Immunolocalization and characterization of two novel proteases in Leishmania donovani: putative roles in host invasion and parasite development.
Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca2+ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.
Choudhury
Rajdeep
R
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Das
Partha
P
De
Tripti
T
Chakraborti
Tapati
T
D000818:Animals; D002460:Cell Line; D004795:Enzyme Stability; D006790:Host-Parasite Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D008264:Macrophages / Q000469:parasitology; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D013379:Substrate Specificity; D013696:Temperature
D015800:Protozoan Proteins; D045726:Metalloproteases; D010447:Peptide Hydrolases; D057057:Serine Proteases