Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS–PAGE. The infected cells produced a number of extra activities at pH 8·0 in the presence of Ca2+. Calcium was found to enhance the activites but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

Expression of metalloprotease activities during metacyclogenesis of a series of strains and clones of Trypanosoma cruzi was investigated using SDS-polyacrylamide gel electrophoresis with a range of different co-polymerized proteolytic substrates. A complex pattern of metalloprotease expression, with considerable quantitative and qualitative variability between strains and life-cycle stages, was apparent. This is in contrast to previously published data, and data presented in this study, which demonstrate a high degree of conservation of expression of cysteine proteases in different strains and clones of T. cruzi. All the metalloprotease activities identified partitioned into the detergent phase of a Triton X-114 extract, suggesting that they are membrane-bound. Developmental regulation of expression during metacyclogenesis, either in terms of quantity, or in terms of difference in relative amounts of different isoforms, was apparent for all isolates studied except CL14. However, a clearly metacyclic-specific/metacyclic-enriched metalloprotease was detected only in T. cruzi Dm28c and 383, and our results demonstrate that a metacyclic-specific metalloprotease common to all isolates of the parasite could not be detected, at least at this level of analysis.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5·5 to 9·0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3′- and 5′-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.