Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

Peptides,Vol. 11, pp. 613-617. ©PergamonPressplc, 1990.Printedin the U.S.A.

0196-9781/90$3.00 + .00

BRIEF COMMUNICATION

Isolation and Microsequence Analysis of a Novel Form of Neuromedin U From Guinea Pig Small Intestine R. MURPHY, *l C. A. TURNER,* J. B. FURNESS,* L. P A R K E R t AND A. G I R A U D t

*Department of Anatomy and Histology and Centre for Neuroscience, School of Medicine Flinders University, Bedford Park, S.A. 5042, Australia and "PDepartment of Medicine, University of Melbourne, Western Hospital Footscray, Victoria 3011, Australia Received 11 December 1989

MURPHY, R., C. A. TURNER, J. B. FURNESS, L. PARKERAND A. GIRAUD.Isolationand microsequenceanalysisof a novel form of neuromedin Ufrom guineapig small intestine. PEPTIDES 11(3) 613--617, 1990.--A multidimensionalchromatographic regimen has been used to isolate and purify a peptide showingimmunoreactivityfor neuromedinU from guinea pig small intestine. Microsequence Edman N-terminalanalysisand C-terminalanalysisby enzymaticdigestionshowed this peptide to be a nonapeptide with the followingsequence:H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2. The C-terminaloctapeptideof this sequenceis the same as porcine NMU-8, and the C-terminalheptapeptide is identicalto rat NMU(17-23). NeuromedinU

Neuropeptides

Isolation

S e q u e n c e Guineapig

NEUROMEDIN U-8 and neuromedin U-25 are two related neuropeptides that have been isolated and sequenced from porcine spinal cord (9). The octapeptide constitutes the C-terminal region of NMU-25, flanked by a dibasic sequence (Arg-Arg) and is thus likely to be derived from the larger peptide. Both peptides show uterine contractile activity and pressor effects (9,13), and NMU-8 enhances water and electrolyte secretion in the porcine intestine, but does not affect gut muscle contractility (3). The distribution of neuromedin U-like immunoreactivity (NMU-LI) has been mapped in rat brain and intestine (1, 2, 5, 8), and in the guinea pig small intestine (1,7), where it has been shown to be localized extensively in neurons and in some endocrine cells. The molecular form of NMU present in rat tissues has been shown to differ from the porcine peptides by chromatographic analyses (1,4), and has recently been isolated and sequenced from rat small intestine (10). The rat peptide is 23 amino acids long and, whilst the C-terminal heptapeptide is identical to the porcine peptide, the remainder of the molecule shares only limited sequence homology with the porcine peptide and the dibasic cleavage point is not present. NMU-LI has also been chromatographically characterized in

Intestine

extracts of human and guinea pig brain and intestine (1,4). NMU-LI from guinea pig brain contains two major components that have similar chromatographic behavior to the porcine peptides, but NMU-LI extracted from the intestine of this species is much more heterogeneous. The major intestinal component elutes in a similar position to porcine NMU-8, and a minor component coelutes with porcine NMU-25, but there are also other immunoreactive components which do not coelute with any known forms of neuromedin U. The present study was undertaken to isolate the major component of NMU-LI present in guinea pig small intestine and to determine its amino acid sequence. METHOD

General Carboxypeptidase Y was purchased from Boehringer (Australia), and dimethylaminonaphthalenesulphonyl chloride (dansyl chloride) was purchased from Sigma Chemical Co. (U.S.A.). All other chemicals were purchased from Ajax Chemicals (Australia)

1Requestsfor reprintsshouldbe addressedto Dr. Roger Murphy,Departmentof Anatomy,Hinders MedicalCentre, BedfordPark, S.A. 5042, Australia.

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FIG. 1. Successive steps in the chromatographic purification of neuromedin U-like immunoreactive material from guinea pig small intestine. Panel A shows the elution profile from the Sephadex G25F column. The abscissa shows elution volume in ml and the ordinate indicates NMU-LI in pmol equivalents of porcine NMU-8. The void volume (Vo) and total volume (Vt) for the column are indicated by the arrows. Panels B to F show the HPLC purification of the lower molecular weight material isolated from the Sephadex column. The abscissa shows retention time in minutes, the left hand ordinate and the solid trace show UV-absorbance at 215 nm, and the right hand ordinate and dashed line indicate the elution gradient. The elution position of NMU-LI is indicated by the black bar below the absorbance trace. Columns used were: (B) analytical C18 column; (C) analytical C8 column; (D) narrow-bore C18 column; (E) cation exchange column; (F) narrow-bore C18 column. The chromatographic details for each step are described in detail in the text.

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ments, Australia (Spherisorb 5-ODS2), and Activon Scientific, Australia (Hypersil 3-C8). Sephadex was purchased from Pharmacia (Australia). Fractions were collected from the chromatograph using a LKB Redirac 2112 fraction collector.

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FIG. 2. Analysis,by reversed-phaseHPLC, of NMU-LIin crude extract of guinea pig ileum. Chromatographic conditionsare described in the text. The elution positionsof porcine NMU-8 and NMU-25 are indicated.

and all chromatography solvents were purchased from Millipore (Australia). All radiochemicals were purchased from Du Pont (Australia). Porcine neuromedin U-8 (NMU-8) was synthesized in this laboratory by standard Fmoc protocols on a CRB Pepsynthesizer I (Cambridge Research Biochemicals). All other peptides were either synthesized in the laboratory by similar protocols, or purchased from Peninsula Laboratories (U.S.A.). High performance liquid chromatography (HPLC) was carded out on a Varian 5020 binary gradient liquid chromatograph (Varian Associates, Australia), equipped with a UV-5 spectrophotometric detector operating at 215 nm, and a Valco C6U injection valve fitted with either a 2.0 ml or 0.5 ml loading loop. Output from the detector was monitored with a Waters M740 data module (Millipore/ Waters Associates, Australia) and a model DP600 chart recorder (I.C.I. Instruments, Australia). HPLC columns were purchased from SynChrom, U.S.A. (SynChropak CM-300), I.C.I. Instru-

A double antibody precipitation assay was used to measure neuromedin U-like immunoreactivity (NMU-LI) in chromatographic fractions. The primary antiserum 21.3 was raised in rabbit against synthetic porcine NMU-8, coupled at its N-terminus to key-hole limpet hemocyanin with glutaraldehyde, and was used at a final dilution of 1:12,000 for assaying chromatography fractions. The antiserum recognized both NMU-8 and NMU-25 (100% cross-reactive), but did not recognize any of the following peptides: neuromedin B, bombesin, Met-enkephalin-Arg-Phe, pancreatic polypeptide(31-36), neuropeptide Y, peptide YY, vasoactive intestinal peptide (all <0.1% cross-reactive). Assays were precipitated by the addition of sheep anti-rabbit secondary antiserum (Silenus Laboratories, Australia) (100 p,1; diluted 1:25 in assay buffer) and normal rabbit serum (100 p,l; diluted 1:100 in assay buffer). Tracer for the assay was 125I-porcine-NMU-8, iodinated by the iodogen method. All components of the assay were diluted in assay buffer (0.04 M sodium phosphate, 0.1 M sodium chloride, 0.01 M EDTA, 0.25% bovine serum albumin, 2 x 10 -3 M bacitracin, 0.1% sodium azide, 0.024% thimerosal, pH 7.4). The assay sensitivity was 18.9-+ 3.2 fmol/tube, with an IC5o of 156 -+25 fmol/tube for NMU-8 (n -- 8).

Tissue Extraction Guinea pig small intestine (336 g) was boiled for 20 min in 1000 ml of water, cooled and then homogenized (Waring blender). Acetic acid and 2-mercaptoethanol were added to final concentrations of 1.0 M and 0.01 M, respectively, and the homogenate was stored at 4°C overnight. Solids were removed by centrifugation (10,000 x g/30 min at 4°C), and the supernatant was then acetone precipitated at 4°(3 overnight at a final concentration of 75% acetone. The precipitate was removed by centrifugation as above and the supernatant concentrated by evaporation in vacuo. The aqueous residue was then lyophilized to give crude, powdered extract.

Chromatographic Purification of NMU-LI TABLE 1 MICROSEQUENCE ANALYSIS OF 20 pmol NMU-8 EQUIVALENTS OF NEUROMEDIN U-LIKE IMMUNOREACTIVE MATERIAL ISOLATED FROM GUINEA PIG SMALL INTESTINE (EQUAL TO 19 pmol OF ISOLATED PEPTIDE)

Cycle 1

2 3 4 5 6 7 8 9 Initial yield= 73%. Repetitive yield= 94%.

Amino Acid

Yield (pmol)

Gly Tyr Phe Leu Phe Arg Pro Arg Asn

13.8 11.5 12.0 10.7 10.7 5.8 7.2 4.8 7.6

A. The dried residue from above was resuspended in 15 ml of 1% aqueous acetic acid, clarified by cenwifugation, and the supernatant loaded onto a column of Sephadex G25F (25 x 1000 mm; bed volume 480 ml), previously equilibrated with 1% aqueous acetic acid. The column was eluted at 30 ml/hr with 1% aqueous acetic acid. Fractions were collected and aliquots dried, reconstituted in assay buffer and assayed for NMU-LI. B. Fractions from the Sephadex column containing NMU-LI were pooled and concentrated in vacuo. The concentrate was filtered and then loaded directly onto an analytical reversed-phase column (Spberisorb 5-ODS2, 4.6 × 250 mm) previously equilibrated with 0.12% aqueous trifluoroacetic acid (TFA). Peptides were eluted with a linear gradient of 0-60% of 0.1% TFA in acetonitrile over 90 min with a final isocratic period of 10 min at 60%. Flow rate was maintained at 1.0 ml/min. Fractions were collected at 1.0-rain intervals and assayed for NMU-LI. C. Those fractions from above that contained NMU-LI were concentrated under a stream of dry nitrogen and the aqueous concentrate was loaded directly onto a second reversed-phase column, Hypersil 3-C8 (4.6× 150 mm) previously equilibrated

616

MURPHY, TURNER, FURNESS, PARKER AND GIRAUD

TABLE 2 SEQUENCECOMPARISONOF PIG,RAT ANDGUINEAPIG (G-P)NEUROMEDINU, WITH SEQUENCEIDENTITIESIN ITALICS Pig NIVIU-25 Pig NMU-8

Phe-Lys-Val-Asp-Glu-Glu-Phe-GlnGly-Pro-Ile-Val-Ser-Gln-Asn-ArgArg-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2 Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2

Rat NMU

Tyr-Lys-Val-Asn-Glu-Tyr-Gln-Gly-Pro-Val-Ala-Pro-Ser-GlyGly-Phe-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2

G-P NMU

Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2

with 0.12% aqueous TFA. Peptides were eluted with a linear gradient of 0-50% acetonitrile, containing 0.1% TFA, over 75 min with a final isocratic step of 15 min at 50%. Flow rate was maintained at 0.7 ml/min, and 1.0-min fractions were collected and assayed for NMU-LI. D. Fractions from the C8 column that contained NMU-LI were concentrated under a stream of dry nitrogen and the aqueous concentrate was loaded directly onto the narrow-bore reversedphase column (Spherisorb 5-ODS2; 2× 150 mm), previously equilibrated with 20% acetonitrile in 0.12% aqueous TFA. Peptides were eluted from the column with a linear gradient of 20-50% acetonitrile, containing 0.1% TFA, over 60 min with a final isocratic period of 10 min at 50%, and at a flow rate of 0.3 ml/min. Fractions were collected (1.0 min) and assayed for NMU-LI. E. Those fractions from the narrow-bore column that contained NMU-LI were pooled and lyophilized. The residue was redissolved in 2 ml of 0,01 M NaH2PO4 and loaded directly onto a cation exchange column (SynChropak CM-300; 4.6 x 250 mm) previously equilibrated with 0.01 M NaH2PO4 containing 10% acetonitrile. Peptides were eluted from the column with a linear gradient over 50 min of 0-25% of 1.0 M NaC1 in starting buffer at a flow rate of 1.0 ml/min. Fractions collected at 1.0-min intervals were assayed for NMU-LI. F. Those fractions from the CM-300 column containing NMULI were pooled and reloaded onto the narrow-bore reversed-phase column, previously equilibrated with 30% acetonitrile in 0.12% aqueous TFA. Peptides were eluted from the column with a linear gradient of 30-50% over 60 min with a final isocratic period of 10 min at 50%, and at a flow rate of 0.3 ml/min. Collected 1.0-min fractions were assayed for NMU-LI. Microsequence Analysis of Isolated NMU-LI Fractions from the final narrow-bore analysis that contained NMU-LI were pooled and a portion was subjected to automated Edman degradation using an Applied Biosystems 470A protein sequencer with on-line PTH-amino acid detection, as previously described (11). C-Terminal Analysis of NMU-LI Analysis of the C-terminal amino acid of the isolated peptide was accomplished by a modification of the method of Tatemoto and Mutt (15). That portion of NMU-LI isolated above that was not used for sequence determination was dried down and then reconstituted in 50 Ixl of 0.1 M sodium citrate buffer, pH 6.0. A solution of carboxypeptidase Y (1 mg/ml; 10 p,1; approx. 0.2 units total) was added and the solution was incubated at 25°C for 150 min. The reaction mixture was dried down and the residue reconstituted in 50 Ixl of 0.2 M Na.2CO3. Dansyl chloride (5 mg/ml; 20 ~1) in acetone was added and the mixture incubated at

25°C for 120 min in the dark. Pyridine (5 p.1) was then added and the mixture allowed to stand for a further 15 rain. The mixture was diluted with 200 p,1 of water and extracted 3 times with 100 Ixl of ethyl acetate. The organic extracts were dried under a stream of dry nitrogen and then reconstituted in 20 p,1 of acetone. The extract was then analyzed on polyamide thin layer plates, developed in 2 dimensions; the first dimension was developed in pyridine/heptane (30/70, v/v) and the second dimension was developed in chloroform/heptane (70/30, v/v). Chromatograms were compared to those for dansyl derivatives of authentic amino acids. Analysis of NMU-LI in Crude Extract Fresh guinea pig ileum (3.2 g) was boiled for 15 min in 5 volumes of 2.0 M aqueous acetic acid containing 0.01 M 2mercaptoethanol. After cooling on ice, the mixture was homogenized and the homogenate stored at 4°(2 overnight. The homogenate was clarified by centrifugation at 10,000 × g for 30 min at 4°C. A portion of the supernatant (2.0 ml) was filtered through a 0.45 ixm disposable filter, and the filtrate was injected directly onto the analytical reversed-phase column. The column was eluted as described in B above. Fractions were collected at 1.0-min intervals and assayed for NMU-LI. Aliquots of the remaining extract were also assayed for NMU-LI. After analysis of the guinea pig ileum extract, the column was calibrated for the elution positions of porcine NMU-8 and NMU-25 by injecting standards onto the column and eluting under the same conditions as those used for the extract. RESULTS Extraction and gel filtration chromatography of the crude extract from guinea pig small intestine gave two peaks of immunoreactive material with Kay of 0.31 (corresponding to a molecular weight of 3000 daltons) and Kav of 0.85 (corresponding to a molecular weight of approximately 1250 daltons), respectively (see Fig. 1A). An attempt was made to chromatograph the higher molecular weight material (approx. 611 pmol NMU-8 equivalents) on the analytical reversed-phase column, but less than 4% of immunoreactive material was recovered in fractions collected from this column (data not shown). No immunoreactive material was found in the eluate either during sample loading or during the column cleaning procedures after the analysis. Analysis of extract from fresh gut by reversed-phase HPLC (see Fig. 2) indicated that there was one major form of NMU-LI present, which eluted earlier than porcine NMU-8. Two other minor peaks of immunoreactive material were also found, one of which eluted slightly before porcine NMU-25. Consequently, the higher molecular weight immunoreactivity was considered to be artifactual and further purification of this material was not attempted. The lower molecular weight material (approx. 574 pmol NMU-8 equivalents) gave only one peak of immunoreactivity when chromatographed on the

GUINEA PIG NEUROMEDIN U

617

analytical C18 reversed-phase column (see Fig. IB), corresponding to 232 pmol NMU-8 equivalents (approx. 40% recovery). The NMU-LI corresponded in retention time to a group of poorly resolved, UV-absorbing peaks. Analysis of the material on a C8 reversed-phase column gave a single peak (see Fig. 1C) of NMU-LI (136 pmol NMU-8 equivalents; 58.6% recovery) corresponding to a large peak of UV-absorbing material. Further purification of this material on a narrow-bore reversed-phase column again gave one peak of NMU-LI (86 pmol NMU-8 equiv.; 63.2% recovery) that corresponded to a group of poorly resolved UV-absorbing peaks (Fig. 1D). Analysis of this material on the cation exchange column gave a single small peak of UV-absorbing material (Fig. 1E) that corresponded to the elution position of the measured NMU-LI (90 pmol NMU-8 equiv.; 105% recovery). This material was finally purified to a single sharp peak of UV-absorbing material (Fig. IF), corresponding to 27 pmol NMU-8 equivalents of NMU-LI (30% recovery). A portion of this material (20 pmol NMU-8 equivalents) was sequenced to completion on the protein sequencer. The sequence determined was the same as porcine NMU-8 extended by a single glycine at the N-terminal (see Table 1 for sequencing data). The C-terminal was found to be amidated by subjecting the remaining isolated peptide to a modification of the method of Tatemoto and Mutt (15), utilizing carboxypeptidase Y to remove the C-terminal amino acid, and dansylation and polyamide thin-layer chromatography to determine the identity of the amino acid thus released. This procedure gave a pale yellow spot with the same chromatographic properties as authentic dansylasparagine amide (Rf in first dimension=0.25; Rf in second dimension=0.10). Thus the sequence was determined to be: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2 (mol.wt. = 1168).

DISCUSSION In this study, the major component of NMU-LI in guinea pig small intestine has been isolated and sequenced and found to correspond to porcine NMU-8 extended by a single glycine at the N-terminal. The C-terminal heptapeptide amide of both rat and porcine NMU is identical to that described here (see Table 2), and it may be postulated that this is a biologically active portion common to NMU from each species. The N-terminal heterogeneity found between rat and porcine peptides may reflect changes in that part of the molecule that is biologically less crucial, and the larger form of the porcine peptide (NMU-25) may serve as the immediate precursor for the true regulatory peptide (NMU-8). The NMU-8 structure contained within porcine NMU-25 is flanked on its N-terminal region by a dibasic cleavage point (Arg-Arg), giving further support to this proposition. This cleavage point is not present in the rat peptide. Lack of evolutionary pressure to retain the sequence beyond the C-terminal heptapeptide may have resulted in this difference between the two species. Consequently the larger form (or forms) of NMU-LI described in extracts of guinea pig tissue (4) may also exhibit differences in amino acid sequence due to lack of evolutionary pressure to conserve any more than the C-terminal heptapeptide. The Nterminal extension of the NMU-8 described here may be evidence for such changes. Indeed, many examples already exist of both substantial and small differences between the guinea pig and other species in the structures of regulatory peptides without any apparent impairment of their biological function (6, 12, 14, 16). ACKNOWLEDGEMENT This work was financially supported by the National Health and Medical Research Council of Australia.

REFERENCES 1. Augood, S. J.; Keast, J. R.; Emson, P. C. Distributionand characterization of neuromedinU-like immunoreactivityin rat brain and intestineand in guinea-pigintestine.Regul. Pept. 20:281-292; 1988. 2. Ballesta, J.; Cariei, F.; Bishop, A. E.; Steel, J. H.; Gibson, S. J.; Fahey, M.; Hennessey, R.; Domin, J.; Bloom, S. R.; Polak, J. M. Occurrence and developmentalpattern of neuromedinU-like immunoreactive nerves in the gastrointestinaltract and brain of the rat. Neuroscience 25:797-816; 1988. 3. Brown, D. R.; Quito, F. L. NeuromedinU octapeptide alters ion transport in porcinejejunum. Eur. J. Pharmacol. 155:159-162; 1988. 4. Domin, J.; Ghatei, M. A.; Chohan, P.; Bloom, S. R. Characterization of neuromedinU like immunoreactivityin rat, porcine, guinea-pigand human tissue extracts using a specific radioimmunoassay,Biochem. Biophys. Res. Commun. 140:1127-1134; 1986. 5. Domin, J.; Ghatei, M. A.; Chohan, P.; Bloom, S, R. Neuromedin U--A study of its distributionin the rat. Peptides 8:779-784; 1987. 6. Du, B.-H.; Eng, J.; Hulmes, J. D.; Chang, M.; Pan, Y.-C. E.; Yalow, R. S. Guinea-pighas a unique mammalianVIP. Biochem. Biophys. Res. Commun. 128:1093-1098; 1985. 7. Furness, J. B.; Pompolo, S.; Murphy, R.; Giraud, A. Projectionsof neurons with neuromedin U-like immunoreactivity in the small intestineof the guinea-pig.Cell Tissue Res, 257:415-422; 1989. 8. Honzawa,M.; Sudoh, T.; Minamino,N.; Tohyama, M.; Matsuo, H. Topographic localizationof neuromedinU-like structures in the rat

9.

10. 11. 12. 13.

14. 15. 16.

brain: an immunohistochemicalstudy. Neuroscience 23:1103-1122; 1987. Minamino,N.; Kangawa,K.; Matsuo, H. NeuromedinU-8 and U-25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 130:10781085; 1985. Minamino, N.; Kangawa, K.; Honzawa, M.; Matsuo, H. Isolation and structuraldeterminationof rat neuromedinU. Biochem. Biophys. Res, Commun. 156:355-360; 1988. Murphy, R.; Furness, J. B.; Costa, M. High-sensitivityphenylthiohydantoin amino acid analysis on-line to a gas phase protein sequencer. J. Chromatogr. 408:388-392; 1987. Smith, L. F. Species variationin the amino acid sequenceof insulin. Am. J. Med. 40:662-666; 1966. Sumi, S.; Inoue, K.; Kogire, M.; Doi, R.; Takaori, K.; Suzuki, T.; Yajima, H.; Tobe, T. Effect of syntheticneuromedinU-8 and U-25, novel peptides identified in porcine spinal cord, on splanchnic circulationin dogs. Life Sci. 41:1585-1590; 1987. Sundby, F. Species variation in the primary structure of glucagon. Metabolism 25:1319-1321; 1976. Tatemoto, K.; Mutt, V. Chemical determination of polypeptide hormones. Proc. Natl. Acad. Sci. USA 75:4115-4119; 1978. Zhou, Z.-Z.; Eng, J.; Pan, Y.-C. E.; Chang, M.; Hulmes, J. D.; Raufman, J.-P.; Yalow, R. S. Unique cholecystokininpeptides isolated from guinea-pigintestine. Peptides 6:337-341; 1985.