Further characterization of the substrate specificity of a TRH hydrolysing pyroglutamate aminopeptidase from guinea-pig brain

Neuropeptides (1990) 15,31-36 0 Longman Group UK Ltd 1990

Further Characterization of the Substrate of a TRH Hydrolysing Pyroglutamate Aminopeptidase from Guinea-Pig Brain M. A. ELMORE”,

E. C. GRIFFlTHSt,

B. O’CONNOR*

Specificity

and G. O’CUINNS

*Department of Biochemistry, University College, Galway, Ireland, tDepartment of Physiological Sciences, University of Manchester, Manchester, Ml3 9PT, &tired Kingdom and #Department of Life Sciences, Regional Technical College, Galway, Ireland (Reprint requests to GOCI

Abstract-In this study the substrate specificity of a pyroglutamate aminopeptidase from synaptosomal membranes of guinea-pig brain was investigated. The enzyme was found to be specific for tripeptides, tripeptide-amides and tetrapeptides which possess the N-terminal sequence Glp-His and as such is specific for Thyrotropin Releasing Hormone or only very closely related peptides. The enzyme was found not to hydrolyse a number of analogues of Thyrotropin Releasing Hormone which have been shown to have therapeutical value in certain neuronal disorders.

range of amino acids in the penultimate position from the N-terminus. Synaptosomal membranes from brain of guinea-pig (6) rat (7, 8) and rabbit (9) also contain a pyroglutamate aminopeptidase [E.C.3.4.19-] whose high molecular weight (231000) and sensitivity to EDTA clearly differentiate this activity from the soluble activity. Preliminary characterization of the substrate specificity of the synaptosomal membrane enzyme indicated an even more restricted substrate specificity than that of the soluble enzyme. The synaptosomal membrane pyroglutamate aminopeptidase while able to remove N-terminal pyroglutamate from Thyrotropin Releasing Hormone (TRH, Glp-HisProNH*) was, unlike the soluble enzyme, unable to remove N-terminal pyroglutamate from a wide range of peptides including LH-RH, neurotensin

Introduction A feature of peptide hydrolases is their broad substrate specificities and where a restriction in substrate specificity occurs there is generally a requirement for a particular amino acid or for one of a group of amino acids on one side or other of the sisile bond. The cytoplasm of guinea-pig brain (1) in common with the cytoplasm of a number of &her tissues (2-5) contains such an enzyme, a low molecular weight pyroglutamate aminopeptidase [E.C.3.4.19.3] whose substrate specificity is restricted to peptides of varying length containing N-terminal pyroglutamate yet containing a wide

Date received 10 May 1989 Date accepted 26 July 1989

31

32 and bombesin (10). These results suggest that the substrate specificity of the synaptosomal membrane pyroglutamate aminopeptidase may be restricted to TRH and closely related peptides. Moreover this enzyme with its synaptosomal location and uniquely restricted substrate specificity represents a possible inactivation mechanism for TRH, which is proposed to play a neurotransmitter (11) and a neuromodulator (12) role within the central nervous system.

Materials and Methods Materials

TRIS, Glp-His-Gly, TRH, pyroglutamate aminopeptidase (bovine liver) and papain (papaya latex type III) were obtained from Sigma Chemical Co. (Poole, Dorset, England), Sephadex G200 and Phenyl-Sepharose CL4B were obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden) Cellulose phosphate P81 chromatography paper was purchased from Whatman (Maidstone, Kent, England) while glycerol was obtained from B.D.H. (Poole, Dorset, England). Glp-His-Pro, Glp-Asn-Gly, Glp-Ala-Glu and Glp-His-3’,4’ dehydro ProNHz were from Bachem Feinchemikalien (Bubendorf, Switzerland), Glp-His and GIp-His-Trp were obtained from U.C.B. Bioproducts (Brussels, Belgium) while all other peptides were from Peninsula Laboratories, Merseyside, England. [Pro-2’,3’,4’,5’-3H] TRH (126.9 Ci/ mMo1) was purchased from New England Nuclear. Orotyl-His-ProNHt (CG 3509) and 6methyl-5-oxo-thio-morpholinyl-3-carbonyl-HisProNH;? (CG 3703) were gifts from Prof. Dr. H. Giertz, Prof. Dr. L. Flohe and Dr. E. Frankus of Grunenthal GmBH, Stolberg, West Germany, Glp-His-[3-methyl]-ProNHz (RX 74355) and GlpHis-[3,3’ dimethyl] ProNH2 (RX 77368) was a gift from Dr. Ian Tulloch, Reckitt and Colman, Kingston-upon-Hull, England, L-pyro-2-aminoadipylHis-thiazolidine-4-carboxide (MK 771) was a gift from Dr. Martin Hichins and Dr. Ruth Nutt, Merck, Sharp and Dohme, West Point, Pa., L-pyro-2-aminoadipyl-Leu-ProNH2 U.S.A., (RGH 2202) was a gift from Dr. T. Szirtes, G. Richter Ltd., Budapest, Hungary, Glp-His-Trp-

NEUROPEPTiDES

Ser-Tyr (LH-RH& was kindly provided by Prof. R. F. Millar, University of Cape Town, South Africa. Purification of synaptosomal membranepyroglutamate aminopeptidase

Synaptosomal membranes were prepared as described before (6) and pyroglutamate aminopeptidase was solubilized by papain treatment and purified by a scheme which involved gel filtration on Sephaex G200 and chromatography on calcium phosphate-cellulose and on Phenyl Sepharose CL-4B as previously reported (10). The active fractions from Phenyl-Sepharose were pooled and stored at -20°C in small aliquots until further use. Pyroglutamate aminopeptidase was assayed by a modification of the method of Bauer and Kleinkauf (2) using [Pro-3H] TRH as substrate. [Pro-3H] TRH was diluted with unlabelled TRH to give a specific activity of 125 mCi/mMole. 10 l~,lof each sample to be assayed was incubated with 10 l.~l of diluted [Pro-3H] TRH. The reaction was terminated after 30 min by the addition of 10 ul of 1 M Acetic Acid and 10~1 aliquots were spotted onto prewashed cellulose acetate P81 paper which was developed using 1M acetic acid. The start segments containing the highly basic His-ProNHz were cut out and placed into scintillation vials to which were added 1 ml of 2M ammonia and 10 min later 10ml of toluene based scintillation cocktail containing 33% Triton X-100. The degradation of TRH could be measured by counting the vials in a scintillation spectrometer. Substrate specificity

The substrate specificity of the synaptosomal membrane pyroglutamate aminopeptidase was tested by incubation of 50 ~1 of the enzyme activity recovered from calcium phosphate cellulose chromatography with 50 ~1 of a 3 mM solution of each peptide or analogue to be tested. Parallel incubations were set up involving 50 l~lof a suspension of commercial soluble calf liver pyroglutamate aminopeptidase in 1OOmM potassium phosphate pH 7.5 containing 2mM EDTA, 2mM dithiothreitol and 5 mM Bacitracin to inhibit contaminating post proline cleaving endopeptidase. After 16h incubation at 37°C aliquots of each

SUBSTRATE SPECIFICITY OF A TRH HYDROLYSING

PYROGLUTAMATE

incubate were spotted onto thin layer plates precoated with Silica gel 60 along with standards representing degradation products where available. The plates were developed in a saturated using chloroform/methanoY30% atmosphere ammonia (60:26:5) as solvent. Peptides and degradation products were visualized using the Pauly stain and by staining with Ninhydrin. Alternatively 50~1 aliquots of the enzyme recovered from Phenyl-Sepharose were incubated with 5Ol.~lof a 3mM solution of peptide or analogue; enzyme activity was terminated by the addition of 10~1 of trifluoroacetic acid (TFA; BDH). After centrifugation at 1OOOghigh-performance liquid chromatography (HPLC) separation of TRH and analogues from their metabolites was carried out using a Varian 5560 UV200 Vista 402 system. HPLC Samples were applied to a reverse-phase column (25cm x 4.6mm i.d.) packed with Whatman Partisil 5/ODS-3 (HPLC Technology Ltd.) protected by a Whatman CSKI guard column. Elution was via two independent gradients at a flow rate of lml/min, with eluent monitored at 206mM. Gradient 1 for TRH and metabolites involved the delivery of a linear gradient of O-50% B over 15 min (Solvent A = 0.08% triflouroacetic acid, TFA; Solvent B = 40% CH3 CN/O.O8% TFA). Gradient 2 for TRH analogues and metabolites was for O-SO% B over 15 min (Solvent A = 0.08% TFA; Solvent B = 70% CH3 CN/O.OB% TFA). Peptides eluted were identified by retention time against known standards and by amino acid analysis (13). HPLC solvents were obtained from Rathburn Chemicals, Peebles, Scotland. Kinetic studies A Stock dilution of [Pro-3H] TRH (0.5 l&i/ 4 nMole/lO ~1) was further diluted to give a range of thyroliberin concentrations for use in kinetic studies. 10 ~1 aliquots of each dilution was added in triplicate to 20~1 aliquots of 50mM potassium phosphate pH 7.5 containing peptide inhibitor or analogue (0.25mM or 0.5mM). In each case the reaction was initiated by addition of 10~1 of the purified post-Phenyl-Sepharose chromatography pyroglutamate aminopeptidase. Separate blanks

AMINOPEI’TIDASE

33

were used for each concentration of [Pro-3H] TRH. The reactions were terminated after 30 min and the conversion of [Pro-3H] TRH was measured as described above. Ki values for each peptide and analogue were derived from Lineweaver-Burk plots.

Results and Discussion

Recent reviews of neuropeptide metabolism have suggested that a limited number of peptide hydrolases located in synaptosomal membranes are capable of degrading a wide variety of neuroactive peptides such as Leu-enkephalin, Met-enkephalin, substance P, bradykinin, neurotensin, luliberin and cholecystokinin (14, 15). These peptide hydrolases are of broad specificity and each peptide hydrolase is capable of hydrolying several neuropeptides while many neuropeptides are capable of being hydrolysed by more than one synaptosomal membrane peptide hydrolase. In the case of TRH only one activity in synaptosomal membranes appears to be capable of introducing a primary cleavage into the molecule. This enzyme had been identified as a pyroglutamate aminopeptidase in synaptosomal membranes of rat brain (7, 8) and of guinea-pig brain (6, 10). Preliminary results on the substrate specificity of this enzyme suggested a more restricted specificity (10) than that obtained with other neuropeptide inactivating peptide hydrolases. The results presented in Table 1 show that only Glp-His-Pro, Glp-His-ProNHz (see Figure), GlpHis-Pro-Gly and Glp-His-Trp of the substrates tested were hydrolysed by the synaptosomal membrane pyroglutamate aminopeptidase. Shortening of Glp-His-Pro by removal of carboxy-terminal proline or lengthening the peptide by addition of GlyNhz or Gly-Lys to the carboxy terminus abolished the ability of the peptide to act as a substrate for the enzyme. No enzyme activity was detected if the Glp residue in position 1 of the TRH sequence was replaced by Glu or if the His residue in position 2 was replaced by either Phe or NVal. as had been previously reported for rabbit brain particulate pyroglutamate aminopeptidase (9). Enzyme activity was conserved by replacement of the carboxy terminal ProNHz of TRH by Trp but

34

NEUROPEPTIDES

Table 1

the peptide substrate. These findings suggest that the enzyme is specific for tripeptides, tripeptideamides and tetrapeptides with an absolute requireGlp-His in the ment for the sequence amino-terminus. Some tolerance for the substitution of carboxy terminal prolineamide by alternative amino acids is indicated by the observation that Trp in this position permitted enzyme activity. However extending the substrate Glp-His-Trp (LH-RHr.3) to Glp-His-Trp-Ser-Tyr (LH-RHr_s) abolished activity. This restricted substrate specificity represents a significant departure from specificities previously reported for peptidases. The substrate specificity of this enzyme raises the possibility that this enzyme represents the inactivating mechanism for neurotransmitter TRH within brain. Further support for this possibility is provided by the recent finding that levels of particulate pyroglutamate aminopeptidase are by far the highest in rat brain of the organs tested in this species (16) and by the observation that pyroglutamate aminopeptidase is sited as an ectoenzyme in rat brain synaptosomal membranes before this enzyme can be (17). However, accepted as the inactivating activity for TRH in the CNS further work is necessary to satisfy criteria such as those previously proposed by Schwartz et al. (18) for neuropeptide inactivating peptidases. Recently attention has been turned to the possibility of using TRH or its analogues in the clinical treatment of a number of neuronal disorders (19,

The effects of substrate length and of substitution in the amino acid sequence of TRH on the activity of synaptosomal membrane pyroglutamate aminopeptidase

Peptide

Whether hydrolysed

Ki (mM)

-

4.5 0.400 0.050 0.125 0.055 0.166 0.610 0.125 0.013 0.170 N.D. 1.650 0.330 0.750 N.D.

Glp-His Glp-His-Pro Glp-His-Pro NH2 Glp-His-Pro-Gly Glp-His-Pro-Gly NH2 Glp-His-Pro-Gly-Lys Glu-His-Pro NH2 Glp-Phe-Pro NH2 Glp-NVal-Pro NH2 Glp-His-Trp Glp-His-Trp-Ser-Tyr Glp-His-Gly Glp-His-Gly NH2 Glp-Asn-Gly Glp-Ala-Glu N.D. t

+ + + + -

Hydrolysis detected by

a a, a, a, a a, a a, a, a, b a, a a a

b b b b b b b b

= not done. = Thin layer chromatography. = High performance liquid chromatography.

not by Gly or GlyNHz. All peptides employed except Glp-His inhibited the enzyme in a competitive manner and it is interesting to note that Glp-His-Pro, Glp-His-Gly and Glp-His-Pro-Gly each displayed higher Ki values than the corresponding amides suggesting that the enzyme prefers an amide group at the carboxy terminus of

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0 I

3

3

5

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1

!

I

0

5

n

15 5

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I

t

15

0

5

limdmin)

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10

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Ti rdmtn)

Figure HPLC separation of peptides after incubation of purified synaptosomal membrane pyroglutamate aminopeptidase II with TRH and CG 3703; Trace A = TRH control sample, enzyme pre-treated with 10 ul TPA; Trace B = TRH + intact enzyme; Trace C = CG 3703 control sample, enzyme pre-treated with 1Oul TPA; Trace D = CG 3703 + intact enzyme. A206 = ultraviolet absorbance at 206mM. Peak 1 = phosphate buffer; Peak 2 = TRH; Peak 3 = endogenous enzyme peak; Peak 4 = Cycle His-Pro; Peak 5 = CG 3703.

SUBSTRATE SPECIFICITY OF A TRH HYDROLYSING

PYROGLUTAMATE

Table 2

Activity of sunaptosomal membrane pyroglutamate aminopeptidase towards analogues of TRH Whether hydrolysed

( m‘h)

CG 3509 CG 3703 RX 74355 RX 77368 MK 771

_ _ -

0.609 0.116 N.D. 0.410 0.044

RGH 2202 Glp-His-3’-4’ dehydro ProNH2 Glp-3 methyl HisProNHz

+

N.D. 1.91

0.500

a. b

Thyroliberin Analogue

Hydrolysis detected by a, b a, b b a, b

a. b b a, b

AMINOPEFTIDASE

35

membrane pyroglutamate aminopeptidase is specific for tripeptides, tripepotideamides and possibly tetrapeptides which commence with the N-terminal sequence Glp-His.

Acknowledgements GO’C acknowledges the receipt of a grant-in-aid from the Health Research Board, Dublin and ECG acknowledges financial support from Proteus Biotechnology Ltd.

References 1. Browne, P. and O’Cuinn, G. (1983). An evaluation of the

N.D. a b

= not done. = Thin layer chromatography. = High performance liquid chromatography.

20) and this has prompted

the investigation of the interaction of synaptosomal me,mbrane pyroglutamate aminopeptidase with a number of TRH analogues. Modifications to the three residues of the TRH structure (Glp-His-ProNH2) have resulted in the production of several analogues. At least two of these, RX 77368 and CG 3703, have already shown considerable potential in the treatment of motorneurone disease and in an experimental model of spinal injury (19, 20). Of the analogues tested (Table 2) none showed any significant inactivation by the purified enzyme indicating a considerable degree of stability to degradation (see Figure). Apart from MK 771, CG 3509 and CG 3703, this has also proved to be the case for cleavage by the soluble proline endopeptidase (13). The enhanced biological activity of these analogues may result from their stability to degradation but it may also reflect their affinity for TRH receptors and certain conformational preferences in their structures (20). From the information obtained, it may be possible to design novel TRH analogues with increased resistance to enzymic inactivation as well as to produce specific enzyme inhibitors for the particulate pyroglutamate aminopeptidase, which may find similar uses to those already suggested for the enkephalinase inhibitors (21). Of the eight analogues presented in Table 2 only Glp-His-3’4’ dehydro ProNH2, which leaves Glp-His unmodified, was hydrolysed supporting the suggestion that the synaptosomal

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

role of a pyroglutamyl peptidase, a post proline cleaving enzyme and a post proline dipeptidyl aminopeptidase. each purified from the soluble fraction of guinea-pig brain, in the degradation of thyrohberin in vitro. Eur. J. Biocheni. 137: 75-87. Bauer. K. and Kleinkauf, H. (1980). Catabolism of thyrohberin by rat adenohypophyseal tissue extract. Eur. J. Biochem. 106: 107-117. Doolittle. R. F. and Armentrout, R. W. (1968). Pyrolidonyl Peptidase: An enzyme for selective removal of pyrrolidone-carboxyflic acid residues from poiypeptides. Biochemistry 7: 516-521. Szewczuk, A. and Kwiatkowska, J. (1970). Pyrrolidonyl peptidase in animal, plant and human tissues: Occurrence and some properties of the enzyme. Eur. J. Biochem. 15: 92-96. Mudge, A. W. and Fellows, R. E. (1973). Bovine pituitary pyrrolidone carboxylyl peptidase. Endocrinology 93: 142% 1434. O’Connor, B. and O’Cuinn, G. (1984). Localisation of a narrow specificity thyroliberin-hydrolysing pyroglutamate aminopeptidase in synaptosomal membranes of guinea-pig brain. Eur. J. Biochem. 144: 271-278. Garat, B., Miranda, J., Chadi, J.-L. and Joseph-Bravo, P. (lY85). Presence of a membrane bound pyroglutamyl aminopeptidase degrading thyroliberin releasing hormone in rat brain. Neuropeptides 6: 27-40. Torres, H., Charh. J.-L., Gonzalez-Noriega, A., Vargas, M. A. and Joseph-Bravo, P. (1986). Subcellular distribution of the enzyme degrading thyrotropin releasing hormone and metabolites in rat brain. Neurochem. Int. 9: 103-110. Wilk, S. and Wilk, E. K. (1989). Pyroglutamyl peptidase II, a thyrotropin releasing hormone degrading enzyme: Purification and specificity studies of the rabbit brain enzyme. Neurochem. Int. (In press). O’Connor, B. and O’Cuinn, G. (1985). Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea-pig brain. Eur. J. Biochem. 150: 47-52. Guillemin, R. (1978). Peptidases in the brain: The new endocrinology of the neuron. Science 202: 390-402.

36 12. Kow, L.-M. and Pfaff, D. W. (1988). Neuromodulatory actions of peptides. Ann. Rev. Pharmacol. Toxicol. 28: 163-188. 13. Griffiths, E. C., Baris, C., Visser, T. J. and Klootwijk, W. (1985). Thyrotropin-releasing hormone inactivation by human postmortem brain. Regul. Pept. 10: 145-154. 14. Turner, A. J., Matsas, R. and Kenny, A. J. (1985). Are there Neuropeptide-specific peptidases? Biochem. Pharmacol. 34: 1347-1356. 15. Turner, A. J. (1986). Processing and metabolism of neuropeptides in Essays in Biochemistry. Vol. 22 ed. R.D. Marshal and K.F. Tipton. Academic Press, London, p. 69-119. 16. Friedman, T. C. and Wilk, S. (1986). Delineation of a particulate thyroliberin-releasing hormone-degrading enzyme in rat brain by the use of specific inhibitors of prolyl endo-peptidase and pyroglutamyl peptide hydrolase. J. Neurochem. 46: 1231-1239.

NEUROPEPTIDES

17 Charli, J. L., Cruz, C., Vargas, M.-A. and Joseph-Bravo, P. (1988). The narrow specificity pyroglutamate aminopeptidase degrading TRH in rat brain is an ectoenzyme. Neurochem. Int. 13: 237-242. 18. Schwartz, J.-C., Malfroy, B. and de la Baume, S. (1981). Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase (“enkephalinase”) as neuropeptidase. Life Sci. 29: 1715-1740. 19. Griffiths, E. C. (1986). Thyrotropin-releasing hormone. New applications in the clinic. Nature (London) 322: 212-213. 20. Griffiths, E. C. (1987). Clinical applications of thyrotropin releasing hormone. Clinical Science 73: 449-457. 21. Waksman, G., Bouboutou, R., Chaillet, P., Devin, _I., Couland, A., Hamel, E., Besselievre, R., Costentin, J., Fournie-Zaluski, M. C. and Roques, B. P. (1985). Kelatorphan: a full inhibitor of enkephalin degrading enzymes. Biochemical and pharmacological properties, regional distribution of enkephalinase in rat brain by use of a tritiated derivative. Neuropeptides 5: 529-532.