21 Molecular and Cellular Biochemistry 175: 21–27, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands. Ribonuclease activity dependent cytotoxicity of Asp fl, a major allergen of A. fumigatus Taruna Madan, Naveen Arora and P. Usha Sarma Centre for Biochemical Technology, Delhi-110007, India Received 13 May 1996; accepted 8 January 1997 Abstract A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC 50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ri- bonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further con- firmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells. (Mol Cell Biochem 175: 21–27, 1997) Key words: Aspergillus fumigatus, ribonuclease, cytotoxicity, Asp fl Address for offprints: P.U. Sarma, Centre for Biochemical Technology, Mall road, Delhi-110007, India Introduction A. fumigatus antigens are complex glycoproteins, glycans and proteins with a variety of biological functions [1]. Antigens with collagenase, elastase and protease activities have been reported from different isolates of the fungi [2–4]. The bio- chemical functions of various antigens and immune responses to some of the antigens contribute significantly to the pathogenesis of Aspergillus induced infections [5]. A detailed investigation on the biochemical properties of immunoreac- tive antigens of A. fumigatus would facilitate understanding on diagnosis and pathogenesis of Aspergillosis. Asp fl, an 18 kDa protein secreted by A. fumigatus, is re- ported to be a major allergen/antigen involved in the patho- genesis of Aspergillus related disorders [6, 7]. An IgE mediated response to Asp fl has been reported in 85% of the patients with A. fumigatus induced allergic disorders [6]. The 18 kDa pro- tein is also a major antigen found in the urine of the patients with invasive aspergillosis [8]. The cytotoxic nature of Asp fl has been established on different eukaryotic cell lines [9]. Recombinant Asp fl was also observed to be cytotoxic to EBV transformed PBMC’s of ABPA patients [10]. Cloning and sequencing of the gene for 18 kDa allergen from two different isolates in different vectors showed ho- mology of the sequence with fungal toxins restrictocin, mitogillin and alpha-sarcin [7] (Sarma et al. unpublished data). These toxins are known to kill cells by inhibiting pro- tein synthesis by hydrolysing the phosphodiester bond of 28 S rRNA [11]. Asp fl inhibited protein synthesis in cell free reticulocyte lysates [6] and produced a c.400 base fragment from the 28S rRNA [10]. Although the mechanism of cytotoxicity of Asp fl appears to be ribosome inactivation, precise mechanism of cytotox- icity is yet to be established. Current study was undertaken to evaluate the effect of Asp fl on protein synthesis in eukaryotic cells, to analyse and characterise ribonuclease activity of Asp fl and to determine the correlation of its ribo- nuclease activity and cytotoxic activity. Understanding on the mechanism of cytotoxicity of Asp fl will also facilitate the use of this cytotoxin in chemotherapy of cancers.