Method: HDM enriched fecal pellets were extracted with PBS or 6 M Urea with 2% SDS and 10 mM DTT. The release of nDer p 23 in the extracts as well as in a commercial mite bodies extract (Greer) was measured by western blot. Recombinant Der p 23 (rDer p 23) and GST-Der p 23 were produced in P. pastoris and E. coli respectively. These two recombinant forms were incubated with activated natural Der p 1, trypsin or HDM extracts. The Der p 23 proteolysis was monitored by SDS-PAGE and western blot.
Results: Very tiny amounts of nDer p 23 is released from fecal pellets upon incubation with PBS. Unexpected high molecular weight immunoreactive bands present also in very low amount were detected when extraction was performed under denaturing and reducing conditions. Intact nDer p 23 was not present in mite bodies extract. Recombinant Der p 23 can be degraded by Der p 1 but not by Trypsin. The Der p 23 O-glycosylation partially reduced the proteolysis. The Der p 23 sensitivity to cysteine proteases from HDM fecal pellets extracts was also demonstrated.
Conclusions: Our results suggest that natural Der p 23, through probably its tight interactions with the peritrophic matrix, is very poorly released from the fecal pellets. According to nDer p 23 very low abundance in mite extracts and high sensitivity to proteolytic degradation, recombinant Der p 23 may be a valuable material for the diagnosis as well as the treatment of Der p 23 sensitivities.