Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • Cysteine protease activities have been suggested to be impor

    2020-11-19

    Cysteine protease activities have been suggested to be important for Giardia’s pathogenesis [[11], [12], [13],16,18,19,21,28,29,35]. Recently several giardial CPs were shown to be released during host cell interactions [9,10]. The three major released CPs have recently been shown to be involved in tight junction disruption and chemokine degradation [11,28]. However, their involvement in the molecular pathogenesis of giardiasis is still not fully understood. Herein, we further characterized these three CPs using phage display and identified their potential roles in degradation of secreted host antibodies and defensins. We used the substrate phage display technology to characterize the substrate specificity of CP16160. The same approach did not work on CP14019 and CP16779 but a number of 2-trx substrates were generated from the phage display alignment and tested with CP14019 and CP16779. Interestingly, we enriched for substrates with several negatively charged IFN-alpha 1, human recombinant protein synthesis from the phage library, but the CPs did not efficiently cleave 2-trx recombinant substrates that included negatively charged amino acids. On the contrary, very few arginines (R) were found in the substrates selected from the phage library, while all three CPs showed efficient cleavage of 2-trx substrates containing R in the P1 or P1´ positions. A preference for R in the P1 position was also seen in an earlier study of the CP14019 substrate specificity using a positional scanning synthetic combinatorial library [26]. These results show that the phage display library we used here might have its limitations in the characterization of this type of proteases. Substrate phage display is very efficient in identifying the most optimal substrate and secondary specificities can thereby easily be overlooked. A difference in activity by only 2–3 times can be sufficient for not detecting the secondary specificities. The cleavage of lysine containing substrates by human neutrophil cathepsin G is one example [33]. Very few cysteine proteases have been studied using substrate phage display and we failed in the analyses of CP14019 and CP16779. Currently we do not know why but these two proteases are very active and relatively non-selective CPs, potentially affecting the phages non-specifically. Other approaches for identification of substrate specificity should be used on CP14019 and CP16779 [36]. Even if a peptide sequence is enriched in the phage display assay it could be inefficiently cleaved in another context due to structural differences. This is important to consider when cleavage motifs are found in putative target proteins. It would be optimal to use structural information from both protease and target proteins when in silico analyses are performed. However, based on results from both phage display and recombinant substrate digestions we can conclude that CP16160 is both a chymase and tryptase with broad cleavage specificity and that there are differences and overlaps in the substrate preferences between the three secreted CPs. It is known that IgA is produced and released in the human small intestine in response to Giardia infections [37] and patients with immunoglobulin deficiencies can develop chronic giardiasis [38]. Anti-Giardia IgA was also detected in human saliva and breast milk and associated with protection against disease [39]. IgA is also required for effective clearance of G. muris and G. intestinalis from the murine host [40]. Many intestinal bacteria and parasites, colonizing the human intestinal tract, secrete proteases that degrade IgA [[41], [42], [43]]. An early report showed that there is an IgA1 degrading activity in Giardia extracts but the protease was never identified [44]. In this report, we showed that the three secreted CPs degrade human IgA1, IgA2 and IgG, suggesting that these Giardia CPs play a role in the interference with the adaptive immune responses induced during Giardia infections.