Gut proteases

Proteolytic activation of Cry toxins is critical not only for protoxin activation, but has also implications for toxin specificity [59,60], receptor binding [61], and insect resistance [62,63]. The absence of a major gut protease in Plodia interpmctdla correlated with its resistance to Cry 1 Ac [62]. Moreover, rapid degradation of Cry toxins was associated with the loss of sensitivity of S instar 5. litoralis larvae to Cry 1C [64], and serine protease inhibitors enhanced the toxicity of some Cry proteins up to 20-fold [65]. 

Pis vary in their ability to inhibit specific proteases. For example, cysteine proteases respond to one set of Pis and serine proteases to another. Some Pis bind strongly to only one type of protease others have dual specificity. The impact of a PI on a particular insect will depend on the insect s gut protease profile and the specific activity (or activities) of the PI... 

Sandoz [70] conducted further long-term tests with SBTI, OC-I, BBI, and a mixture of OC-I and BBI fed continuously to 2-day-old bees at concentrations of 1, 0.1, or 0.01 mg/ml. Significant mortality occurred only for bees fed SBTI, BBI, or the OC-I/BBI mixture at the highest dose level. These findings were confirmed by Jouanin et al. [71] who reported that OC-I (1,0.1, or 0.01 mg/ml) had no effect on short- or long-term honey bee mortality. BBI at 1 mg/ml, however, reduced bee survival, altered olfactory learning performance and resulted in overproduction of the gut proteases, trypsin and chymotrypsin. 

Thus, research with Pis and bees so far suggests that adult bee gut protease activities may be reduced, with a resultant impact on bee longevity, when bees ingest these proteins. However, the effects will depend on the specificity of the particular inhibitor and the concentration to which the bee is exposed. PI effects on bee larvae have not yet been ascertained. 

B. thurigiensis is a common Gram-positive, spore-forming soil bacterium that produces inclusion bodies, microcrystalline clusters of many different proteins. These crystalline proteins, called 5-endotoxins, are the ion channel toxins that are sold commercially for pest control. Most such endotoxins are protoxins, which are inactive until cleaved to smaller, active proteins by proteases in the gut of a susceptible insect. One such crystalline protoxin. 

Desai PV, Patny A, Sabnis Y, Tekwani B, Gut J, Rosenthal P, Srivastava A, Avery M. Identification of novel parasitic cysteine protease inhibitors using virtual screening. 1. The ChemBridge database. / Med Chem 2004 47 6609-15. 

The promise of the isolation and production of therapeutic polypeptides and proteins demands that for treatment of a chronic disease state an oral delivery system be developed which will protect these valuable agents from the hostile gastric environment. Subsequently, the drugs will have to be completely released in the intestine, preferably in a state that will enhance their rapid dissolution and transport across the gut wall minimizing interaction with intestinal proteases. 

Fig. 13.4. Comparison of the predicted three-dimensional structure of an H. contortus gut-derived cysteine protease (HMCP1) with that of human cathepsin B.

Longbottom, D., Redmond, D.L., Russell, M., Liddell, S., Smith, W.D. and Knox, D.P. (1997) Molecular cloning and characterisation of an aspartate protease associated with a highly protective gut membrane protein complex from adult Haemonchus contortus. Molecular and Biochemical Parasitology 88, 63-72. 

Maefarlane, G.T., Hay, S., and Gibson, G.R., Influence of mucin on glycosidase, protease and arylamidase activities of human gut bacteria grown in a 3-stage continuous culture system, J. Appl. Bacteriol., 66 407-417 (1989). 

There are mechanisms other than the complexlng of proteins that may prevent nutrients from passing across the gut wall. Protease inhibitors decrease the availability of nutrients preventing the break-down of proteins into their component amino acids. The effects of protease inhibitors on Insects have been reviewed (38, 39, 40 paper by C. A. Ryan in this symposium). 

Proteomics in parasitic flatworms can be completed on intracellular fractions (e.g. microsomal or cytosol) or at the host-interface on excretory-secretory (ES) products. ES analysis can be completed during in vitro culture or in vivo by, for example, bile or gut content analysis. In all cases, a rapid and careful preparation is vital to prevent altered pro-teomic profiles due to stress responses (upreg-ulation of heat shock proteins) and action of proteases. Parasitic flatworms are best extracted from fresh host material, washed with a buffered saline solution at approximately the host s body temperature. In F. hepatica, for example, this will allow regurgitation of gut contents to remove digested material from, and removal of host material adherent to the outer surfaces of the parasite (Jefferies et al., 2001), both of which can subsequently complicate separation and identification. 

MEROPS database published by Rawlings et ai., 2004). Each of these has overlapping but distinct preferences for peptide bonds and, hence, together can affect the efficient breakdown of proteins. As cysteine proteases generally operate at slightly acid pH (5.0-6.5), they are suitable for functioning within the low pH of the gut lumen (for S. mansoni) where initial cleavage steps take place (Dresden et ai., 1981 Dalton et ai., 1996 Brindley et ai., 1997 Tort et ai., 1999 Sajid and McKerrow, 2002). 

A number of reports have suggested a relationship between cysteine protease activity and excystment of metacercariae. Cysteine proteases may act alone or in concert with extrinsic host factors such as trypsin in the host gut (Intapan and Maleewong, 2001 Li et al., 2004). In vitro, Paragonimus ohirai metacercarial cysteine protease activity is induced by addition of the bile salt, sodium cholate and parasite... 

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