As shown in Figure 1, a highly pure (purity ?98%) radiolabeled human 125I-Ang-(1-12) substrate was used in CPA and hrChymase-mediated hydrolysis studies. The purity of radiolabeled 125I-Ang-(1-12) was routinely checked on the HPLC to make sure that 125I-Ang-(1-12) peptide was not degraded at the time it was used for enzymatic hydrolysis.
Figure 2, illustrates the HPLC chromatogram of the hydrolytic products generated from 125I-Ang-(1-12) substrate by CPA alone. We found that CPA sequentially metabolized the 125I-Ang-(1-12) into Ang-(1-9) [53% (major product)], Ang II (22%) and Ang-(1-7) [11%] (Figure 2).
Hydrolysis of 125I-Ang-(1-12) by hrChymase alone is shown in Figure 3. hrChymase enzyme directly metabolized the 125I-Ang-(1-12) substrate into Ang II (89%) and no further hydrolysis of Ang II was detected (Figure 3). Ang II was still the major product generated from Ang-(1-12) (68%) when the substrate was incubated with both CPA + hrChymase (1:1 ratio) for 5 min (Figure 4). In addition to Ang II, small amount of Ang-(1-9) and Ang-(1-7) products (11% and 10%, respectively) were also detected in the reaction mixture.
When CPA and hrChymase, at 1:? ratio, was incubated with Ang-(1-12) substrate for 5 min, Ang II was still a major product (65%). Ang-(1-9) and Ang-(1-7) production from 125I-Ang-(1-12) amounted to only 27% and 6%, respectively (Figure 5). To determine of Ang-(1-9) hydrolysis by hrChymase, human 125I-Ang-(1-12) substrate was first incubated with CPA (0.325 µg/mL) for 5 min, next the CPA activity was stopped by adding 50 µM of benzylsuccinate [Lyons, 2008 #38] and then this reaction mixture was further incubated with hrChymase (0.325 µg/mL) for additional 5 min.
In these experiments, we found that the hydrolytic product of CPA “Ang-(1-9)” was not metabolized by hrChymase (data not shown).
Kinetic analysis (Km, Vmax and catalytic efficiency) of CPA and hrChymase enzymes for Ang-(1-12) substrates was also determined. A representative curve showing the hydrolytic products generated by CPA and hrChymase with increasing concentrations of Ang-(1-12) substrate is shown in Figure 6. The Km and Vmax were 150 ± 5 µM and 384 ± 23 nM/min/mg of CPA/Ang-(1-12) and 40 ± 9 µM and 116 ± 23 µM/min/mg of hrChymase/Ang-(1-12) reactions, respectively [Table 1]. The catalytic efficiency (the ratio of Vmax/Km ratio) was higher for hrChymase/Ang-(1-12) (2.97 ± 0.1) compared to CPA/Ang-(1-12) (2.56 ± 0.1) [Table 1].
The discovery of an Ang I upstream precursor [Ang-(1-12)], which serves as an alternate substrate for biologically active Ang II formation in the human heart radically altered our understanding of Ang II production.[Ahmad, 2011 #24] Our laboratory has done pioneer works in unraveling the complexity of the biotransformation pathways that account for the formation of on the Ang II hormone from angiotensinogen,.[Ahmad, 2016 #48;Ferrario, 2010 #50;Ferrario, 2016 #51;Ferrario, 2014 #27;Ferrario, 2016 #29;Ferrario, 1991 #49;Ferrario, 1996 #52;Reyes, 2017 #28] Although chymase-mediated Ang II formation from Ang I has a long-standing history in human,[Urata, 1993 #35][Lorenz, 2010 #36] the clinical importance of the Ang II-forming pathways from human Ang-(1-12)/MC proteases axis is largely unknown. Earlier, we shwed that Ang-(1-12) is rapidly hydrolyzed by human cardiac chymase to generate directly Ang II.[Ahmad, 2011 #24] But the specific role of MC CPA in human Ang-(1-12) substrate hydrolysis has not been demonstrated yet. In this study, we have investigated the individual and combined effects of two MC proteases (CPA and hrChymase) to hydrolyze the human Ang-(1-12) substrate.
Incubation of CPA alone with human Ang-(1-12) substrate for 15 min at 37?C, yielded the generation of Ang-(1-9) (53%, major product), Ang II (22%) and Ang-(1-7) (11%, end product). No further hydrolysis of Ang-(1-7) was detected by CPA (Figure 7). These data are consistent with our previous studies in human cardiac tissues,[Ahmad, 2011 #24;Ahmad, 2013 #25] where the hrChymase rapidly hydrolyses the human Ang-(1-12) substrate directly into Ang II (89%). No further hydrolysis of Ang II was detected by hrChymase. Since, the angiotensin processing peptidases (CPA and chymase) are stored in cardiac MC secretory granules and are co-released into the extracellular environment after activation/degranulation,[Pejler, 2010 #32] we investigated the combined effects of CPA and hrChymase on human Ang-(1-12) substrate hydrolysis. In the presence of both CPA and hrChymase (1:1 and 1:? ratio), we found that human Ang-(1-12) was rapidly metabolized into Ang II (69% and 65%, respectively) within 5 min. In both conditions, relatively small amounts of Ang-(1-9) and Ang-(1-7) products were generated. Although, human Ang-(1-12) is rapidly metabolized into Ang II directly, our current investigation show that CPA-mediated generated Ang-(1-9) product has negligible substrate affinity for hrChymase enzyme to generate Ang II. These findings clearly indicate that human Ang-(1-12) is primarily hydrolyzed by chymase into Ang II. Further, the enzyme kinetics results (lower Km value and higher catalytic efficiency) confirm the specificity and primacy of chymase over CPA to generate directly Ang II product from human Ang-(1-12) substrate.
The classical view of the biochemical pathways for the formation of biologically active angiotensin peptides continues to undergo significant revision as new data uncovers the existence of important alternate non-renin dependent mechanisms of Ang II formation from the novel dodecapeptide Ang-(1-12) (an Ang I upstream angiotensinogen precursor peptide) by MC proteases (chymase and CPA). In contrast to rodents, in human only the ?-form of the chymase (a chymotryptic serine endopeptidase) is stored in MC secretory granules in large amount.[Caughey, 1991 #22] In addition of chymase, MC secretory granules also stores CPA in large quantity. The MC CPA resembles bovine pancreatic CPA in cleaving COOH-terminal aromatic and aliphatic amino acid residues.[Goldstein, 1989 #14] CPAs cleave newly exposed COOH-terminal residues after endopeptidase cleavage by chymase, thereby sequentially degrading common proteins and peptides substrates.[Kokkonen, 1989 #16;Kokkonen, 1986 #39] Our recent finding suggests that the CPA has low affinity to cleave the newly exposed -COOH-terminal amino acid of Ang II generated from human Ang-(1-12) hydrolysis by chymase. Once Ang-(1-9) is cleaved by CPA from Ang-(1-12), chymase has negligible substrate affinity to hydrolyze the Phe8-His9 bond of the Ang-(1-9) to generate Ang II product.
The hydrolytic potential of an enzyme depends on the amino acid sequence of the peptides/proteins at the cleavage site. In Table 2, we document the characteristic differences between chymase and CPA enzymes. CPA1 and CPA2 isolated from rat mesenteric arterial bed perfusate (identical with their pancreatic counterparts), hydrolyze rat Ang-(1-12) substrate differently.[Pereira, 2012 #34] CPA1 has negligible affinity to hydrolyze the C-terminal amino acid of the rat Ang-(1-12) sequence. However, CPA2 rapidly hydrolyzes the rat Ang-(1-12) into Ang I. Further hydrolysis of Ang I into Ang-(1-9) by CPA2 was negligible. The amino acids sequence of the rat and human Ang-(1-12) are different [rat sequence, Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Leu11-Tyr12 and human sequence, Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Val11-Ile12]. In human, at the C-terminal end -Val11-Ile12 is present whereas, in rat -Leu11-Tyr12.[Ferrario, 2014 #27] Both, human Ang-(1-12) and Ang I substrates have very high specificity for human cardiac chymase to generate Ang II. We showed for the first time that rat cardiac chymase has much higher substrate affinity for rat Ang-(1-12) substrate compared to Ang I to generate Ang II.[Ahmad, 2016 #48] Our current findings show that CPA hydrolyzes the human Ang-(1-12) substrate into Ang-(1-9) (major product) and Ang II and Ang-(1-7) products. No intermediate products [Ang-(1-11) and Ang-(1-10)] was detected in the reaction mixture suggesting that CPA sequentially cleaves the human Ang-(1-12) C-terminal three peptide bonds (Val11-Ile12, Leu10-Val11 and His9-Leu10 bonds) very rapidly. Once Ang-(1-9) is generated (after the cleavage of C-terminal His9-Leu10 bond of Ang I) by CPA, the formation of Ang II and Ang-(7) is markedly delayed, indicating that CPA has less substrate affinity for Ang-(1-9) to generate Ang II and further decrease for Ang II to generate Ang-(1-7) (Phe8-His9 and Pro7-Phe8 bonds, respectively).
CPA3 (the MC CPA) shares significant homology with the other CPA subfamily. CPA3 resembles to pancreatic CPA1 in cleaving the C-terminal end of aromatic (Phe, Tyr and Trp) and aliphatic (Ala, Leu, Ile and Val) amino acids. CPA3 functions together with endopeptidases (chymases and tryptases) secreted from mast cells to degrade proteins and peptides, including Ang I.[Kokkonen, 1986 #39][Lundequist, 2004 #40] CPA3 may be involved in host defense against certain parasites, snake venom toxins, and the vasoconstricting peptide endothelin 1.[Sanglas, 2009 #41][Metz, 2006 #42][Maurer, 2004 #43] CPA3 is highly upregulated and makes it a potential diagnostic parameter of allergic inflammation and autoimmune disease models.[Benoist, 2002 #44][Dougherty, 2010 #45][Takabayashi, 2012 #46][Guo, 2015 #47] CPA3 mRNA was not detected in human normal tissues (including lung, heart, and kidney), but its expression could be induced in disease subjects.[Huang, 1999 #18] Our studies clearly show that both chymase and Ang-(1-12) substrate were predominantly expressed intracellularly in human atrial cardiac myocytes obtained from diseased patients.[Ahmad, 2011 #24] The expression and precise role of CPA in disease heart remains to be established.
Overall, our study suggests that Ang II generation form Ang-(1-12) was primarily mediated by chymase rather than CPA. Our studies also suggest that selective inhibition of chymase may provide greater benefit in the management of adverse cardiac remodeling than the MC CPA therapeutic approaches.