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Volume 196, number2 FEBS 3363 February 1986

Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein

Hans Meisel

Institut fir Chemie und Physik der Bundesanstalt fzZrMilchforschung, 2300 Kiel, FRG

Received 17 October 1985; revised version received 4 December 1985

The in vivo formation of an (exorphin) derived from p-casein has been proved for the first time. It was isolated from duodenal thyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a /?-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named /I--l l, displayed substantial opioid activity in an opiate receptor-binding assay.

Exorphin Casomorphin @Casein (Duodenal digest) Opioid activity

1. INTRODUCTION cipitated casein (Biogen-~asein, Bayr. Milchver- sorgung, N~rnberg) after overnight fasting. Post- The existence of opioid peptides has been de- prandial duodenum samples were taken for 8 h, scribed in partial enzymatic digests of proteins frozen immediately in liquid nitrogen and freeze- derived from foodstuffs [l-3]. These peptides are dried. A control experiment was performed after called exorphins because of their exogenous origin feeding with a maize starch diet composed as in [9] and -like activities. without casein. Such opioid peptides have been discovered re- The extraction of 110 g freeze-dried duodenal cently in enzymatic digests of whole bovine casein thyme from 5 collection periods with chloroform/ and were designated as Bcasomorphins because methanol (65 : 35, v/v), and the following batchwise their sequences identified them as fragments of adsorption/desorption procedure was performed bovine fl-casein [4]. as in [I]. The oily residue from the last desorption The physiological role of and step was diluted up to 5 ml with methanol. other exorphins is not yet understood. Neverthe- less, evidence exists that exorphins do affect in- testinal motility as well as the release of insulin and 2.2. Thin-layer analyses and preparative glucagon [5-S]. separations This paper reports on the isolation and charac- Aliquots (1 ~1) of the extraction residue were terization of an opioid peptide from duodenal subjected to the fingerprint technique on silica gel thyme of minipigs after feeding with casein. 60 sheets (20 x 20 cm, 0.2 mm, Merck). First, elec- trophoresis was carried out in pyridine/acetic acid/water (100:4:900, by vol.), pH 6.5, at 10 mA 2. MATERIALS AND METHODS for 1 h. The second dimension was ascending 2.1. Collection and extraction of digesta chromatography in chloroform/methanol/2O~o Two male adult G~ttingen minipigs (35 kg) fit- ammonia (60: 30: 5, by vol). The peptides were ted with T-shaped canulas at the duodenum, 20 cm detected by a tyrosine-specific spray reagent ac- from the pylorus, were fed with 100 g acid-pre- cording to [lo] or by spraying with 0.05% fluores-

Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50 0 1986 Federation of European Biochemical Societies 223 Volume 196, number 2 FEBS LETTERS February 1986 camine (Serva) in acetone, All ~-casomorphin 2.6. Opiate receptor-binding assay standards were obtained from Sigma. Opiate receptor binding in rat brain membrane The conditions for preparative dectrophoresis preparations was assayed by competition with ( - )- were the same as for fingerprinting but the sample D-13H] (Amersham, 51 Ci/mmol) ac- was applied as a streak (80&‘12 cm). Aliquots (12 cording to [14]). The membranes from brains, pi/17 cm) of the material from several electro- without cerebellum, of 3 male rats (Wistar) were phoretic separations were applied to silica ge1 60 reconstituted in 30 ml of 0.05 M Tris-HCI buffer, HPLC plates with ~uorescent indicator (10 x 10 pH 7.4, at 37°C. cm, Merck) which had been activated at 100°C for Protein concentration was determined by a 1 h. After development in the solvent system as microprotein assay with the Coomassie G-250 described above the main peptide band was visual- reagent as in [15] using bovine serum albumin as ized under UV light (254 nm), eluted with chloro- standard. Aliquots (200/1rl) of the membrane pre- form/methanol (1: 1, v/v), evaporated to dryness paration were incubated for 5 min at 37”C, pH and redissolved in a small volume of methanol. 7.4, with peptides (I-20 nmol), and the incubation The peptide concentration was determined spec- continued for 15 min after the addition of [3H]- trophotometrically with 0.5 ml of an o-phthaldial- naloxone (0.55 pmol, 25 000 cpm) in a final volume dehyde @PA, Serva) reagent as in [l l] using L- of 0.5 ml. After rapid filtration through glass fiber leucine and &casomorphin-7 as standards (J&O = circles (Whatman, 2.4 cm) and washing with four 6000 M-’ . cm-’ for calculations). 5-ml portions of ice-cold Tris buffer, the filters were transferred into counting vials, immersed in 1 ml TS-1 tissue solubilizer (Zinsser) and kept over- The purified peptide (2-3 nmol, based upon night at room temperature. The filters were ex- OPA spectrophotometric assay) was first hydro- tracted by shaking with 15 ml Ready-SoIv MP lyzed in 6 M HCI for 18 h at 105’C; the released (Beckman) for 1 h. The radioactivity was counted amino acids were dansylated and identified as at 40% efficiency after addition of 100 ~1 acetic described below. acid/water (1: 1, v/v) and equilibration at $*C in a liquid scintillation spectrometer (Packard Tri- Carb 2660). Nonspecific binding was determined in the presence of a large excess (1 ~mol/l) of A solution of the purified peptide (0.5-i nmol) unlabelled naloxone (Sigma). was dried in vacua and the dansyiation carried out using 3 ~1 dansyl chloride (Merck) solution in acetonitrile and 6 ~1 lithium carbonate buffer, pH 3. RESULTS 9.5 [12]. After reaction for 30 min at 30°C an ali- 3.1. Purification quot (OS ~1) was withdrawn to check the purity by The peptide map of the duodenal extract displays thin-layer chromatography (TLC). Acid hydrolysis at least 23 tyrosine-containing peptides (fig. 1). No and identification of the N-terminal dansyl amino tyrosine-containing peptides were detectable in the acid by two-Dimensions TLC on 5 x 5 cm micro- duodenal extract after feeding with a protein-free polyamide sheets (Schleicher & Schiill) was carried diet. out as in 1131. After preparative electrophoresis the main band including the positions of standard P-casomor- 2.5. C-terminal amino acid analysis phin-7 and -5 (1. dim. in fig.1) was eluted. With The purified peptide (3-5 nmol) was dissolved in HPLC of the electrophoretically prepurified sam- 60~1 sodium acetate, pH 5.5, buffer, and after ad- ple on silica gel a more refined preparative separa- dition of 0.5 pg carboxypeptidase Y (Sigma) in- tion of the hydrophobic peptides (Rr>0.4) was cubated at 37°C. Timed aiiquots (10 pl) were achieved (fig.2). The detection with ~uorescamine removed and immediately heated for 3 min in a (not shown) revealed that the bulk of interfering boiling water bath. Dansylation and identification peptides emerged with & values less than 0.35. of the liberated C-terminal amino acid was per- The application of the tyrosine-specific reagent formed as described above. gave 14 peptides with the main band in the position

224 Volume 196, number 2 FEBS LETTERS February 1986

85 @7 X

I m. 0 - 2. dell * I c3 x 4 amide t3 7 g x 5

Fig.1. Fingerprint of the tyrosine-containing peptides from duodenal thyme extract on silica gel. Synthetic caso- morphins were applied as standards. 1. dim., electrophor~is; 2. dim., ascending chromatography. For detaiis see section 2.2. of ,&casomorphin-5 (RF 0.42) and two minor experimental conditions for C-terminal analysis bands corresponding to P-casomorphin-7 (RF 0.63) enabled the slow sequential release of the first 3 and ,&casomorphin-4 amide (RF 0.88), The main amino acids from synthetic P-casomorphin-5 and peptide band as indicated by an arrow in fig.2 was -7. By treatment of the purified peptide with car- eluted from several HPLC plates for further char- boxypeptidase Y for 30 min only leucine was acterization. After rechromatography, the eluted released and consequently recognized as the C- peptide (77 nmol) was homogeneous as proved terminus. after dansylation and by two-dimensional TLC on As the purified peptide had been isolated from micropolyamide layer. a duodenal digest of bovine casein, the sequences of as-, ,& and x-casein were examined for regions 3.2. Characterization and opioid activity containing the determined end-groups and amino Because of the small quantities of the purified acid residues. It was indeed discovered that the peptide the hydrolysed sample was dansylated and composition found is only compatible with a frag- analysed for qualitative amino acid composition ment of ,&casein (genetic variant A2 or A3) 1161 by TLC. The constituents found were Pro, Tyr, originating at Tyr 60 and extending to Leu 70: Tyr- Phe, Gly, Ile, Leu, Asx, Ser. When subjected to N- Pro-Phe-Pro-Gly-Pro-Be-Pro-Asn-Ser-Leu. terminal group analysis by the dansyl chloride Our results allow the conclusion that the purified method it revealed tyrosine as the N-terminus. peptide contains the whole P-casomorphin-7 se- In preliminary experiments it was found that the quence (N-terminal) plus the next 4 amino acids in

225 Volume 196, number 2 FEBS LETTERS February 1986

the sequence of &casein. Moreover, it displayed opioid activity in an opiate receptor-binding assay - - 4 am/de (fig.3) and hence was referred to as fi-casomor- phin-Il. As shown in fig.3, the isolated p-caso- morphin-ll exhibited a weaker but substantial (micromolar) affinity for opiate receptors as com-

- -7 pared with synthetic fl-casomorphin-5 which is known to be a very potent opioid peptide in several bioassays [17,18].

Z 1; *- 4. DISCUSSION The present results show that a peptide with opioid activity, termed &casomorphin-1 1, can be isolated in vivo from duodenal thyme of minipigs after feeding with casein whereas no casomorphin- like peptides could be detected after a casein meal in the gastric thyme (H. Meisel, unpublished). $___--______---, c----l ,&Casomorphins are hidden in an inactive state duodenal peptides fl-casomorphins inside the casein structure. To exert biological ef- fects in vivo, they must be produced in the in- Fig.2. Thin-layer chromatogramm of the electrophoreti- testinal tract but resist complete degradation by in- tally prepurified sample from duodenal thyme extract. Synthetic casomorphins were applied for comparison. testinal proteases. Because of the known specificity For details see section 2.2. of pepsin and pancreatic peptidases, cleavage of peptide bonds releasing significant amounts of ,& casomorphins from the corresponding @casein se- quence is unexpected. Furthermore, the proline- rich fi-casomorphin structure itself was described as being highly resistant towards proteolysis by gastric and pancreatic enzymes [4,17]. However, the intestinal brush border contains high levels of peptidase activity [19], so that the action of several peptidases from the intestinal epithelium could contribute to the rise of free casomorphins. Authors in [20] and [21] have shown that the in vitro digestion of buffalo ,&casein released neither fi-casomorphin-7 nor a fragment of it but a putative precursor (decapeptide) which was further digested by incubation with brush border pep- tidases, especially with dipeptidyl peptides IV from I I OkI 1o-6 10d human intestine; the peptides were not checked for /, -cosomorphm s (molii ) their opioid activities. The question arises as to whether the isolated ,& Fig.3. Displacement of specifically bound [3H]naloxone casomorphin-1 1 may be considered as a precursor from particulate fractions of rat brain homogenates by for smaller casomorphin fragments, because two ,&casomorphin-1 1 in comparison to synthetic P-caso- further peptides from duodenal thyme have indeed morphin-5. The incubation volume (0.5 ml) contained 0.7 mg protein, 1.1 nmol/l [3H]naloxone and the shown a chromatographic behaviour correspond- indicated concentrations of casomorphins. Control ing to standard ,&casomorphin-7 and -4 amide, specific binding was 1806 cpm (77% of the total bound). respectively (fig.2). Values are means of 3 separate determinations. It seems likely that fl-casomorphin-1 1 itself can

226 Volume 196, number 2 FEBS LETTERS February 1956 interact with receptors in the intestinal tract, or 141 Henschen, A., Lottspeich, F., Bra&, V., Tesche- may be degraded to further opioid fragments in the macher, H. (1979) Hoppe-Seyler’s Z. Physiol. brush border. Such highly hydrophobic fragments Chem. 360, 1217-1224. are expected to be absorbed into the bloodstream ISI Morley, J.E., Levine, A.S., Yamada, T., Gebhard, and to bind to brain opiate receptors as well as to R.K., Prigge, W.F., Shafer, R.B., Goetz, F.C. and Silvis, S.E. (1983) Gastroenterology 84, 1517-1523. those of peripheral organs [2,3]. Both rapid brush [61 Schusdziarra, V., Henrichs, I., Holland, A., Klier, border degradation and high absorption rate could M. and Pfeiffer, E.F. (1981) Diabetes 30, 362-364. be possible explanatations for the relatively low I71 Schusdziarra, V., Schick, A., De La Fuente, A., amounts of ,&casomorphin-I 1 found in duodenal Specht, J., Klier, M., Bran& V. and Pfeiffer, E.F. thyme (77 nmol isolated vs about 1.0 mmol in- (1983) Endocrinology 112, 885-889. gested). (81 S~husdziarra, V., Schick, R., De La Fuente, A., In the present study the formation of an opioid Holland, A., Brantl, V. and Pfeiffer, E.F. (1983) peptide has been proved for the first time in vivo, Endocrinology 112, 1948-1951. and under physiological conditions. however, it is [91 Hagemeister, H., Scholz, K., Kinder, E., Barth, not yet clear whether the opioid @-casomorphins CA. and Drochner, W. (1985) in: Digestive Phys- iology in the Pig (Just, A. et al. eds) pp. 124-127, could reach concentrations of physiological signi- Report from the National Institute of Animal ficance. Thus, their possible physiological signi- Science, Copenhagen. ficance has to be elucidated by further in vivo I101 Scoffone, E. and Fontana, A. (1975) in: Protein studies. Sequence Determination (Needleman, S.B. ed.) pp. 162-203, Springer, Berlin. 1111Church, F.C., Porter, D.H., Catignani, G.L. and ACKNOWLEDGEMENTS Swaisgood, H.E. (1985) Anal. Biochem. 146, This work was supported by the European Com- 343-348. munity (VO 271/82-12.3). The author wishes to WI Tapuhi, Y., Schmidt, D.E., Lindner, W. and thank Dr A. W&hen for his helpful advice con- Karger, B.L. (1981) Anal. Biochem. 115, 123-129. Narita, K., Matsuo, H. and Nakajima, T. (1975) cerning the radiochemical methods, Professor H. 1131 in: Protein Sequence Determination (Needleman, Hagemeister for providing duodenal samples from S.B. ed.) pp. 30-113, Springer, Berfin. minipigs and Dipl.oec.troph. N. Roos for the 1141 Pert, C.B. and Snyder, S.H. (1973) Science 179, preparation of rat brains. Sincere appreciation is 101 l-1014. expressed to Professor E. Schlimme and Dr W. WI Bradford, M.M. (1976)Anal. Biochem. 72,248-254. Bu~hheim for carefulIy reading the manuscript, WI Swaisgood, H.E. (1982) in: Developments in Dairy The technical assistance of Miss B. Hansen is Chemistry - 1 (Fox, P.F. ed.) pp. l-59, Applied gratefully acknowledged. Science Publishers, London. 1171&anti, V., Teschemacher, H., Blisig, J., Henschen, A. and Lottspeich, F. (1981) Life Sci. 28,1903-1909. [IS] Chang, K.-J., Killian, A., Hazum, E. and Cuatre- REFERENCES casas, P. (1979) Science 212, 75-77. 1191 Adibi, S.A. and Kim, Y-S. (1981) in: Physiology of HI Brantl, V., Teschemacher, H., Henschen, A. and the Gastrointestinal Tract (Johnson, L.R. ed.) pp. Lottspeich, F. (1979) Hoppe-Seyler’s 2. Physiol. 1073-1095, Raven, New York. Chem. 360, 1211-1216. [20] Petrilli, P., Picone, D., Caporale, C., Addeo, F., PI Zioudrou, C., Streaty, R.A. and Klee, W.A. (1979) Auricchio, S. and Marino, G. (1984) FEBS Lett. J. Biol. Chem. 254, 2446-2449. 169, 53-56. [31Loukas, S., Varoucha, D., Zioudrou, C., Streaty, [21] Caporale, C., Fontanella, A., Petrilli, P., Pucci, R.A. and Klee, W.A. (1983) 22, P., Molinaro. M.F., Picone, D. and Auricchio, S. 4567-4573. (1985) FEBS Lett. 184, 273-277.

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