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Cloning of the gp63 surface protease of Leishmania infantum. Differential post-translational modifications correlated
G González-Aseguinolaza1, F Almazán, J F Rodríguez
1Centro de Investigaciones Biológicas, CSIC. Velázquez, Madrid, Spain.
Abstract:
The Leishmania cell surface virulence factor gp63 is a protease family that plays an important role in the survival of the parasite protozoon into the host macrophages. We have cloned and characterised the gp63 gene from L. infantum. The sequence analysis of the gene indicates the existence of a high degree of conservation with the other old world species L. major and L. donovani. The similarity is lower with new world species with the exception of L. chagasi which shows a strikingly high percentage of identity (99-100%). In L. infantum the gp63 gene expresses two polypeptides of 58 and 60 kDa, respectively, which show a similar proteolytic activity. The 60 kDa polypeptide is expressed during the whole life cycle of the promastigote form of the parasite with a moderate increase at the stationary phase of growth while the 58 kDa product, although slightly present in the logarithmic phase, notable increases its expression during the highly infectious stationary phase. RNA analysis showed that the presence in L. chagasi of these two polypeptides correlates with two RNA molecules and with the degree of parasite infectivity, whereas in the case of L. infantum a single 3 kb messenger RNA is detected through the whole promastigote life cycle. Our data indicate that in L. infantum, the differences in gene expression of the gp63 protease family according to parasite phase of growth seem to be due to a differential pattern of glycosilation of the polypeptides which correlates with the different infective forms of the promastigote form of the parasite.
Insights
The Leishmania infantum gp63 protease gene expresses two key polypeptides, gp63 (58 kDa) and gp63 (60 kDa), crucial for parasite survival. Differential glycosylation patterns in L. infantum correlate with infectivity, impacting Leishmania virulence.
Area of Science:
- Molecular Parasitology
- Protozoology
- Biochemistry
Background:
- The gp63 gene encodes a Leishmania cell surface protease vital for parasite survival within host macrophages.
- Understanding gp63 gene expression is critical for elucidating Leishmania virulence mechanisms.
Purpose of the Study:
- To clone and characterize the gp63 gene from Leishmania infantum.
- To investigate the expression patterns and functional significance of gp63 polypeptides during the parasite life cycle.
Main Methods:
- Gene cloning and sequence analysis of L. infantum gp63.
- Polypeptide expression analysis using SDS-PAGE.
- RNA analysis to correlate gene expression with polypeptide production.
Main Results:
- Sequence analysis revealed high conservation of gp63 in Old World Leishmania species and striking identity (99-100%) with Leishmania chagasi.
- L. infantum gp63 gene expresses two polypeptides (58 and 60 kDa) with similar proteolytic activity.
- Differential expression of 58 kDa and 60 kDa polypeptides correlates with parasite growth phases and infectivity, potentially due to glycosylation.
Conclusions:
- Leishmania infantum gp63 gene expression exhibits phase-specific regulation impacting parasite infectivity.
- Differential glycosylation of gp63 polypeptides is implicated in the distinct infective forms of Leishmania promastigotes.
- The study provides insights into the molecular basis of Leishmania virulence and host-parasite interactions.