3 RESULTS
3.2 Identification of enzymes generating BNP1-30
3.2.2 Identification of BNP1-30-generating enzymes in heart
Figure 23 shows the amount of BNP1-30 accumulated through incubation with membrane preparations of the heart in the presence of enzyme family-specific inhibitors (white bars) or enzyme-specific inhibitors (grey bars). The amount of BNP1-30 generated with the membranes in the absence of peptidase inhibitors was set to 100% (black bar). One-Way ANOVA was used to determine statistical differences, which was rather insensitive for small changes in BNP1-30 accumulation caused by the multitude of groups analysed in parallel. However, this was thought reasonable, since the enzyme inhibitors can also influence BNP metabolism of cleavage products of BNP1-30. Thus, inhibitors can have influence on accumulation of BNP1-30, which is not caused by the inhibition of the particular peptidase generating the metabolite but on other peptidases e.g. proteolyzing BNP1-30. It was anticipated that rather big decreases of BNP1-30 concentrations are caused mainly by the inhibition of peptidases generating the metabolite. The generation of BNP1-30 using membrane preparations of heart could be strongly reduced by two enzyme inhibitors.
The serine-type peptidase inhibitor aprotinin decreased the generation of BNP1-30 by 2/3 and the carboxypeptidase inhibitor (CP inhib.) by 80%. Other family-specific inhibitors and specific inhibitors for enzymes known to be involved in the generation
of vasoactive peptides like ACE or ACE2, and peptidases known to cleave BNP (Meprin A, DPPIV, NEP) did not show significant effects on BNP1-30 accumulation.
To get a more detailed insight into the metabolism of BNP1-32 in the heart, atrium and ventricle have been investigated separately.
The use of the inhibitors showed a slightly different profile for the two heart tissues (Figure 24a&b). Whereas the carboxypeptidase inhibitor decreased the formation of BNP1-30 by 80% in the heart, it showed a much higher effect in the ventricle alone, where the generation of BNP1-30 was completely abolished. Vice versa, the effect in atrium was less pronounced. Aprotinin had approximately the same effect in both tissues. Since aprotinin and also the carboxypeptidase inhibitor reduced BNP1-30 generation, it was anticipated that a serine-carboxypeptidase is responsible for the generation of BNP1-30 in heart membranes.
Heart
roche coc ktail
EDTA chymostat
in pepstatin
aprotini n leupeptin
trp.inh ib.
CP inhib. GEMSA
SM19 712 actinoni
n diprotin A
candoxatrilat captopril
F480 ZPP aliskiren
kalli stop 0
25 50 75 100 125 150
enzyme family-specific
inhibitors enzyme-specific
inhibitors no inhibitor
*** ***
generation of BNP1-30 [%]
Figure 23: Quantification of the accumulation of BNP1-30 in percent in the presence of family-specific enzyme inhibitors (white bars) and enzyme-specific inhibitors (grey bars) using heart membrane preparations. The initial concentration of BNP1-30 generated in the incubations with heart membrane preparations without the use of inhibitors was set to 100% (black bar). For abbreviations of the inhibitors see Table 2. Significances were calculated with One-way ANOVA; *** P < 0.001 vs. no inhibitor.
In the numerical classification scheme for enzymes four different serine-carboxypeptidases are described. One of them, the serine-type D-Ala-D-Ala carboxypeptidase (EC 3.4.16.4) can be found in bacteria only and cleaves
Atrium
roche coc ktail
EDT A
chymos tatin
pepsta tin aproti
nin leupeptin
trp.inh ib.
CP i nhib.
GEMSA SM19
712 actinon
in diproti
n A candoxatrila
t captop
rilF480 ZPP aliskiren
kallistop 0
25 50 75 100 125 150
no inhibitor enzyme family-specific
inhibitors enzyme-specific inhibitors
*** ***
a
generation of BNP1-30 [%]
Ventricle
roche coc ktail
EDT A
chymo statin
pepsta tin aproti
nin leupeptin
trp.inh ib.
CP i nhib.
GEMSA SM1
9712 actinon
in diproti
n A
candoxatrila t captop
rilF480 ZPP aliskiren
kallistop 0
25 50 75 100 125 150
***
***
b
generation of BNP1-30 [%]
Figure 24: Quantification of the accumulation of BNP1-30 in percent in the presence of family-specific enzyme inhibitors (white bars) and enzyme specific inhibitors (grey bars) using a) membrane preparations of atria or b) membrane preparations of ventricles. The initial concentration of BNP1-30 generated in the incubations with heart membrane preparations without the use of inhibitors was set to 100% (black bar). For abbreviations of the inhibitors see Table 2. Significances were calculated with One-way ANOVA; *** P < 0.001 vs. no inhibitor. When no error bar is seen, variations are within the bar.
preferentially after alanin230. The second candidate, Prolylcarboxypeptidase (Angiotensinase C, PRCP, EC 3.4.16.2), belongs to the S28 class of serine proteases firstly isolated from swine kidney lysosomal fractions231. It cleaves bradykinin and angiotensin II231. Because of its actions, PRCP is considered to be a cardioprotective enzyme232, also since PRCP polymorphism is linked to hypertension and pre-eclampsia233, 234 and its depletion leads to vascular dysfunction and hypertension in mice235. PRCP favorably proteolyzes penultimate C-terminal prolines and is thus not an optimal candidate to cleave BNP1-32 which harbors a penultimate leucine.
Moreover, the prolyloligopeptidase inhibitor Z-Pro-Prolinal (ZPP), which is also described as a PRCP inhibitor235 had no effect on BNP1-30 generation (Figure 24).
Additionally, recombinant PRCP has been incubated with BNP1-32 and no formation of BNP1-30 could be observed (Figure 25a).
The other two candidates are Carboxypeptidase C and D. CPD (EC 3.4.16.6) is involved in the processing of growth factors and hormones in Drosophila236 and human237, however, it preferentially releases C-terminal arginines or lysines, which are not present at the C-terminus of BNP1-32. The CPC or better known as Cathepsin A (CathA, EC 3.4.16.5) is widely distributed in mammalian tissues on the cell surface but is also present in lysosome and is secreted from the cell238. CathA has a broad substrate spectrum, since this enzyme works multifunctional as deamidase, esterase, and carboxypeptidase with a preference for substrates with hydrophobic amino acid residues at the C-terminus239. CathA can hydrolyse several regulatory peptides,
Recombinant PRCP
0 15 30 45 60
0 1 2 3 4 5
time [min]
remaining BNP1-32 [µM]
Figure 25: Quantification of the remaining concentration of BNP1-32 after incubation of 5 µM BNP1-32 with a) 2.5 mU Prolylcarbocypeptidase (PRCP) and b) 0.15 mU Cathepsin A (CathA) over time.
Recombinant CathA
0 h 2 h 4 h
0 1 2 3 4 5
remaining BNP1-32 [µM]
a b
including substance P, met-enkephalin, and oxytocin and has a high activity against the vasoactive peptide endothelin-1240 and also against angiotensin (Ang) I by releasing a C-terminal leucine and converting it to Ang-(1-9)241. The mouse model of CathA deficiency shows significantly increased arterial blood pressure240. To test its actions on BNP1-30 formation, recombinant CathA was incubated with BNP1-32. No formation of BNP1-30 was observed (Figure 25b).
Since none of the serine-carboxypeptidases investigated was able to generate BNP1-30 from BNP1-32, it was hypothesized that the sought after peptidase might be a carboxypeptidase that is not a serine peptidase but is nevertheless in part inhibitable by aprotinin. Aprotinin is a single-chain polypeptide with a molecular weight of 6.5 kDa. It is a typical Kunitz-type protease inhibitor and inhibits especially trypsin and related proteolytic enzymes by the tight binding of its lysine in the active site of such peptidases. Aprotinin gained special importance in the treatment of influenca, since it targets trypsin-like proteases responsible for influenza virus hemagglutinin cleavage and virus activation in infected organism242. Moreover, it has a long history in medical practice in the treatment of pancreatitis and post-operative bleeding243, 244, and several licensed compounds of aprotinin are available including TrasylolTM (Bayer AG, Germany) or AntagosanTM (Sanofi Aventis, France)245. Since aprotinin seems to be involved in many pathways it might be possible that it can interfere with a carboxypeptidase not being a typical serine peptidase. For this purpose other recombinant carboxypeptidases have been tested for their ability to form BNP1-30, subsequent to this chapter. However, prior, enzymes generating BNP1-30 in lung, liver and spleen have been investigated.