Peptides,Vol. 12, pp. 563-567. ©PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Catabolism of Calcitonin Gene-Related Peptide and Substance P by Neutral Endopeptidase M. K A T A Y A M A , * J. A. N A D E L , * t § N. W. BUNNETT,:~ G. U. DI MARIA,* M. HAXHIU* AND D. B. B O R S O N * t 1

Cardiovascular Research Institute, tDepartment of Physiology, SDepartment of Surgery §Department of Medicine, University of California San Francisco Received 1 October 1990

KATAYAMA, M., J. A. NADEL, N. W. BUNNETT, G. U. DI MARIA, M. HAXHIU AND D. B. BORSON. Catabolismof calcitonin gene-relatedpeptide and substance P by neutral endopeptidase. PEPTIDES 12(3) 563-567, 1991.--Calcitonin generelated peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidase (NEP), a membrane-boundpeptidase, cleaves many peptides including SP, thereby limiting their biological actions. Recombinant NEP cleaved CGRP1 approximately 88-fold less rapidly than it cleaved SP. The slow cleavage by NEP of CGRP compared to SP suggests that this enzyme is likely to have weaker physiologic effects on CGRP than have been demonstrated for SP. Enkephalinase CALLA CD10 Neutralmetalloendopeptidase Peptidases Enzymekinetics HPLC

CALCITONIN gene-related peptide (CGRP), a peptide of 37 amino acids, is widely distributed in sensory neurons, including those of the lungs of guinea pigs and humans (29,34). There is evidence that CGRP is colocalized with substance P in some capsaicin-sensitive sensory nerves (13,25). Calcitonin gene-related peptide is a potent vasodilator (5), and it potentiates tachykinin-induced increases in vascular permeability in the skin (4) and in the airway (15). Some investigators have reported that it also constricts airway smooth muscle of humans (29), but others have not confirmed these observations (27). In the digestive tract CGRP is a potent inhibitor of gastric and pancreatic secretion (17) and is a stimulator of somatostatin release (7). Although there is one report of CGRP degradation in the cerebrospinal fluid (23), little is known about its metabolism in the periphery. The only published study so far demonstrated that CGRP was cleaved effectively by human lung mast cell tryptase (36). Many target cells for CGRP and other peptides contain a ceU surface enzyme, neutral endopeptidase (NEP, also known as neutral metalloendopeptidase, enkephalinase, or E.C. 3.4.24.11). It is present in airway smooth muscle, epithelium, submucosa, and nerves (3,31). It is also present in skin (19), kidney (20), brain (24,26), gastrointestinal tract (6,8) and lung (3,24). Previous studies have demonstrated that NEP can cleave a variety of peptides including tachykinins such as substance P (28,32), neurokinin A (18), as well as the beta chain of insulin (20), opioids (28), kinins (14,28), neurotensin (32) and other peptides (14, 28, 30, 37). In the present studies, we examined whether NEP cleaves

E.C.3.4.24.11

Peptidaseinhibitors

CGRP. We determined the kinetics and sites of cleavage of CGRP 1 by recombinant human NEP, and we found that NEP cleaved CGRP~ at several locations. However, the kinetic results suggested a weak substrate enzyme interaction. We also confirmed that NEP cleaved SP similarly to other preparations of NEP purified from the kidney. METHOD

Materials Human calcitonin gene-related peptide, phosphoramidon, and SP were purchased from Peninsula Laboratories (Belmont, CA). Recombinant human NEP was provided by B. Bridenbaugh of Genentech Inc. (South San Francisco, CA). The enzyme was produced in 293 cells (16), a permanent line of kidney-derived cells. The NEP was purified from these cells using modifications of previously published methods (24). The enzymatic activity was measured under initial velocity conditions (less than 10% substrate degradation) using published methods (24) and expressed in ng, where 0.1 ng cleaves 4.0% of the substrate [3H-Tyr,D-Ala2]leucine enkephalin at 37°C in 30 min (22). Leucine-thiorphan was a gift of Squibb Pharmaceuticals (Princeton, NJ), and it was dissolved in 100% ethanol and diluted in buffer prior to use. The buffer used consisted of 5 mM Hepes and 147.5 mM NaCI at pH 7.40.

Experimental Methods HPLC. Degradation products of CGRP and SP were sepa-

1Requests for reprints should be addressed to D. B. Borson, Ph.D., Cardiovascular Research Institute, Box 0911, University of California, San Francisco, CA 94143-0911.

563

564

KATAYAMA ET AL.

rated on a Vydac C-18 reverse-phase column using a Waters 540 pump system. Products were eluted using a linear gradient (1 or 2% min) of acetonitrile in water each containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 rnl/min. Peaks were detected by their absorbance at 214 nm and were quantified by integration. Degradation of peptides. In the first study, to determine whether NEP cleaves CGRP] and the time course of its cleavage, 2 nmole (10 -5 M) CGRP~ was incubated with 40 ng NEP in 5 mM Hepes buffer pH 7.4 in 100 Ixl total volume at 37°C. Incubation times were 15', 30', 60' and 120'. After stopping the reaction by boiling, 400 I~1 0.1% TFA in H20 was added for analysis by HPLC. A parallel sample was incubated without NEP and served as a control for calculations of the percent degradation. Then, to determine the kinetic parameters of cleavage, we used 10 -6 M to 3.3 × 10 -4 M CGRP] incubated in duplicate with 40 ng of NEP in a volume of 40 txl for 30 min. The reaction was stopped by adding 20 ~1 of 2 N HC1. Parallel tubes containing each concentration of CGRP] incubated without NEP served as controls for calculations of reaction velocities. We compared these results with those obtained using a wellstudied substrate, SP. We determined the kinetics of cleavage of SP by recombinant human NEP by incubating 2.0 ng of NEP with concentrations of SP from 4 x 10 -6 M to 10 -3 M for 30 min in a volume of 100 ill. The reaction was then stopped by adding 50 Ixl 2 N HC1. Parallel samples were incubated in the absence of NEP for calculations of reaction velocities. To show that the cleavage of the peptide was due to the enzymatic properties of NEP, we incubated parallel samples in the presence of leucine-thiorphan (10 -6 M). To determine the sites of cleavage of CGRP 1, 20 nmoles of peptide (10 - 4 M) was incubated with 200 ng NEP for 1 h in a volume of 100 I~1. The products were eluted in a l%/min gradient of acetonitrile, collected in 1.5 ml Eppendorf tubes, dried in a Speed-Vac, and the amino acid compositions of the recovered peptides were determined using a Beckman 6300 analyzer by comparison to authentic amino acid standards (33). To show that the cleavage of CGRP was due to enzymatic properties of NEP, we incubated 10 -5 M peptide with 40 ng NEP in the presence of 10 -6 M leucine-thiorphan in a volume of 100 Ixl for 1 h to prevent the action of NEP. RESULTS Recombinant human neutral endopeptidase cleaved CGRP L in a time-dependent fashion, with 80% of the peptide being degraded by 2 h (Fig. 1A). For determination of kinetic parameters, incubating different concentrations of CGRP for 30 rain with 40 ng of NEP resulted in degradation of CGRP1 which followed Michaelis-Menten kinetics. Transformation of the data using the Lineweaver-Burk method (Fig. 1B) resulted in a linear plot (r2 =0.944) from which first order kinetic parameters were determined. The values of Km and Kcat were 105 IxM and 56/ min, respectively, and the Kcat/Km ratio was 0.53. Moreover, neutral endopeptidase cleaved substance P with linear Lineweaver-Burk kinetics with a Km of 15 IzM and a Kcat of 4654/ min (Fig. 2). Leucine-thiorphan (10 -6 M) prevented the degradation of SP by NEP (data not shown). Thus the kinetic parameters of cleavage of SP by recombinant human NEP were similar to those of previous studies (28) using NEP purified from the kidney. Neutral endopeptidase cleaved CGRP, and generated 4 major product peaks (Fig. 3A). Leucine-thiorphan (10 -6 M) prevented CGRP, degradation of NEP (Fig. 3B). Amino acid analyses of the recovered metabolites showed, in most cases, integral numbers of identified residues (Table 1), enabling the most probable

100

%

Breakdown

J

50

2"

1/V 1" (min/prnole)

,

f

r

J

i

30

60

90

120

TIME

(min)

150

~~

m in M 0.53

-I

-0.01

0.020.040.060.08 1/[S]

0.1

0.12

(L/~mole)

FIG. 1. Kinetics of calcitonin gene-related peptide degradation by recombinant human NEP at 37°C. (A) Time course of degradation of 2 nmole (10 -5 M) CGRP~ by 40 ng NEP. NEP cleaved CGRP, in a timedependent fashion, with 80% degradation by 2 hours. (B) LineweaverBurk plot of the kinetics of CGRP1 degradation by NEP obtained using 40 ng NEP and 30-min incubationperiods.

identification of the metabolites (Fig. 4). The most probable sites of cleavage were between amino acid positions 11-12, 14-15 or 15-16, and 27-28. Furthermore, with the exception of the N-terminal fragment of CGRP~, each metabolite contained a hydrophobic amino acid at its N-terminus. DISCUSSION

We conclude that NEP cleaves CGRP~ at several sites. This conclusion is based on kinetics and studies of recovered metabolites. The finding that leucine-thiorphan prevented degradation suggested that the cleavage of CGRP~ was due to the enzymatic properties of NEP. The identification of the sites of cleavage is based on the amino acid analyses of collected peaks after cleavage by NEP. The identification of the peaks shown in Fig. 3 (top) is likely to be correct. Peak 1 is identified on the basis of single alanine, glycine, and leucine residues. The amino-terminus of this peak is identified by the lack of arginine or histidine. The carboxyterminus is likely to be residue 14 because only a single leucine

4 3(min/nmole) 21/V

i -0.1

.2

0.0

~



.

i

, 0.1

1/[S]

.

4654/min 5 ~M = 310 .

,



.

0.2

j 0.3

(L/mole)

FIG. 2. Kinetics of substance P degradation by NEP at 37°C. Lineweaver-Burk plot of data obtained using 2.0 ng NEP and 30-min incubation periods.

NEUTRAL ENDOPEPTIDASE, CGRP AND SP

565

Peak Number

2

1

Amino acid position: t

4

11l~--t,

3

16

2r ~

a7

ACNTATCVTHRLAGLLSRSGGWKNNFVPTNVGSKAF-NH2

f

¢ ,0

¢

I t

A214

FIG. 4. Structure of CGRP~ and the probable sites of its cleavage by NEP. Amino acid analysis (Table 1) suggested strongly the identities of the peaks. i

I 40

2O

'

I 6O

r

TIME (rain)

spite of some differences in the expected and observed amino acid composition of the peaks. In peak 2, 3 threonine residues were expected and 2.4 were observed. This difference, however, does not bring into question the identity of the peak. The presence of the expected single histidine and arginine residues and no leucine residue identify the carboxy-terminus of the fragment. The presence of the single alanine and asparagine residues identify the amino-terminus. Similarly, in peak 3, the difference between the one expected and 1.4 observed glycine residues does not bring into question the identity of the peak. Because this peak contains the only proline, only two (1.86) valines, only 1 phenylalanine, and contains only a single asparagine, this peak does not contain any portion of the peptide on the amino-terminal side of position 28. Because of the presence of single alanine, serine, and asparagine residues, the peptide contains the carboxy-terminal region of CGRP. Thus the single phenylalanine observed is most likely to be the one at position 37 and not the one at position 27. Likewise, in peak 4, the differences between expected and observed arginine and serine residues does not bring the identity of this peak into question. Because this peak contains single phenylalanine and lysine residues, 2 (1.71) asparagine residues, and only 2 (1.93) valine residues, the carboxy-terminus of this fragment is at position 27. Because this peak contains only 1 leucine residue, the amino-terminus is most likely to be at position 16. Finally, in peak 5, the difference in the expected and observed numbers of valine residues does not bring the identity of the peak into question. All of the other amino acids were recovered in their expected ratios, and because this peak comigrates with intact CGRP (Fig. 3 bottom), the identity of the peak is not in question.

A 2t4 ~

20

40 TIME

60

(min)

FIG. 3. Degradation of CGRP 1 by NEP. (A) Chromatogram of CGRPj and its metabolites generated by NEP. Five major, well-separated peaks were collected for amino acid analysis, with peak 5 representing the intact CGRP molecule. (B) Leucine-thiorphan (10 - 6 M) prevented the degradation of CGRP l (10 -s M) by 40 ng NEP and resulted in the appearance of no product peaks.

residue was recovered. The exact site of the cleavage generating the N-terminus of peak 4 is uncertain because of the absence of a second leucine residue in either peak 1 or peak 4. However, it is likely that the leucine residue in peak 1 represents Leu t2, because peak 2 contains no leucine, and thus a cleavage at the Leul2-Ala 13 bond is unlikely. The identification of the other peaks is likely to be correct in

TABLE 1 AMINO ACID ANALYSISOF PEPTIDESOF CGRP GENERATEDBY RECOMBINANTHUMANNEUTRALENDOPEPTIDASE Peak: Amino Acid A G L N T V H R P S K F

#1

#2

#3

#4

#5

Exp

Obs

Exp

Obs

Exp

Obs

Exp

Obs

Exp

Obs

1 1 1 0 0 0 0 0 0 0 0 0

1.0 1.09 0.96 0 0 0 0 0 0 0 0 0

2 0 0 1 3 1 1 1 0 0 0 0

1.88 0 0 1.24 2.43 1.12 1.0 1.04 0 0 0 0

1 1 0 1 1 2 0 0 1 1 1 1

1.10 1.37 0 1.19 0.97 1.86 0 0 1.0 0.98 1.15 0.98

0 2 1 2 0 2 0 1 0 2 1 1

0 1.97 1.06 1.71 0 1.93 0 0.43 0 1.22 1.09 1.0

4 4 3 4 4 5 1 2 1 3 2 2

3.89 4.26 3.04 4.05 3.44 3.60 1.00 1.98 1.00 2.39 1.98 1.94

Exp: No. of residues expected. Obs: No. of residues observed.

566

KATAYAMA ET AL.

Although CGRP 1 is cleaved, the kinetic parameters of the process are characteristic of a weak substrate-enzyme interaction. The cleavage of CGRP by human lung mast cell tryptase is different in that tryptase cleaves the peptide at two sites, between Argla-Ser19 and between Lys24-Asn25 (36). Furthermore, tryptase cleaves CGRP with different kinetics; the Kcat is 1116/min and the Km is 69 IxM (36). By comparison with CGRP1, NEP can cleave many other peptides more efficiently. Most of the differences between the kinetics of degradation of different peptides by NEP are in the Kcat values, because the Kms for most peptides are in the 10-100 ~M range. For example, the Kcats of the tachykinins SP and NKA, kinins and enkephalins are several thousand per min, and the Kcat for neurotensin is 1280/min (28). Our results with recombinant human NEP confirm previous studies using NEP purified from the kidney (28). Thus the kinetic parameters of the cleavage of SP by recombinant human NEP (Kcat = 4654/min, Km = 15 IxM) were similar to those obtained using kidney NEP (Kcat = 5088/min, Km = 32 txM) (28). The finding that the cleavage of CGRP results in metabolites whose N-termini consist of hydrophobic amino acids is also similar to the findings for different peptides (14,32). In the cerebrospinal fluid, CGRP 1 and SP are degraded by an endopeptidase that cleaves CGRP~ at the Leul6-Ser ~7 bond (23). Because the sites of cleavage are different for NEP, this enzyme is most likely not NEP. Of the two fragments resulting from this cleavage, CGRP(I-16) showed little or no binding affinity for CGRP receptors in the spinal cord, and the other, CGRP(17-37), interacted only weakly with these binding sites (23). These results suggest that the biological effects of CGRP~ are likely to be decreased by cleavage at this site. Because NEP cleaves CGRP1 generating even shorter fragments, these results suggest that the cleavage of CGRP~ by NEP would also result in the production of fragments with decreased binding affinity for CGRP receptors and therefore decreased biological activity compared to the intact molecule. Although the cleavage of CGRP 1 is slow, endogenous NEP might possibly modulate some of the physiological or pathophysiological effects of CGRP. This may be so if the NEP is close

to the sites of release or action of CGRP. This idea is possible because CGRP and SP colocalize in certain sensory nerves (13) and NEP appears to modulate many of the effects of SP released from these nerves. Additionally, NEP appears to modulate the effects of other peptides. For example, inhibitors of NEP potentiate the effects of endogenously released tachykinins on bronchoconstriction (10), cholinergic neurotransmission (31), cough (21), vascular permeability (38), and neutrophil adhesion (38). Neutral endopeptidase inhibitors also potentiate SP-induced mucus secretion in airways (2), vascular permeability responses in the skin (19) and airways (1), and contractile responses in the airways (31,35) and the gastrointestinal tract (11). Inhibitors of NEP also potentiate the effects of kinins (12) and neurotensin (9) on airway smooth muscle contraction. However, if NEP does modulate the effects of CGRP, the modulation is likely to be less substantial than for more rapidly cleaved substrates such as SP, the kinins, or neurotensin. Because NEP is present in the skin where it modulates vascular permeability responses to SP (19), and because CGRP is a potent vasodilator in this tissue (5), it is possible that the vascular effects of CGRP could be modulated by endogenous NEP. Furthermore, NEP is also present in the endothelium of arteries (37). It is possible that systemic vascular effects of CGRP are also modulated by NEP. Finally, because NEP is present in the gastrointestinal tract (6,8), it is possible that the effects of CGRP in this organ system are also modulated by NEP. However, because the cleavage of CGRP by NEP is relatively slow, it is also possible that other enzymes may modulate the actions of CGRP. In summary, the degradation of endogenous peptides by NEP may play significant roles in the modulation of their physiological effects. Thus NEP appears to play roles in modulating peptide-induced responses analogous to those of acetylcholinesterase in modulating the cholinergic nervous system. ACKNOWLEDGEMENTS The authors thank B. Raymond and I. F. Ueki for technical assistance. Study supported in part by HL-38947, HL-24136, and DK-39957.

REFERENCES 1. Borson, D. B.; Brokaw, J. J.; Sekizawa, K.; McDonald, D. M.; Nadel, J. A. Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections. J. Appl. Physiol. 66:2653-2658; 1989. 2. Borson, D. B.; Corrales, R.; Varsano, S.; Gold, M.; Viro, N.; Caughey, G.; Ramachandran, J,; Nadel, J. A. Enkephalinase inhibitors potentiate substance P-induced secretion of 35SO4-macromolecules from ferret trachea. Exp. Lung Res. 12:21-36; 1987. 3. Borson, D. B.; Malfroy, B.; Gold, M.; Ramachandran, J.; Nadel, J. A. Tachykinins inhibit enkephalinase activity from tracheas and lungs of ferrets. Physiologist 29:174; 1986. 4. Brain, S. D.; Williams, T. J. Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin. Br. J. Pharmacol. 97:77-82; 1989. 5. Brain, S. D.; Williams, T. J.; Tippins, J. R.; Morris, H. R.; MacIntyre, I. Calcitonin gene-related peptide is a potent vasodilator. Nature 313:54-56; 1985. 6. Bunnett, N. W.; Debas, H. T.; Turner, A. J.; Kobayashi, R,; Walsh, J. H. Metabolism of gastrin and cholecystokinin by endopeptidase 24.11 from the pig stomach. Am. J. Physiol. 255:G676G684; 1988. 7. Bunnett, N. W.; Helton, W. S.; Debas, H. T.; Ensinck, J. W. CGRP stimulates the release of pro-somatostatin-derived peptides from gastric fundus. Am. J. Physiol. 258:G316-319; 1990. 8. Bunnett, N. W.; Turner, A. J.; Hryszko, J.; Kobayashi, R.; Walsh, J. H. Isolation of endopeptidase-24.11 [EC 3.4.24.11, "enkephali-

nase"] from the pig stomach. Gastroenterology 95:952-957; 1988. 9. Djokic, T. D.; Dusser, D. J.; Borson, D. B.; Nadel, J. A. Neutral endopeptidase modulates neurotensin-induced airway contraction, J. Appl. Physiol. 66:2338-2343; 1989. 10. Djokic, T. D.; Nadel, J. A.; Dusser, D. J.; Sekizawa, K.; Graf, P. D.; Borson, D. B. Inhibitors of neutral endopeptidase potentiate electrically and capsaicin-induced noncholinergic contraction in guinea pig bronchi. J. Pharmacol. Exp. Ther. 248:7-11; 1989 11. Djokic, T. D.; Sekizawa, K.; Borson, D. B.; Nadel, J. A. Neutral endopeptidase inhibitors potentiate substance P-induced contraction in gut smooth muscle. Am. J. Physiol. 256:(Gastrointest. Liver Physiol. 19):G39-G43; 1989. 12. Dusser, D. J.; Nadel, J. A.; Sekizawa, K.; Graf, P. D.; Borson, D. B. Neutral endopeptidase and angiotensin converting enzyme inhibitors potentiate kinin-induced contraction of ferret trachea. J. Pharmacol. Exp. Ther. 244:531-536; 1988. 13. Franco-Cereceda, A.; Henke, H.; Lundberg, J. M.; Petermann, J. B.; Hokfelt, T.; Fischer, J. A. CGRP in capsaicin-sensitive substance P immunoreactive sensory neurons in animals and man. Peptides 8:399-410; 1987. 14. Gafford, J. T.; Skidgel, R. A.; Erdos, E. G.; Hersh, L. B. Human kidney "enkephalinase," a neutral metalloendopeptidase that cleaves active peptides. Biochemistry 22:3265-3271; 1983. 15. Gamse, R.; Saria, A. Potentiation of tachykinin-induced plasma protein extravasation by CGRP. Eur. J. Pharmacol. 114:61-66; 1985. 16. Gorman, C. M.; Gies, D.; Schofield, P. R.; Kado-Fong, H.; Mal-

NEUTRAL ENDOPEPTIDASE, CGRP AND SP

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25.

26. 27.

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Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase.

Calcitonin gene-related peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidas...
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