β-D-galactofuranosidase: Purification of the Penicillium fellutanum enzyme and its detection in Trypanosoma cruzi

Author(s)

Luiz Claudio Miletti

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2001-10-05

Examining board members

Maria Julia Manso Alves; Pio Colepicolo; Helio Kiyoshi Takahashi; Walter Ribeiro Terra; Ivarne Luis dos Santos Tersariol

• Advisor: Maria Julia Manso Alves

Abstract

β-D-galactofuranose is a component of several galactofuranose containing macromolecules of many organisms, including bacteria, protozoa and fungi. Interestingly, this unusual sugar is absent in mammalian glycoconjugates. An alternative and fast method for the purification of β-D-galactofuranosidase was developed using p-nitrophenyl-β-D-galactofuranoside as substrate. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by an affinity chromatography column of 4-aminophenyl-1-thio-β-D-galactofuranoside-Sepharose which yielded two separate peaks of enzyme activity when elution was performed with 10mM D-galactonic acid-γ-lactone in a 100-500 mM NaCl gradient. Both peaks rendered a single 70 kDa protein as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable to immunoprecipitate 77 % units out of total units of the enzyme from the crude extract. One of the excised bands was submited to microsequencing and the sequence for 3 peptides (65, 73, 101) was obtained. Antibodies were raised against the corresponding synthetic peptides and used to immunoprecipitade a polypeptide with β-D-galactofuranosidase activity. To verify the presence of β-D-galactofuranosidase in T. cruzi we used the same 4-aminophenyl-1-thio-β-D-galactofuranoside-Sepharose affinity column. An extract of epimastigotes metabolically labelled with 35S-methionine was applied on the affinity colunm. After washing, the putative enzyme was eluted by 10 mM D-galactonic acidy-γ-lactone and 100-500 mM NaCl gradient. SDS-PAGE analysis of the eluted material show a polypeptide with an apparent molecular mass of 50-60 kDa that is recognized by all the antibodies raised against β-D-galactofuranosidase from P. fellutanum. Also the antibodies react with p-formaldehyde fixed parasites as detected by indirect immunofluorescence. The 50-60 kDa polypeptide was then employed in enzymatic assays. Almost no enzymatic activity was observed when p-nitrophenyl galactofuranoside was employed as substrate. LPPG (lipopeptidophosphoglycan), a galactofuranose-con taining glycoconjugate isolated from epimastigotes of T. cruzi was then employed as substrate. Galactopyranose, the product of the enzymatic activity, was detected by high pH anion-exchange chromatography, suggesting the presence of β-D-galactofuranosidase in T. cruzi. (AU)