prevalent in South America and caused by a dimorphic fungus, P. brasiliensis. The infection occurs by inhaling airborne propagules, that reach the pulmonary alveolar epithelium and differentiate into the pathogenic yeast form. The infective forms may either be eliminated by immune competent cells or disseminate to tissues by the lymphatic or hematogenous route. In a preliminary report, Franco et al. showed that P. brasiliensis yeast cells lysate (PbAg) induced proliferation and in vitro granuloma formation of PBMC from PCM patients with distinct clinical forms. Recently our laboratory has fractionated PbAg using a FPLC system and investigated its protective activity. Two fractions, termed F0 and FII, have been demonstrated to confer protection in mice, whereas another fraction, FIII, exacerbates the disease. It appears that F0 and FII contain vaccine candidate molecules. Since the F0 fraction is composed by several molecules, it becomes important to purify and test some components present in this fraction to identify proteins involved in its protective property. In the present work we report a more detailed study about F0 using FPLC methodology. Table 1: N-terminal sequence of the identified proteins and homology analysis in data bank. Methodology 7 days culture on 35 ºC YEAST CELLS Ultracentrifugation at 37.000 rpm at 4 ºC for 1:30 h CEPA Pb 18 Soluble supernatant fluids (PbAg) FPLC (seven fractions) FPLC (three peaks) SDS-PAGE and Western-blot with sera from PCM patients F0 Amino acid sequencing Mechanical disintegration of yeast cells with glass beads (30 to 40 cycles) Homology analysis Discussion and Conclusion In this work we could conclude that the first and second fractionation methodology adopted were reproducible and effective in separating complex preparations into samples, each one containing a slightly different range of proteins in aqueous form, and in small volume. We were able to obtain three distintic peaks (1 to 3) and two of them (1 and2) showed a characteristic protein profile in 10% SDS-PAGE. The peak 1 separated by 10% SDS-PAGE showed multiple proteic bands, most of their molecular mass ranging from 18,4 to 120 kDa. The peak 2 also showed multiple proteic bands, most of their molecular mass ranging from 18,4 to 66,0 kDa. The peak 3 didn’t show any detectable protein band. The peak two that was obtained in the fractionation of the fraction 0 of PbAg allowed the identification of a new protein with a molecular weight about 81 Kda, recognized by sera from PCM patients. In the same way two more proteins with molecular weight of 27 kDa, equivalent to the protein of the same molecular weight described and characterized by McEwen et al. (1996) as a hypothetical protein specific of the fungus, and 43 kDa, equivalent to the immunodominant protein of the fungus (gp43), respectively. Yeast cells Micelium