Characterization of Recombinant Microsporidian Methionine Aminopeptidase Type 2

Microsporidia are obligate intracellular protists related to fungi that are pathogens in both vertebrate and invertebrate hosts [21,23,25]. In humans microsporidiosis most often presents as a diarrheal illness, but keratoconjunctivitis, disseminated disease, hepatitis, myositis, sinusitis, renal infection, prostatitis, ascites, and cholangitis have also been described [21]. Of the over 144 genera of microsporidian the following species are human pathogens: Nosema, Vittaforma, Pleistophora, Encephalitozoon (Septata intestinalis is now Encephalitozoon intestinalis), Enterocytozoon, Brachiola, Trachipleistophora and Microsporidium [21]. Benzimidazoles, such as albendazole, are therapeutic agents of choice for infections due to Encephalitozoon sp., however, they are not very efficacious for infections due to Enterocytozoon bieneusi [2,24]. Fumagillin and its derivatives, have been effective for the treatment of microsporidiosis in fish (Pleistophora sp., Loma salmonae, Nucleospora fsyn Enterocytozoong salmonis) and in insects (Nosema kingi, Nosema apis, Octosporea muscaedomesticae) [2,7] Fumagillin has also been demonstrated to have in vitro and in vivo activity against Encephalitozoon sp. and Vit. corneae [2,3,14,17]. Importantly, fumagillin also had efficacy in the treatment of AIDS patients that had Ent. bieneusi infection [13]. We have demonstrated that TNP470 and other fumagillin derivatives are also active both in vitro and in vivo against microsporidia [3,7,22]. Fumagillin and TNP470 bind irreversibly to a common bifunctional protein identified by mass spectrometry as methionine aminopeptidase type 2 (MetAP2) [5,18]. These drugs selectively block the aminopeptidase, but not the elF-2 phosphorylation activity, of human MetAP2 and do not bind or inhibit methionine aminopeptidase type 1 (MetAP1). We have previously demonstrated that microsporidia have MetAP2 activity using the substrates methionine-para-nitroanilide and L-methionine-AFC. In addition, we have previously used homology PCR to obtain partial MetAP2 genes from Enc. cuniculi, Enc. hellem, Enc. intestinalis, Glugea americanus and Brachiola algerae [22] (Weiss, L.M. & Zhang, H. 2002. Microsporidian Methionine Aminopeptidases as Therapeutic Targets, Woods Hole Molecular Parasitology Meeting XIII, 284A, abstract). Phylogenetic analysis of these genes suggested that the microsporidian MetAP2 genes, while related to fungal MetAP2, formed a distinct clade (Weiss, L.M. & Zhang, H., 2002, Woods Hole Molecular Parasitology Meeting XIII, 284A, abstract). We have used 59 and 39 RACE techniques to obtain the fulllength MetAP2 sequences from Enc. cuniculi (GeneBank AF440270), Enc. intestinalis (GeneBank AY224693), and Enc. hellem (GeneBank AY224694). By homology PCR we could not demonstrate MetAP1 in Enc. cuniculi [22] and the subsequently published Enc. cuniculi genome confirms that this organism lacks MetAP1 [8]. We have now cloned Enc. cuniculi MetAP2 into a baculovirus expression system and have been able to purify enzymatically active microsporidian MetAP2. We also used data on the crystal structure of the human MetAP2 protein to model the Enc. cuniculi MetAP2 sequence.