3 RESULTS
3.1 Metabolism of BNP1-32
3.1.1 Metabolism of BNP1-32 by the proteolytic activity of different organs
3.1.1.1 Comparison of the proteolytic activity of organs towards BNP1-32 BNP1-32 has been synthesized as recombinant peptide via solid-phase synthesis (see Method section 2.2.1). The quality and purity of the synthetic peptide has been proven by HPLC and mass spectrometry. Recombinant BNP1-32 was incubated with murine membrane preparations of different organs harboring intact peptidase activities (see Method section 2.2.2). In particular, preparations of heart, lung, liver, kidney, and spleen were used. The remaining concentration of BNP1-32 in these incubations was quantified by HPLC and compared between the different organs at one distinct point of time. The experiment has been designed to get a qualitative overview about the proteolytic activity of the organ preparations towards BNP1-32.
Figure 9 illustrates that proteolytic activity towards BNP1-32 is very divergent between the organs investigated. Although protein concentration of the membrane preparations has been analyzed before and was normalized for each sample, the proteolysis of BNP1-32 was unequal. Hardly any proteolysis of BNP1-32 occurred in incubations with heart and spleen preparations, while only a small part of the initial peptide concentration was left when incubated with kidney preparations. The proteolysis of BNP1-32 observed in lung and liver preparations was similar.
However, both organ preparations showed higher proteolytic activity towards BNP1-32 compared to heart and spleen but much less compared to kidney.
This first dataset highlights that proteolytic activities towards BNP1-32 diversifies between the organs indicating that the amount of proteolyzing enzymes differs from organ to organ. However, at the same time it is also likely that each organ does harbor different peptidases responsible for BNP1-32 proteolysis. Thus, emerging products of BNP1-32 proteolysis might vary between the preparations due to different enzymatic activity present in these organs. Such BNP products have been determined in each organ preparation in the following investigations.
Proteolysis of BNP1-32
heart lung live
r
kidne y
sple en 0
20 40 60 80 100
remaining BNP1-32 [%]
Figure 9: Quantification of the remaining concentration of BNP1-32 in percent after incubation with membrane preparations of heart, lung, liver, kidney and spleen for 90 min. The initial BNP1-32 concentration before incubation was set to 100%. Membrane preparations were normalized to protein concentration.
3.1.1.2 Investigation of initial proteolytic products of BNP1-32
The formation of initial products emerging from the proteolysis of BNP1-32 has been investigated for each organ. The peptide has been incubated with the membrane preparations until proteolysis of 20 to 25 % was achieved. The emerging BNP1-32 cleavage products have been separated by HPLC (Figure 10).
Datafile Name:16.11.2010_mBNP32+heartM_90min_005.lcd Sample Name:mBNP32+heartM_90min Sample ID:UNK-007
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:15.11.2010_mBNP32+kidneyM_004.lcd Sample Name:mBNP32+kidneyM Sample ID:UNK-006
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:16.11.2010_mBNP32+liverM_45min_003.lcd Sample Name:mBNP32+liverM_45min Sample ID:UNK-005
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
PM1
Datafile Name:16.11.2010_mBNP32+lungM_45min_002.lcd Sample Name:mBNP32+lungM_45min Sample ID:UNK-004
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
PM1
PM3 PM1 PM2
Datafile Name:16.11.2010_mBNP32+lungM_45min_002.lcd Sample Name:mBNP32+lungM_45min Sample ID:UNK-004
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:15.11.2010_mBNP32+kidneyM_004.lcd Sample Name:mBNP32+kidneyM Sample ID:UNK-006
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:12.12.2011_bnp32+spleen 90min_003.lcd Sample Name:bnp32+spleen 90min Sample ID:UNK-067
11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
0 50 100 150 200
mAU
PM1
Datafile Name:15.11.2010_mBNP32+kidneyM_004.lcd Sample Name:mBNP32+kidneyM Sample ID:UNK-006
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:12.12.2011_bnp32_002.lcd Sample Name:bnp32 Sample ID:UNK-066
11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150
mAU
no membranes
heart membranes
lung membranes
liver membranes
kidney membranes
spleen membranes
BNP1-32
BNP1-32
BNP1-32
BNP1-32
BNP1-32
BNP1-32
Datafile Name:16.11.2010_mBNP32+lungM_45min_002.lcd Sample Name:mBNP32+lungM_45min Sample ID:UNK-004
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:16.11.2010_mBNP32+lungM_45min_002.lcd Sample Name:mBNP32+lungM_45min
Sample ID:UNK-004
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
Datafile Name:16.11.2010_mBNP32+lungM_45min_002.lcd Sample Name:mBNP32+lungM_45min Sample ID:UNK-004
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min 50
100 150 200 mAU
Dataf ile Name:15.11.2010_mBNP32+kidneyM_004.lcd Sample Name:mBNP32+kidneyM Sample ID:UNK-006
11,00 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 min
50 100 150 200
mAU
a
b
c
d
e
f
Figure 10: HPLC chromatograms of (a) BNP1-32 alone, and BNP1-32 incubated with membrane preparations of (b) heart, (c) lung, (d) liver, (e) kidney, and (f) spleen until 20-25% of the initial BNP1-32 concentration was proteolyzed. According to their retention times, initial cleavage products are labeled with P 1, P 2 and to P 3. HPLC method 1 was used to separate peaks.
An optimal HPLC method has been established beforehand to allow distinct peak separation (see Methods section 2.2.3). For the identification of significant products, an automatic threshold for the integration of the peaks was set.
Figure 10a shows the peak of pure BNP1-32. Its retention time (rt) emerging from its interaction with the column with the set conditions is 17.7 min. Besides this peak, new peaks with different retention times occurred in the chromatograms obtained from the incubations with the different organ preparations (Figure 10b-f). These peaks represent cleavage products of BNP1-32. Two peaks were generated through incubation with heart (PM1&2), and one each in incubations with lung (PM1), liver (PM1), kidney (PM3) and spleen (PM1).
Among these six product peaks, three had the same retention time. Peak PM1, eluting first, (rtPM1 = 12.3 min) occurred in all approaches except for kidney. Conversely to PM1, peak PM3 (rtPM3 = 19.0 min) occurred in kidney only. Peak PM2 (rtPM2 = 13.9 min) was found in heart only.
It could be revealed that the generation of PM1 was highest in liver compared to other organ preparations used. A comparison of the amount of PM1 generated in the organs within 90 min, is shown in Figure 11, in which the peakarea of PM1 in liver was set to 100%. The absolute peakarea of PM1 in liver approximates 40% of the initial peak of BNP1-32.
Generation of PM1
heart lung
liver
kidne y
sple en 0
20 40 60 80 100
PM1 [%]
Figure 11: Quantification of the generation of PM1 from BNP1-32 in percent after incubation with membrane preparations of heart, lung, liver, kidney and spleen for 90 min. Membrane preparations were normalized to protein concentration. The highest amount of PM1 generation was set to 100%.
The occurrence of these different product peaks indicates that the enzymatic processing of BNP1-32 varies between the organs. The identification of the amino acid sequence of these products and hence the cleavage sites of BNP1-32, is described in the next chapter.
3.1.1.3 Identification of the initial proteolytic products of BNP1-32
Each peak has been manually fractionated after chromatographic separation for analysis with MALDI-TOF MS to obtain masses (see Methods section 2.2.4).
As expected, the six peaks could be identified as three different BNP peptide fragments varying in their sequence lengths. Figure 12a shows the MALDI-TOF spectra of the three products with their corresponding masses.
Figure 12b displays the peptides’ amino acid sequences conducted from their masses. For comparison also BNP1-32 is shown. The BNP fragments represented by the peaks PM1 and PM2 are C-terminally truncated, while the peptide of peak PM3 is N-terminally truncated compared to the mature BNP1-32. PM1, which occurred in all
PM1 PM2 PM3
a
30 32 1
dissulfide bond-linked ring 7
BNP1-32
N S K V T H I S S C FG H K I D R I G SVS R LG C NA L K L L
PM1 (BNP1-30)
N S K V T H I S S C FG H K I D R I G SVS R LG C NA L K
PM2 (BNP1-29)
N S K V T H I S S C FG H K I D R I G SVS R LG C NA L
PM3 (BNP7-32)
N K V T H I S S C FG H K I D R I G SVS R LG C NA L K L L
29
N-terminal part C-terminal part
10 26
Figure 12: a) MADLI-TOF-MS spectra of the three different BNP products generated through incubation with organ membrane preparations. Each spectrum shows the isotope distribution of the mass peaks; b) Amino acid sequences of BNP1-32 and the three identified BNP products PM1, PM2 and PM3 conducted from their masses. Relevant amino acids are numbered according to their order, beginning with the N-terminus of the mature BNP1-32.
b
incubations except for the one with kidney, is truncated by the two C-terminal leucine (L), decreasing the number of amino acids to 30. Subsequently, this peptide is named BNP1-30. The peptide represented by PM2 is additionally shortened by lysine (K), reducing the number of amino acids to 29. Thus, the peptide is named BNP1-29. The third product, PM3, is truncated by six amino acids at the N-terminal side and is named BNP7-32. BNP7-32 has been described before in 2007 by an approach of Pankow et al. as the renal meprin A (EC 3.4.24.18) cleavage product of BNP1-32160. According to the monoisotopic peptide masses obtained from MALDI-TOF-MS and the comparison with the linear forms of the peptides, it was revealed that the two cysteines at positions 10 and 26 are present in a non-reduced form and are involved in a disulfide bridge (C10-C26). Thus, all truncated forms obtained in this approach still harbor the disulfide-linked ring structure, which is the prerequisite for biological activity of natriuretic peptides39 (see Introduction section 1.1.2). Hence, the three identified BNP products are referred to as metabolites rather than degradation products.
3.1.1.4 Overview of the proteolysis of BNP1-32 by organ membrane preparations
The investigation of the initial metabolism of BNP1-32 revealed that proteolytic activities towards the peptide vary between the organs. Kidney preparations had the highest proteolytic activity against BNP1-32, whereas the peptide was relatively stable in heart and spleen preparations (Figure 9). Moreover, the formation of three BNP metabolites was observed (Figure 10). The C-terminally truncated BNP1-30 was generated through the proteolytic activity of all organs except kidney. Comparing all organs, BNP1-30 was accumulating the most using liver membrane preparations (Figure 11). BNP7-32, the N-terminally truncated BNP metabolite was emerging in kidney only. The third BNP metabolite, BNP1-29 was generated in heart only. These results highlight that BNP metabolism is organ-dependent.
Moreover, it is possible that the BNP metabolites are further metabolized. To complement the results, the metabolites found have been further investigated for their metabolism.
3.1.1.5 Metabolism of the identified BNP metabolites
The identified metabolites BNP1-30, BNP1-29, and BNP7-32 have been synthesized via solid-phase synthesis and were incubated with the organ preparations to see if they are further processed.
Table 13 summarizes the results of this approach. Incubation of BNP1-30 led to a dominant formation of BNP1-29 in all organ preparations except for kidney. BNP1-29 has also been present in heart preparations incubated with BNP1-32, thus, BNP1-30 might function as an intermediate product for the formation of BNP1-29. The significant accumulation of BNP1-29 in heart which is initially not present in other organs also generating BNP1-30 might be due to an increased concentration of the peptidase responsible for the generation of BNP1-29 or a decreased concentration of the peptidase degrading BNP1-29.
Proteolysis of BNP1-29 led to the formation of another new BNP metabolite, BNP7-29, occurring in heart, lung and kidney but not in liver and spleen. Moreover, also BNP1-30 was metabolized to another new BNP metabolite, BNP7-30, in the same tissues, indicating that a proteolytic activity is able to remove the six N-terminal amino acids from both substrates similar to the formation of BNP7-32 in these tissues.
Incubation of BNP7-32 with organ preparations led to the generation of BNP7-30 in lung, heart, liver, and spleen. In kidney itself, the C-terminally truncation forming BNP7-30 was, similarly to BNP1-30, not happening.
Table 13: Summary of BNP metabolites occurring after incubation of the initial BNP metabolites BNP1-30, BNP1-29 and BNP7-32 with membrane preparations of heart, lung, liver, kidney and spleen.
Formation of metabolites with membrane preparations of:
heart lung liver kidney spleen
BNP1-30 1-29 7-30
1-29 7-30
1-29 -
- 7-30
1-29 -
BNP1-29 7-29 7-29 - 7-29 -
BNP7-32 7-30 7-30 7-30 - 7-30
In conclusion, BNP1-32 and BNP metabolites can be C-terminally and N-terminally truncated in heart and lung. On the contrary, in kidney, only an N-terminal truncation is possible, whereas in liver and spleen only a C-terminal truncation of the peptides is
observed. This evolves the hypothesis that BNP metabolism is organ-specific and that full proteolysis of BNP1-32 needs the coactions of different organs and different peptidases.
To get a more physiological insight in the metabolism of BNP1-32 and to confirm the above results, it was decided to perform an organ perfusion in situ, exemplarily done in liver.