In contrast to Mc-cpa^–/– mast cells (Fig. 1 A, lane 7), Mc-cpa^Y356L,E378A mast cells (Fig. 1 A, lane 6) expressed Mc-cpa and Mcp-5 protein. Titrated Western blots showed that Mc-cpa^Y356L,E378A mast cells expressed 80% of the amount of Mc-cpa protein compared with wild-type mast cells (Fig. 1 A). To exclude a dose effect of reduced Mc-cpa expression in Mc-cpa^Y356L,E378A mast cells, we compared, where appropriate, heterozygous (Mc-cpa^+/–) mice to Mc-cpa^Y356L,E378A mice. To test whether the introduced mutations ablated catalytic activity, enzyme function of Mc-cpa^Y356L,E378A mast cells was analyzed in vitro (Fig. 1 B). Titration experiments showed that Mc-cpa enzyme activity was detectable in as few as 1,000 Mc-cpa^+/+ mast cells, but undetectable in 20,000 Mc-cpa^Y356L,E378A mast cells (not depicted), the highest cell number tested, and equivalent to approximately one half of the total mast cell number present in one peritoneal cavity. Hence, Mc-cpa^Y356L,E378A mast cells expressed Mc-cpa protein lacking enzyme activity, and this was sufficient for Mcp-5 expression.

Mast cells express ET-1 receptors on their surface, and binding of ET-1 to ET-1 receptors is a potent stimulus for release of secretory granules (10, 11). Using RT-PCR, we compared the expression of two ET-1 receptors, the ET_A and ET_B, in purified peritoneal mast cells. ET_A is the receptor type that is predominantly, if not exclusively, expressed on peritoneal mast cells (unpublished data), which is consistent with the requirement for ET_A expression for ET-1–induced degranulation of peritoneal mast cells (11). Next, ET-1–induced degranulation, which is measured by release of ß-hexosaminidase, was analyzed in mast cell–containing peritoneal exudate cells (PECs). With the exception of mast cell–deficient Kit^W/Wv mice (26), numbers of peritoneal mast cells were comparable in all genotypes analyzed (unpublished data). Relative to maximal signaling by ionomycin, ET-1 stimulated strong degranulation in mast cell–containing PECs from Mc-cpa^+/+ and Mc-cpa^–/–, but not in mast cell–deficient PECs from Kit^W/Wv mice (Fig. 1 C). Hence, ET-1 specifically stimulated mast cells for degranulation among PECs, and loss of Mc-cpa and Mcp-5 did not impair ET-1–stimulated granule release in mast cells.

Susceptibility of Mcp mouse mutants to ET-1
ET-1, which is the strongest endogenous blood pressure–regulating peptide, has been implicated in many pathological processes, including shock and sepsis (for reviews see references 6, 7). In an in vivo model of the pathological effects of ET-1, intraperitoneal injection of 3 µg ET-1 per mouse caused a rapid drop in body temperature and the death of two thirds of Kit^W/Wv, but not wild-type, mice (11). In our hands, injection of 7.5 µg ET-1 into mast cell–positive Kit^+/+ mice (designated as Mc-cpa^+/+ mice in Fig. 2 A) showed a transient temperature drop, and 14/15 mice fully recovered (Fig. 2 A). In contrast, the same dose caused a massive temperature drop within 20 min, and the death of 9/9 Kit^W/Wv mice by 60 min (Fig. 2 B). Hence, this dose was used for all subsequent experiments.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Susceptibility of Mc-cpa–/– and Mc-cpaY356L,E378A mutant mice to ET-1. ET-1 was injected intraperitoneally into Mc-cpa+/+ (A), KitW/Wv (B), Mc-cpa–/– (C), Mc-cpa+/– (D), and Mc-cpaY356L,E378A (E) mice, and the body temperature was tracked by rectal measurements at the indicated time points. Summarized total numbers of dead per injected mice are indicated for each genotype. For clarity, the temperature kinetic is shown for representative mice. Mc-cpa+/+ and Mc-cpa+/– showed a mild and transient temperature drop (A and D), whereas mast cell–deficient KitW/Wv (B), and mice lacking Mc-cpa and Mcp-5 (Mc-cpa–/–; C) succumbed to ET-1 injection within 60 min. Mice lacking only catalytically active Mc-cpa (Mc-cpaY356L,E378A; E) were also largely susceptible to ET-1, but the kinetic of the temperature drop was delayed compared with KitW/Wv or Mc-cpa–/– mice.

Mast cell–mediated degradation of ET-1
To address a mechanistic link between mast cells and survival of the mice in response to exogenously applied ET-1, peritoneal mast cells were incubated with ET-1 in vitro. Initially, we measured ET-1 degradation by an ET-1–specific ELISA. This assay has previously been reported to measure mast cell–mediated degradation of ET-1, and to reveal the mechanism behind how mast cells limit ET-1 toxicity (11). ET-1 was incubated for 60 min with PECs from Mc-cpa^+/+, Mc-cpa^–/–, or Kit^W/Wv mice (Fig. 3 A). Compared with input, the amount of ET-1 after incubation with Mc-cpa^+/+ PEC was reduced by >90%. Surprisingly, a similarly strong reduction in ELISA-reactive ET-1 was found after incubation with Mc-cpa^–/– PECs. Degradation of ET-1 was mostly mast cell–mediated, as shown by the inability of Kit^W/Wv PECs, which lack mast cells, to substantially reduce the amount of ET-1. These data suggested that not only normal mast cells but also mast cells lacking Mc-cpa and Mcp-5 proteins could degrade ET-1, a notion that is not easily reconciled with the observation that Mc-cpa^+/+ mice were protected (Fig. 2 A), whereas Mc-cpa^–/– mice were fully susceptible (Fig. 2 C) to ET-1. This discrepancy implied that the loss of ELISA-reactivity did not indicate whether or not ET-1 remained biologically active.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Degradation of ET-1 by mast cells. (A) ELISA measurements of "residual" ET-1 after incubation of ET-1 with no cells (open bar), or with PECs from Mc-cpa+/+ (solid bar), Mc-cpa–/– (striped bar), or KitW/Wv (shaded bar) mice. Mast cell–containing PECs from Mc-cpa+/+ (P = 0.030 [significant] comparing Mc-cpa+/+ versus KitW/Wv mice), and Mc-cpa–/– (P = 0.037 [significant] comparing Mc-cpa–/– versus KitW/Wv mice) mice abolished ET-1 reactivity by ELISA, suggesting that ET-1 was degraded regardless (P = 0.78 [not significant] comparing Mc-cpa+/+ vs. Mc-cpa–/–) of the mast cell genotype. Data summarize the mean ± one standard deviation for three independent experiments. (B–F) Mass spectrometric analysis of ET-1 degradation (left), and schematic depiction of substrate products (right). ET-1 was left untreated (B) or incubated with ionomycin-stimulated purified peritoneal mast cells from Mc-cpa+/+ (C), Mc-cpa–/– (D), or Mc-cpaY356L,E378A (E and F) mice. C-terminal degradation, evident by the appearance of ET-1 fragments corresponding to the molecular masses of 1–19 and 1–20, was mediated by Mc-cpa+/+ (C), but not by Mc-cpa–/– (D) or Mc-cpaY356L,E378A (E), mast cells. However, after incubation of ET-1 with Mc-cpa–/– (D) or Mc-cpaY356L,E378A (E) mast cells, the molecular mass increased from 2,492 (ET-1; B) to 2,510 daltons (ET-1 plus water; D and E). Hydrolysis of ET-1 was demonstrated after reduction of the samples by the disappearance of the ET-1 1–21 peak, and the appearance of new fragments with molecular masses of 1,639 and 1,786 daltons (see Materials and methods) corresponding to ET-1 1–13 and 1–14, respectively (F). Data are representative of three independent experiments.

In the presence of Mc-cpa^+/+ mast cells, most ET-1 (1–21) was converted into shorter fragments with masses of 1–20 and 1–19 (Fig. 3 C). C-terminal degradation products were not generated by Mc-cpa^–/– mast cells (Fig. 3 D). Thus, mast cells can degrade ET-1 at the C terminus, and this degradation requires expression of Mc-cpa or Mcp-5. To directly analyze the role of Mc-cpa in this process, ET-1 was incubated with Mc-cpa^Y356L,E378A mast cells (Fig. 3 E). Interestingly, ET-1 (1–21) was also maintained in the presence of these mutant mast cells lacking only catalytically active Mc-cpa. These experiments unequivocally identify Mc-cpa as the nonredundant enzyme required for removal of C-terminal amino acids from ET-1.

ET-1 can also be cleaved at multiple positions by endopeptidases (27). Along this line, Mcps other than Mc-cpa, notably chymases (11, 28), were reported to contribute to ET-1 degradation, and cleavage by chymases has, in fact, been proposed as the protective mechanism (11) (for review see reference 29). Mast cells from Mc-cpa^–/– and Mc-cpa^Y356L,E378A mice could not degrade the C terminus of ET-1, but we noticed a shift in the molecular mass of ET-1 from 2,492 (ET-1) to 2,510 daltons (Fig. 3, D and E). This increase in molecular weight suggested addition of a water molecule (ET-1 plus one H₂O = 2,510 daltons), which pointed at hydrolysis of one peptide bond within the ET-1 molecule. ET-1 harbors two intramolecular disulfide bridges linking the cysteine in position 1 with that in position 15, and the cysteines in positions 3 and 11 (27). In the absence of C-terminal degradation, the overall maintained length of the peptide could mask internal cleavage of ET-1 between the disulfide bridges by a mast cell–derived endopeptidase. To address this possibility, ET-1 was incubated with Mc-cpa^Y356L,E378A mast cells, and half of the supernatant was kept under nonreducing conditions in which ET-1 remained full length (Fig. 3 E). The second half was reduced to open the disulfide bridges. Under reducing conditions, ET-1 (1–21) disappeared, and shorter peptide fragments became apparent (Fig. 3 F), which is consistent with an ET-1 cleavage at positions 13 and 14, i.e., within the two cysteines in positions 11 and 15. Thus, mast cell products attack ET-1 internally, causing a nick in the peptide that could only be revealed under reducing conditions. This internal nicking was not mediated by Mcp-5 because the same pattern of degradation products was generated by Mc-cpa^–/– mast cells lacking both Mc-cpa and Mcp-5.

These data strongly suggested that the Mc-cpa–catalyzed removal of a single amino acid (tryptophan) from the C terminus of ET-1 was necessary and sufficient for the mast cell–mediated inactivation of ET-1. To prove that C-terminal degradation of ET-1 was sufficient for protection, synthetic peptides corresponding to full-length ET-1 (1-21) or C-terminally shorter peptides were injected into mast cell–deficient Kit^W/Wv mice (Fig. 4). Of these, only full-length ET-1 (Fig. 4 A), but neither the 1–20 (Fig. 4 B), nor the 1–19 (not depicted) amino acid long peptides induced a temperature drop or lethality in Kit^W/Wv mice.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Removal of tryptophan from the C terminus renders ET-1 nontoxic for mast cell–deficient mice. Synthetic peptides of ET-1 (1–21; A) or of ET-1 minus tryptophan in position 21 (1–20; B) were injected intraperitoneally into mast cell–deficient KitW/Wv mice, and the body temperature was followed by rectal measurements at indicated time points. Mast cell–deficient KitW/Wv mice were resistant to ET-1 (1–20), but succumbed to ET-1 (1–21).

Catalytic activity of Mc-cpa is essential to protect against snake venom sarafotoxin
Snake venom sarafotoxins share significant amino acid sequence similarity with endogenous ET-1 (27, 30). This sequence homology appears to be immunologically relevant because mast cells can enhance resistance not only to ET-1 but also to snake venom sarafotoxins (12). The molecular mechanism underlying the mast cell–mediated degradation of sarafotoxins has not been fully uncovered. Given the similarities in sequence and structure involving two disulfide bridges, it appears likely that the mechanisms of ET and sarafotoxin degradation are similar, if not identical. However, the specific role of Mc-cpa could not easily be addressed up to now because ablation of Mc-cpa expression by gene targeting, as in Mc-cpa^–/– mice (16), or by RNA interference (RNAi), as in Mc-cpa^+/+ BM-derived mast cells transplanted into mast cell–deficient mice (12), inevitably also lead to abrogation of Mcp-5 expression (see Introduction and Fig. 1 A) (12, 16).

To address whether Mc-cpa or Mcp-5 are involved in the resistance against S6b, which is the most toxic representative of this group of snake venom peptides, the different Mc-cpa mouse genotypes (Mc-cpa^+/+, Mc-cpa^+/–, Mc-cpa^–/–, and Mc-cpa^Y356L,E378A), as well as Kit^W/Wv mice, were injected with 7.7 µg S6b (Fig. 5). Mc-cpa^+/+ and Mc-cpa^+/– mice were protected (Fig. 5 A), but mast cell–deficient Kit^W/Wv mice were not (Fig. 5 B), thereby confirming a crucial role of mast cells in the protection against S6b in vivo (12). Mice lacking Mc-cpa and Mcp-5 (Mc-cpa^–/–) showed a rapid temperature drop similar to Kit^W/Wv, and all Mc-cpa^–/– mice succumbed to S6b injection (Fig. 5 C). Interestingly, Mc-cpa^Y356L,E378A mice were also fully susceptible to S6b (Fig. 5 D). In contrast to the delayed ET-1–induced lethality of Mc-cpa^Y356L,E378A mice when compared with Kit^W/Wv or to Mc-cpa^–/– mice (Fig. 2), Mc-cpa^Y356L,E378A mice were equally vulnerable to S6b as Kit^W/Wv or Mc-cpa^–/– mice (Fig. 5). These experiments establish an essential role for Mc-cpa in the mast cell defense against S6b.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Susceptibility of Mc-cpa mutants to S6b. The S6b was injected intraperitoneally into Mc-cpa+/+ (A), Mc-cpa+/– (only numbers shown in A), KitW/Wv (B), Mc-cpa–/– (C), and Mc-cpaY356L,E378A (D) mice, and the body temperature was followed by rectal measurements at the indicated time points. Summarized total numbers of dead per injected mice are indicated for each genotype. The temperature kinetic of Mc-cpa+/– mice (not depicted) was similar to the one from Mc-cpa+/+ mice. All strains except for Mc-cpa+/+ and Mc-cpa+/– mice succumbed to S6b injection within 60 min. The full susceptibility of Mc-cpaY356L,E378A mice demonstrates the essential role of Mc-cpa enzyme activity for survival of snake venom S6b.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Degradation of S6b by mast cells. (A–D) Mass spectrometric analyses (left side) of degradation products of S6b. Resulting peptides are schematically depicted on the right side. S6b was left untreated (A) or incubated with ionomycin-stimulated purified mast cells from Mc-cpa+/+ (B) or Mc-cpaY356L,E378A (C and D) mice. C-terminal degradation, which is evident from the appearance of fragments corresponding to the molecular mass of S6b (1–19; 2,266 daltons), was mediated by Mc-cpa+/+ (B), but not by Mc-cpaY356L,E378A (C) mast cells. The peak marked by the asterisk in B does not correspond to a fragment of S6b, and it was not observed in a second experiment. The molecular mass of S6b (1–21) increased after incubation with Mc-cpaY356L,E378A mast cells from 2,565 (1–21; A) to 2,583 daltons (S6b 1–21 plus water; C). This indicated hydrolysis of S6b, which was proven after reduction of the samples by the disappearance of the S6b 1–21 peak, and the appearance of residual fragments with molecular masses of 1,710 and 1,858 daltons (see Materials and methods) corresponding to S6b 1–13 and 1–14, respectively (D). Data are representative for two independent experiments.

	DISCUSSION

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In contrast to the notoriously harmful role of mast cells in allergic disease, new mast cell functions in immunity are emerging. Mast cell properties such as protection against the lethal toxicity of snake venom sarafotoxins raise questions as to which mast cell effector molecules mediate these innate immune responses, and by what mechanism. Mcps are good candidates for relevant effector molecules because they represent major protein components in mast cell granules that are secreted during signaling-dependent degranulation. However, Mcps have been commonly viewed as tissue damaging (31). In contrast, the high degree of evolutionary conservation of Mcps in various species, as well as the strong expression of proteases, suggests positive functions. Impaired worm expulsion in mice lacking a mucosal Mcp, Mcp-1 (32), is an example of a protease contributing to protection.

Recent reports demonstrated that mast cells must be present in the peritoneal cavity for mice to survive a challenge with the blood pressure regulating peptide ET-1 (11), and the related S6b (12). Protection against these peptides is initiated by binding of ET or sarafotoxin to ET receptors that induce rapid and potent degranulation of peritoneal mast cells (Fig. 7, A-C) (10, 11). ET_A is the major ET receptor expressed on peritoneal mast cells (not depicted) (11), and expression of ET_A on mast cells is important, as ET_A-deficient mast cells are nonprotective (11).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Molecular mechanism of ET family peptide degradation by Mc-cpa. (A–C) ET-1 and S6b, shown here for ET-1, stimulate mast cells via binding to ET receptors. The receptor type most prominently expressed on peritoneal mast cells is ETA. ETA activation leads to massive degranulation, which is comparable to stimulation via ionomycin, and release of mast cell granule content. These early events are identical in mast cells from Mc-cpa+/+ (A–C; left) and Mc-cpaY356L,E378A (A–C; right) mice. (D–H) Mast cells secrete a set of proteases that include one carboxypeptidase A (Mc-cpa) and several chymases that include Mcp-5. Mc-cpa attacks the C terminus of ETs, and removes amino acids in position 21 (Trp; D) and 20 (Ile; F). These modifications render ETs and related toxins biologically inactive by three orders of magnitude (G; 34), and the mice survive (H). Mc-cpaY356L,E378A mice, which selectively lack active Mc-cpa, are unable to attack the C terminus of ET-1 (I). Mast cell products (possibly Mcp-4) other than Mc-cpa and Mcp-5 attack ET-1 internally by hydrolysis (E and I). Although the molecular structure (46) of position 13–nicked ET-1 is likely altered, ET-1 fragmentation is prevented by the disulfide bridge (1 to 15; E and I), and the molecules retain their toxicity (J and K), unless the C terminus is truncated (E). Mc-cpaY356L,E378A mutant mice are unable to do so, and succumb to exogenous ET-1 and related toxins, thereby demonstrating the essential role of Mc-cpa for this innate immune pathway. Whether or not Mc-cpa is also involved in the degradation of endogenously produced ETs remains to be investigated.

To address the exclusive function of Mc-cpa, we generated a mouse homozygous for a mutated Mc-cpa allele. Based on data from previous structure–function experiments that addressed the catalytic mechanism in pancreatic Cpa (24, 25), we mutated Y356 to L356 and E378 to A378. The observation that Mc-cpa^Y356L,E378A mast cells expressed Mc-cpa protein that lacked detectable carboxypeptidase activity (Fig. 1) showed that pancreatic and mast cell carboxypeptidases are not only homologous in their amino acid sequences but that homologous amino acids are also functionally conserved. One important aspect of Mc-cpa^Y356L,E378A mast cells was the stoichiometrically normal expression of Mcp-5 (Fig. 1), which demonstrates a structural, rather than an enzymatic, Mc-cpa requirement for Mcp-5 expression. This, in turn, was a prerequisite to specifically evaluate the function of Mc-cpa.

Previous experiments addressing the mast cell–mediated degradation of ET-1 led to the conclusions that chymases and Mc-cpa (28), or chymases but not Mc-cpa (11), or Mc-cpa but not chymases (12) degrade ET-1. Challenging Mc-cpa^Y356L,E378A mice by ET-1 and S6b now revealed directly that the enzyme activity of Mc-cpa is essential for protection against ET-1 (Fig. 2) and S6b (Fig. 5). Identification of Mc-cpa as the functionally relevant enzyme also helped to clarify which proteolytic mechanism is protective. Combining the genetic defects of Mc-cpa^–/– or Mc-cpa^Y356L,E378A mast cells with mass spectrometric analyses of substrates showed two sites of attack. The first is C-terminal removal of one (Fig. 7 D) or two (Fig. 7 F), or more (28) amino acids. This step is exclusively catalyzed by Mc-cpa as shown by intact C termini after contact with Mc-cpa^Y356L,E378A mast cells (Fig. 7 I). That this is the protective reaction was further supported by the lack of toxicity of synthetic ET-1 (1–20) in mast cell–deficient mice (Fig. 4). The second site of attack is internal cleavage at positions 13 or 14 (Fig. 7 E). This could be conclusively detected in the absence of C-terminal degradation, i.e., in the presence of mutant mast cells lacking Mc-cpa protein (Mc-cpa^–/–; not depicted) or activity (Mc-cpa^Y356L,E378A; Figs. 3 F and 6 D) under reducing conditions. Because hydrolysis was seen using Mc-cpa^–/– mast cells (Fig. 3 D), the reaction is not catalyzed by Mcp-5. The consequence of internal attack on ET-1 by mast cells remains elusive because only C-terminal degradation correlated with abrogation of in vivo toxicity. It remains to be determined whether internal cleavage has absolutely no effect on the biological activity of ET-1 in vivo.

The in vivo toxicity of mast cell–mediated degradation products of ET-1 is in agreement with previous structure–function analyses of ETs. ETs nicked by endopeptidase maintains toxicity (27), whereas the two C-terminal residues (Ile and Trp) are important for receptor binding and biological activity of ETs (34, 35). A note of caution should be made regarding ELISA measurements of ET-1 degradation. This assay, used before to correlate in vitro degradation with in vivo toxicity (11), was, in our hands, misleading because it indicated degradation even by mast cells that could not protect mice in vivo (Fig. 2 versus Fig. 3). It is possible that immunoreactivity in this assay was abrogated by the internal nicks, or by conformational changes. In any case, it may be prudent to consider the biological activity of ET-1 when further studying the mast cell–mediated degradation of ET-1 in the future.

Collectively, we have shown that Mc-cpa is an essential effector molecule within the "mast cell–ET link" providing a very rapid (minutes) and life-saving response of toxin neutralization in vivo. Several sites on ET-1 are attacked by mast cells but only the C-terminal degradation is a safeguard. It is remarkable that the protective Mcp, Mc-cpa, "targets" exactly that amino acid in snake venom sarafotoxin that is essential for toxicity. This substrate specificity of Mc-cpa, as well as the fast circuit composed of binding of sarafotoxin to ET receptors, expressed on the cell surface of mast cells, followed by the rapid degranulation, release of the protective enzyme, and cleavage of the toxin, may represent an evolutionarily well conserved, and hence old, function of mast cells. ET-1 levels in plasma, liver, and peritoneal cavity rapidly rise during experimental sepsis, such as in cecal ligation and puncture models (36–38). Moreover, in human sepsis patients, high levels of ET-1 are correlated with morbidity and mortality (4, 39). ET-1 is thought to be involved in the pathological manifestations of sepsis in many organs, including heart, lung, liver, kidney, and intestines (4, 9). Massive local or systemic ET-1–mediated vasoconstriction (40) is a major pathological mechanism in sepsis as shown by beneficial effects of ET receptor antagonist treatment in models of sepsis (41). The observation that mast cells are closely located, among other sites, to blood vessels, and present in the peritoneal cavity, suggests that mast cells are exposed systemically or locally to changes in ET-1 levels (42). Mast cells are not only activated by ET-1 via ET_A (10) but also have a potent mechanism to inactivate ET-1 via secretion of Mc-cpa. Hence, mast cells may play an important role in either promoting an inflammatory cascade in response to ET_A stimulation by releasing mediators such as TNF or in dampening the deleterious pathophysiological effects of ET-1 on the vascular system by Mc-cpa–mediated rapid cleavage of ET-1. It remains to be determined under which conditions of local or systemic inflammation the effect of ET-1 on mast cells and the reciprocal inactivation of ET-1 by mast cells prevail.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mice and gene targeting.
WBKit^W/+, C57BL/6Kit^Wv/+ mice (Japan-SLC) and WBB6F1Kit^W/Wv mice were bred as previously reported (43). Mc-cpa^–/– mice (16) were backcrossed for 12 generations to the C57BL/6 strain, and maintained on this background. Mc-cpa^Y356L,E378A mice were generated by gene targeting ("knock-in") in E14.1 ES cells (129/OlaHsd). The targeting construct was assembled in a pBSK-based vector bearing a loxP-flanked neomycin (Neo) resistance gene and the HSV thymidine kinase gene (TK; provided by H. Luche and H.J. Fehling, University of Ulm, Ulm, Germany). The NotI-linearized vector consisted, from 5' to 3', of a 1.6-kb homologous "short arm" (nucleotides 24,438 to 26,059 of the Mc-cpa gene, according to Ensembl gene number ENSMUSG00000001865), the Neo gene under the TK promotor in the opposite orientation to the Mc-cpa locus, a 5.6-kb homologous "long arm" (nucleotide 26,077 to +5,141 of Mc-cpa gene) and the TK gene in the orientation of the Mc-cpa locus. The long arm harbored exon 11, which was mutated from tacttg (nucleotide 1,072–1,074, according to National Center for Biotechnology Information Entrez mRNA number NM_007753), resulting in a substitution of tyrosine 356 to leucine, and from gaggcg (nucleotide 1138–1140 [NM_007753]), resulting in a substitution of glutamic acid 378 to alanine. PCR-based mutagenesis was done as previously described (44). Gene targeting led to insertion of the Neo gene into intron 10. Gene targeting, ES cell selection, Neo gene excision in ES cells, and the generation of chimeric mice were done as previously described (16). All animal experiments were performed in accordance with institutional and government regulations and were approved by Regierungspräsidium Tübingen, Germany.

Western blots.
Protease expression was analyzed as previously described (16). Briefly, proteins were separated by 11.5% SDS-PAGE, followed by blotting onto PVDF membranes (Millipore). Rat anti–mouse antisera specific for Mc-cpa and Mcp-5 (provided by Lars Hellman, Uppsala University, Sweden) were used at 1:200 dilutions. Blots were developed by anti–rat–horseradish peroxidase (1:100,000 dilution; GE Healthcare) as the second step, followed by ECL substrate (Pierce Chemical Co.). Actin expression was analyzed using monoclonal mouse anti–ß-actin (clone AC-15; 1:3,000 dilution; Sigma-Aldrich), followed by polyclonal rabbit anti–mouse– horseradish peroxidase (1:1,000 dilution; Dako Cytomation).

Mc-cpa enzyme assay.
Mast cells were lysed in PBS, 2 M NaCl, and 1% Triton X-100 for 15 min on ice. The lysate volume corresponding to 10,000 cells/well was adjusted to 60 µl with lysis buffer and mixed with 40 µl water and 20 µl chromogenic substrate (1.8 mM; N-[4-methoxyphenylazoformyl]-Phe-OH; Bachem). Substrate degradation was measured as the change over time in absorbance at OD₄₀₅ with a SpectraMax 250 (MWG Biotech) spectrophotometer.

Mast cell degranulation assay.
PECs were resuspended in 120 µl Tyrode's buffer (10 mM Hepes, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.1% BSA, pH 7.4) and stimulated with either 2 µM ionomycin or 1.25 µg ET-1 for 15 min at 37°C. The cells were pelleted at 500 g for 3 min before collection of the supernatants. To measure ß-hexosaminidase release, 50 µl of the supernatant was mixed with an equal volume of substrate (p-nitrophenyl-N-acetyl-ß-D-glucosaminide). A 25-mM stock of p-nitrophenyl-N-acetyl-ß-D-glucosaminide in DMSO was diluted 1:25 in 0.1 M citrate buffer, pH 4.5, before use. After 90 min of incubation at 37°C, the reaction was stopped by addition of 125 µl of 1 M Tris, and the substrate conversion was measured photometrically at OD₄₀₅. Data were analyzed by Student's t test.

In vivo toxicity.
Mice were injected intraperitoneally with ET-1 (7.5 µg in 300 µl/mouse; 1–21), ET-1 (6.9 µg in 300 µl/mouse; 1–20), or S6b (7.7 µg in 300 µl/mouse). To verify proper intraperitoneal injection, the injected fluid contained 0.1 µg/ml Evans blue. At the end of the experiment, the peritoneal cavity was opened in each mouse to identify free blue peritoneal fluid as an indicator of successful injection. Mice lacking this fluid were excluded from the analysis. Changes in body temperature were followed by periodic measurements of rectal temperature using a Qtemp 200 (VWR International) thermometer.

Mass spectrometry.
15,000 PMCs (Kit⁺ PEC) in 120 µl acetate buffer (150 mM NH₄Ac, pH 7.4, 130 mM NaCl, and 1.4 mM CaCl₂) were prewarmed for 10 min at 37°C, and stimulated by the addition of 2 µM ionomycin and 1.25 µg ET-1, or by the addition of 2 µM ionomycin and 1.25 µg S6b. After incubation at 37°C for 60 min, the cells were pelleted at 500 g for 3 min and 100 µl of supernatant was split into 2 50-µl aliquots. For reducing conditions, the sample was dried again under vacuum, resuspended in 10 µl DTT solution (2.5 mM DTT in 10 mM NH₄HCO₃), and incubated overnight at 4°C. After addition of 2.5 µl IAA solution (10 mM IAA in 10 mM NH₄HCO₃), the sample was incubated for 30 min in the dark. Under reducing conditions, the molecular mass of the fragments was increased by the binding of one IAA molecule (molecular weight, 58 daltons) to each sulfate group. Nonreduced and reduced samples were desalted with PerfectPure C18 tips (Eppendorf), eluted with 10 µl acetonitril/0.1% TFA, and mixed 1:1 with -cyano-4-hydroxycinnaminic acid matrix before analysis. Mass spectrometry was done on a REFLEX IV MALDI-TOF mass spectrometer (Bruker-Daltonics). Full-length ET-1 was purchased by Sigma-Aldrich or custom made by Bachem (ET-1 in the length of 1–19, 1–20, and 1–21), and S6b was purchased from Alexis.

ET ELISA.
To measure the potential degradation of ET-1 by ELISA, mast cell–containing PECs were resuspended in 120 µl Tyrode's buffer (see Mast cell degranulation assay), and incubated with 1.25 µg ET-1 for 30 min at 37°C to stimulate degranulation, as shown in Fig. 1 C. At the end of the incubation period that allowed mast cell products to "attack" ET-1, cells were pelleted at 500 g for 3 min at 4°C, and supernatants were collected. Samples of these supernatants were tested in the ß-hexosaminidase assay to control for degranulation, and analyzed by ELISA for residual ET-1. The ELISA kit (Biomedica) was used according to the manufacturer's instructions. Serial dilutions of the samples were analyzed in parallel. The amount of input versus output ET-1 was taken as a measure for mast cell–mediated ET-1 degradation. Data were analyzed by Student's t test.

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Protease protects from snakes and sepsis

Hema Bashyam
J. Exp. Med. 2007 204: 2496. [Full Text] [PDF]