Leishmania panamensis: a 44bp deletion in gp63 gene is found in cDNA and genomic libraries.

Leishmanolysin (EC 3.4.24.36) (gp63) is a zinc metalloprotease (ND Rawlings et al. 1995 Methods Enzymol 248: 183-228) abundantly expressed in the promastigote form of Leishmania parasites, where it is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (P Schneider et al. It has been suggested that gp63 becomes active at multiple steps during the process of macrophage invasion and its function seems to be required for the parasite's intraphagolysosomal survival (GD Rusell et al. The protein is encoded by genes present in multiple copies that are clustered in two to three tandem repeats usually located on a single chromosome (JR Webb et al. 1991 The gp63 familiy is constituted by a group of proteins with different molecular weights depending upon the species. The importance of the major surface glycoprotein of Leishmania promastigotes, gp63, in the binding of promastigotes to macroph-ages has been inferred largely due to its abundance, surface localization, and proteolytic activity (J Bouvier et al. In the present work, we report for the first time the molecular characterization of gp63 gene in L. panamensis, which is the most prevalent Leishma-nia species in Colombia, and also the presence of copies containing deletions of this gene either on cDNA or DNA material. In order to identify the gp63 gene from L. panamensis, an internal 940 bp long probe was generated by PCR, using two consensus primers designed from the alignment of all Leishmania gp63 nucleotide sequences reported to date. Lpan1: 5'TACGTCGCCTCGGTGCCGA3' and Lpan2: 5´GCACCTGGACGCTGTACG3' primers were synthesized by the solid phase phosphito-triester method. This probe (U62634) was radiolabelled and used for hybridization assays and library screening. A cDNA library was constructed from L. panamensis infective clone M/HOM/PA/71/LS/74 stationary phase promastigotes. cDNA was synthesized using a cDNA synthesis kit (Amersham) and ligated into the Eco RI site of bacteriophage λgt11. Several clones were isolated and sequenced either by manual (Sequenase v.2.0; United States Biochemical) or automatic (Applied Biosystems 373; Perkin Elmer) sequencing using both DNA strands. Of those, Lp63c1, represents a 2648-nt long cDNA sequence that is homologous to other gp63 sequences and includes the 1728 nucleotides coding region, 79 nt of the 5' untranslated region, and 841 nt of the 3' untranslated region (Figure). However, this clone exhibits a 44 bp deletion 26 nt downstream to the start codon causing a frame-shift compared to other previously described gp63 genes from Leishmania spp. In order to detect the presence …