Biomedical Engineering Reference
In-Depth Information
a signal peptide (Figure 4.2B). Functional studies of Sar s 3 confirmed that it
was an active serine protease with trypsin-like activity.
56
In silico analysis
predicted that the protease would have a preference for proteases with RSG or
RSA sites in the P1-P2
0
position and generated a list of prospective targets.
Among the top 100 predicted human substrates were several skin proteins,
including filaggrin. The skin protein filaggrin which contains several RSG sites
was tested and found to be effectively digested by Sar s 3.
56
Apart from Sar s 3, all other scabies mite homologs to the group 3 house dust
mite allergens are missing one pair of cysteine residues, and some or all of the
catalytic triad residues have been substituted by various non-synonymous
residues (Figure 4.2B). Such dramatic changes in the catalytic site indicate that
these proteins cannot function as active serine proteases by any known
mechanism. Hence, they have been named Scabies Mite Inactive Protease
Paralogues (SMIPPs).
53
Another family of inactivated cysteine proteases was
discovered recently.
57
To distinguish between the two families, they have been
termed SMIPP-Ss and SMIPP-Cs respectively. SMIPP-Cs will not be addressed
further in this chapter.
4.4 Scabies Mite Inactive Protease Paralogues
(SMIPP-Ss)
The first biological studies of the scabies mite inactive proteases determined
their localization within the mite gut and the fecal pellets excreted into burrows
in the host epidermis. Previous localization work by Rapp et al. had already
established that scabies mites ingest host immunoglobulins, as demonstrated by
the presence of human IgG in the mite gut.
58
In further studies, the detection of
human IgG was used as a marker to localize the mite gut in serial sections of
scabies mite infested human skin. Adjacent sections were probed with SMIPP-S
antibodies and found to co-localize with IgG to the gut and fecal pellets (Figure
4.3).
44
Further studies confirmed the localization of the active serine protease
Sar s 3 to the mite gut and feces as well (Figure 4.3).
56
Complementary to the functional studies outlined below, structural char-
acterization studies on two SMIPP-Ss have identified the mechanism behind the
inactivation of these proteases.
59
To understand the structural basis for
SMIPP-S function, two crystal structures at 0.185 and 0.20 nm resolutions were
generated. The lack of the third disulfide bond in the SMIPP-Ss, which is
present in the active Sar s 3, is thought to enforce a conformational shift. This
leads to occlusion of the S1 subsite by a conserved large tyrosine residue,
thereby blocking substrate ingress. In contrast to the active serine protease Sar
s 3, all SMIPP-Ss discovered to date lack the third disulfide bond (Figure 4.2B).
It was concluded that the cumulative effects of the structural changes together
with the mutations in the catalytic triad rendered the SMIPP-Ss proteolytically
inactive. We postulate that SMIPP-Ss lost the ability to bind substrates in a
classical 'canonical' fashion. Instead, the SMIPP-Ss may have evolved an
exosite that may be used as alternative binding site for non-proteolytic
