Biology Reference
In-Depth Information
4.3.2 Substrates Cleaved by Meprin A and B
(In Vitro and In Vivo)
Meprins are capable of cleaving a wide variety of substrates including extracellular
proteins, cytokines, hormones, and growth factors. All isoforms of meprins are
capable of hydrolyzing some extracellular matrix (ECM) proteins such as collagen
IV, gelatin nidogen, fibronectin, and laminins. These proteins are probably not
normally substrates for the kidney and intestinal meprins because they are not
present at brush border membranes at the apical surface of epithelial cells or in
the lumen of these organs where meprins are localized. However, there are several
circumstances where the meprins are redistributed to other portions of the cell, and
then ECM substrates are relevant. In addition, in cancer cells and immune system
cells that move through cell barriers, ECM and other extracellular proteins (junc-
tional proteins) become physiologically relevant substrates.
There is evidence implicating meprins in the hydrolysis of cell junctional pro-
teins from cell-based studies. E-cadherin was shown to be a meprin B substrate in a
cell culture system, and cells expressing meprin B exhibited weaker intercellular
contacts (Huguenin et al.
2008
). Meprin B also hydrolyzes sites in tenascin-C, an
adhesion-modulating ECM protein, and hydrolysis interferes with the oligomeri-
zation and adhesion properties of the protein (Ambort et al.
2010
). There have
been attempts to use proteomic analysis to find novel meprin substrates by looking
at protein changes between cells expressing meprin vs. cells with no meprin
expression. In one such approach, 17 potential novel meprin substrates have
been identified (Ambort et al.
2008
). For example, clusterin, lysyl oxidase, and
vinculin were identified as meprin substrates. These substrates implicate meprins
in processes such as immune response, tissue remodeling, and cell maintenance
and growth.
Many bioactive peptides including several naturally present in the gastrointesti-
nal tract are substrates for the meprins. For example, gastrin-releasing peptide frag-
ment (GRP-14-27) and sulfated cholescystokinin (sCCK8
NH2
) are substrates for
both meprins A and B (Bertenshaw et al.
2001
). The Km values are in the range of
50-350
10
5
M
1
s
1
. These affinity and
specificity constants are not particularly high; however, considering the high con-
centrations of meprins at the cell surface, the substrates are likely hydrolyzed if
M, and the kcat/Km range from 0.6 to 7
m
Fig. 4.3 (continued) between 5 and 10% digested. The incubation was in 25 mMHEPES, 100 mM
NaCl, 5 mM CaCl
2
, pH 7.4, at 37
C. Amino-terminal sequencing of the resulting products allowed
for the elucidation of amino acid preference for meprin B at each primed site. The data in each
sequencing cycle were normalized to the total molar amount of amino acids in that cycle so that a
value of 1 indicates the average value. (b) The same acetylated dodecamer peptide library mixture
(1 mM) was incubated with meprin A (33 nM) until the library was between 5 and 10% digested.
Amino-terminal sequencing of the resulting products allowed for the characterization of amino
acid preference for meprin A at each primed subsite (Bertenshaw et al.
2001
)
