Biomedical Engineering Reference
In-Depth Information
protein and peptide substrates for DP4 have been identified; hence it is
speculated that substrates of all DPs will be limited to a maximum of 100-200
amino acids in length,
1,143
thus limiting the potential substrate repertoire.
However, FAP is known to cleave a
2
AP, which is 464 amino acids in length.
28
This suggests that it is also possible for larger proteins with a free, non-
restricted N-termini to be in vivo substrates of DPs.
In comparison with DP4, the DP substrate specificity of FAP at the P2
position is much more restricted with FAP displaying a preference for iso-
leucine, proline, or arginine in the P2 position.
14
Edosada et al. characterized
the P4-P2
0
substrate specificity of FAP endopeptidase activity, revealing a
strong requirement for the presence of glycine or small amino acids in the
P2 position in order for the endopeptidase activity of FAP to occur.
14
A reduction in specificity was observed at positions P3, P4, P1
0
,andP2
0
with a
wider range of residues being accepted.
14
Considering that this specificity of
FAP has been determined in vitro, it will be of interest in future to determine if
a broader specificity is observed within the in vivo complex biological setting
or against true endogenous substrates where other features interacting with
the structure/conformation of FAP may come into play. Homology models
reveal a high degree of similarity in structure of DP8 and DP9 compared with
DP4 and FAP, suggesting that substrate access would be similar with slight
variations in specificity.
138,144
Differences in kinetics of purified recombinant
DP8 to those of DP4 indicate the likelihood of there being subtle differences
in substrate-binding pockets between these two enzymes.
145
AlargerS1
pocket size would potentially allow for larger residues to be accommodated in
the S1 position of DP8/9 compared to DP4. Elucidation of DP8/9 substrate
specificities has been determined in vitro using combinatorial peptide libraries.
In vitro analysis reveals a preference for DP8 and DP9 towards substrates
containing small, basic, or large hydrophobic residues in the P2 posi-
tion.
123,124,145
Lee et al. confirmed DP8's strong preference for Pro in the P1
position and detected activity against substrates with alternate alanine,
methionine, or threonine in the P1 position
123
. DP4 is well known to cleave
substrates with Ala, Ser, and Gly in the P1 position such as GLP-1. In vitro,
DP8 and DP9 cleavage of GLP-1 has been demonstrated.
122
Acidic residues in
the P2 position were the least favorable for both DP8 and DP9 cleavage.
123,124
In regards to these findings, consideration must be given to the fact that while
substrate specificity is being determined in vitro, the specificity of DP8/9 may
be considerably different in the complex biological setting in vivo.Interest-
ingly, during our own research involving DP8/9 substrate discovery, utilizing
stable cell lines and terminal anity labeling of substrates (TAILS) technol-
ogy
146
we have identified a potential biological substrate of DP8/9 with one of
the least favored acidic residues in the P2 position using a peptide library
(Wilson et al., in preparation). While structural studies and determination of
in vitro substrates specificity are useful, they tell us very little about the bio-
logical roles of a protease within the human body. Of more use is the iden-
tification of true biological substrates that allow for elucidation of the
pathophysiological roles of a given protease.
