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Immunoregulatory properties of synthetic peptides, fragments of a proline-rich polypeptide (PRP) from ovine colostrum
MolecularImmunology,Vol. 24, No. Printed in Great Britain
10,pp. 1029-1031,1987
0161-5890/87$3.00+0.00
Pergamon Journals Ltd
IMMUNOREGULATORY PROPERTIES OF SYNTHETIC PEPTIDES, FRAGMENTS OF A PROLINE-RICH POLYPEPTIDE (PRP) FROM OVINE COLOSTRUM* MARIA JANUSZ,~ ZBIGNIEW WIECZOREK,~ KRYSTYNA SPIEGEL,~ ALEKSANDRA KUBIK,$ ZBIGNIEW SZEWCZUK,§ IGNACY SIEMION$ and J~ZEF LISOWSKI~ TDepartment of Immunochemistry and SLaboratory of Immunobiology of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland; §Institute of Chemistry, Wroclaw University, Wroclaw, Poland (Received 11 March 1987; accepted 23 March 1987)
Abstract-It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.
INTRODUCTION
of PRP with chymotrypsin (StaroScik et al., 1983). The nonapeptide showed the full spectrum of biological activities of PRP. The results presented in this paper showed that synthetically obtained nonapeptide is as active as the peptide obtained by chymotryptic digestion of PRP. The C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, also showed immunoregulatory activity similar to PRP and the nonapeptide. digestion
A proline-rich polypeptide (PRP) was isolated in our laboratory from ovine colostral whey (Janusz et al., 1974; Janusz et al., 1981). It contains a high number of proline residues (22%). In the absence of dissociating agents, the apparent molecular weight of PRP is 18,000. The polypeptide is built of noncovalently linked subunits of apparent molecular weight 6000. PRP has a regulatory activity stimulating or suppressing the immune response (Wieczorek et al., 1979). It induces maturation of murine thymocytes into functionally mature helper or suppressor cells (Zimecki et al., 1984a; Zimecki et al., 19846). PRP induces, in vivo and in vitro, formation of helper cells from PNA+ thymocytes concomitant with an increased of cortisone-resistance. When PNA- thymocytes are incubated with PRP, a formation of suppressor cells is observed and the number of cortisone-sensitive cells increases (Zimecki
MATERIALS AND
METHODS
Animals CBA mice (6-8 weeks old) were obtained from the Animal Breeding Center of our Institute. PRP The polypeptide was prepared from ovine colostrum according to the method of Janusz et al. (198 1).
et al., 1982). We also presented evidence that murine thymocytes have on their surface a receptor specific for PRP and showed that the polypeptide does not need to enter the cell to exert biological functions (Janusz et al., 1986). A nonapeptide fragment: Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro was isolated from products of Address all correspondence to: Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Czerska, 53-l 14 Wrodaw, Poland. *This work was supported by the Polish Academy of Sciences, Grant 6;. 10.5 and 06.01. Abbreviations: PRP, proline-rich polypeptide; PFC, plaque-forming cells; SRBC, sheep red blood cells; BSA, bovine serum albumin.
Synthesis of nona- and hexa-peptides The synthesis of the partial sequences of PRP: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro and Tyr-ValPro-Leu-Phe-Pro followed the classical solution method. The purity of peptides was controlled using TLC plates. The structure of synthesized peptides was confirmed by elemental analysis, amino acid analysis and ‘H-NMR spectra. Bioassay systems Effect of the nonapeptide and hexapeptide on the immune response was investigated in mice, both in vivo and in vitro, by determination of PFC-19s. The in vitro assay was performed according to the procedure of Chan et al. (1980). Mice were primed
1029
MARIA
1030
JANUSZ
with SRBC. Four days later, the animals were killed and cells isolated from their spleens were cultured in the absence and in the presence of synthesized peptides. PFC-19s (per lo6 splenocytes) were determined in 4-day cultures according to the method described by Mishell and Dutton (1967). For the in vivo assay, mice were injected intraperitoneally with the synthesized peptides 3 hr before immunization with SRBC. After 4 days, the animals were killed and the spleens were isolated. Spleen cell suspensions were prepared and PFC-19s were determined in the same way as described for the in vitro assay. In control experiments, neither BSA nor poly-LPro showed effects on the immune response.
RESULTS
s 6
V c 0; IOOCO-
SOOO-
7coO“0
6Oco-
T z1 a
5000-
4000
-
3m2OOo-
lw-tl
6
V
c?
; Fig. 2. Effect of the synthetic nonapeptide: Val-Glu-Ser-TyrVal-Pro-Leu-Phe-Pro and its C-terminal hexapeptide fragment: Tyr-Val-Pro-Leu-Phe-Pro on the primary humoral immune response in uiuo measured by determination of PFC-198 Mice were treated intraperitoneally with 1.0 or 10pg of the hexapeptide (2 and 3) or with 1.0 or 10pg of the nonapeptide (4 and 5) or with 1.0 or 10pg of PRP (6 and 7) 3 hr before immunization with SRBC. (1) Control mice. The values presented are averages of five determinations. Bars denote standard error.
s
11 1 0
-
0
AND DISCUSSION
The synthetic nonapeptide: Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro and its C-terminal hexapeptide: Tyr-Val-Pro-Leu-Phe-Pro showed similar stimulation of the immune response as PRP, both in vitro and in vivo (Fig. 1 and Fig. 2). The immunoregulatory activity of the synthetic nonapeptide is the same as
9000
et al.
!I
I 4
Fig. 1. Effect of the synthetic nonapeptide: Val-Glu-Ser-TyrVal-Pro-Leu-Phe-Pro, its C-terminal hexapeptide fragment: Tyr-Val-Pro-Leu-Phe-Pro, and PRP on the primary hu&oral immune response in vitro measured by determination of PFC-19s. Mice were mimed with SRBC. (1) Control mice; (2, 3 and 4) spleen- cells cultured with 0: i, 1.O and 10 pg of the hexapeptide, respectively; (5, 6 and 7) spleen cells cultured with 0.1, 1.0, and 10 pg of the nonapeptide, respectively; (8 and 9) spleen cells cultured with 1.0 and 1Opg of PRP, respectively. The results are the mean of four determinations f standard error. The experiments were performed twice.
that of the nonapeptide obtained by chymotryptic digestion of PRP (StaroScik et al., 1983). This confirms the correctness of amino acid sequence of the nonapeptide given by Staroscik et al. (1983). Control experiments excluded the possibility of contamination of PRP with endotoxin. There also was a possibility that the biological effects of PRP were due to a contamination of PRP with a very low concentration of a substance with a very high biological activity. However, the experiments showing that the synthetic nonapeptide has similar immunoregulatory activity to PRP and the nonapeptide obtained from the chymotryptic digest of PRP presented evidence that the observed effects on the immune response are caused by PRP and not by contaminations. It has recently been found that N-terminal tripeptide, Val-Glu-Ser, was inactive whereas the Cterminal pentapeptide fragment, Val-Pro-Leu-PhePro, showed immunoregulatory activity (Kubik et al., 1984). This confirms our results with the C-terminal hexapeptide that C-terminal region of the nonapeptide is responsible for its immunoregulatory activity. Preparation of biologically active fragments of PRP with lower molecular weight will be of greater help in studies of the mechanism of action of PRP and also the mechanism of the immune response.
Synthetic immunoregulatory REFERENCES
Chan E. L., Mishell B. B. and Shiigi S. M. (1980) Secondary immunization to heterologous erythrocytes. In Selected Methorls in Cellular Immunology (Edited by Mishell B. B. and Shiigi S. M.), pp. 43-45. Freeman and Company, San Francisco. Janusz M., Lisowski J. and Fransk F. (1974) Isolation and characterization of a proline-rich polypeptide from ovine colostrum. FEBS Lett. 49, 276-279. Janusz M., Staroscik K., Zimecki M., Wieczorek Z. and Lisowski J. (1981) Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum. Biothem. J. 199, 9-15. Janusz M., StaroScik K., Zimecki M., Wieczorek Z. and Lisowski J. (1986) A proline-rich polypeptide (PRP) with immunoregulatory properties isolated from ovine colostrum. Mu&e thymocytes have on their surface a receptor soecific for PRP. Arch. Immun. Ther. Exe. 34. 427-436. K&k A., KliH W. A., Szewczuk Z., Siemion I. Z., Janusz M., StaroScik K., Zimecki M., Lisowski J. and Wieczorek Z. (1984) Proline-rich polypeptide (PRPt-_A new peptide immunoregulator and its partial sequences. In Peptides 1984. Proceedings of the 18th European Peptide Symposium (Edited by Ragnarsson U.), pp. 457460.
Almqvist and Wigzell, Stockholm. Mishell R. J. and Dutton R. W. (1967) Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med. 126, 423-442.
fragments of PRP
1031
Staroscik K., Janusz M., Zimecki M., Wieczorek Z. and Lisowski J. (1983) Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory prop_erties. Molec. Immuk 20, 1277-1282. Wieczorek 2.. Zimecki M.. Janusz M.. StaroHcik K. and Lisowski J.’ (1979) Proline-rich polypeptide from ovine colostrum: its effect on skin permeability and on the immune response. Immunology 36, 875-88 1. Zimecki M., Janusz M., Staroscik K., Lisowski J. and Wieczorek Z. (1982) Effect of a proline-rich polypeptide on donor cells in graft-versus-host reaction. Immunology 47, 141-147.
Zimecki M., Lisowski J., Hraba T., Wieczorek Z., Janusz M. and Staroscik K. (1984a) The effect of a proline-rich polypeptide (PRP) on the humoral immune response I. Distinct effect of PRP on the T-cell properties of mouse glass-nonadherent (NAT) and glass-adherent (GAT) thymocytes in thymectomized mice. Arch. Immun. Ther. Exp. 32, 191-196.
Zimecki M., Lisowski J., Hraba T., Wieczorek Z., Janusz M. and StaroScik K. (19846) The effect of a proline-rich polypeptide (PRP) on the humoral immune response II. PRP induces differentiation of helper cells from glassnonadherent thymocytes (NAT) and suppressor cells from glass-adherent thymocytes (GAT). Arch. Immun. Ther. Exp. 32, 197-201.
10,pp. 1029-1031,1987
0161-5890/87$3.00+0.00
Pergamon Journals Ltd
IMMUNOREGULATORY PROPERTIES OF SYNTHETIC PEPTIDES, FRAGMENTS OF A PROLINE-RICH POLYPEPTIDE (PRP) FROM OVINE COLOSTRUM* MARIA JANUSZ,~ ZBIGNIEW WIECZOREK,~ KRYSTYNA SPIEGEL,~ ALEKSANDRA KUBIK,$ ZBIGNIEW SZEWCZUK,§ IGNACY SIEMION$ and J~ZEF LISOWSKI~ TDepartment of Immunochemistry and SLaboratory of Immunobiology of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland; §Institute of Chemistry, Wroclaw University, Wroclaw, Poland (Received 11 March 1987; accepted 23 March 1987)
Abstract-It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.
INTRODUCTION
of PRP with chymotrypsin (StaroScik et al., 1983). The nonapeptide showed the full spectrum of biological activities of PRP. The results presented in this paper showed that synthetically obtained nonapeptide is as active as the peptide obtained by chymotryptic digestion of PRP. The C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, also showed immunoregulatory activity similar to PRP and the nonapeptide. digestion
A proline-rich polypeptide (PRP) was isolated in our laboratory from ovine colostral whey (Janusz et al., 1974; Janusz et al., 1981). It contains a high number of proline residues (22%). In the absence of dissociating agents, the apparent molecular weight of PRP is 18,000. The polypeptide is built of noncovalently linked subunits of apparent molecular weight 6000. PRP has a regulatory activity stimulating or suppressing the immune response (Wieczorek et al., 1979). It induces maturation of murine thymocytes into functionally mature helper or suppressor cells (Zimecki et al., 1984a; Zimecki et al., 19846). PRP induces, in vivo and in vitro, formation of helper cells from PNA+ thymocytes concomitant with an increased of cortisone-resistance. When PNA- thymocytes are incubated with PRP, a formation of suppressor cells is observed and the number of cortisone-sensitive cells increases (Zimecki
MATERIALS AND
METHODS
Animals CBA mice (6-8 weeks old) were obtained from the Animal Breeding Center of our Institute. PRP The polypeptide was prepared from ovine colostrum according to the method of Janusz et al. (198 1).
et al., 1982). We also presented evidence that murine thymocytes have on their surface a receptor specific for PRP and showed that the polypeptide does not need to enter the cell to exert biological functions (Janusz et al., 1986). A nonapeptide fragment: Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro was isolated from products of Address all correspondence to: Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Czerska, 53-l 14 Wrodaw, Poland. *This work was supported by the Polish Academy of Sciences, Grant 6;. 10.5 and 06.01. Abbreviations: PRP, proline-rich polypeptide; PFC, plaque-forming cells; SRBC, sheep red blood cells; BSA, bovine serum albumin.
Synthesis of nona- and hexa-peptides The synthesis of the partial sequences of PRP: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro and Tyr-ValPro-Leu-Phe-Pro followed the classical solution method. The purity of peptides was controlled using TLC plates. The structure of synthesized peptides was confirmed by elemental analysis, amino acid analysis and ‘H-NMR spectra. Bioassay systems Effect of the nonapeptide and hexapeptide on the immune response was investigated in mice, both in vivo and in vitro, by determination of PFC-19s. The in vitro assay was performed according to the procedure of Chan et al. (1980). Mice were primed
1029
MARIA
1030
JANUSZ
with SRBC. Four days later, the animals were killed and cells isolated from their spleens were cultured in the absence and in the presence of synthesized peptides. PFC-19s (per lo6 splenocytes) were determined in 4-day cultures according to the method described by Mishell and Dutton (1967). For the in vivo assay, mice were injected intraperitoneally with the synthesized peptides 3 hr before immunization with SRBC. After 4 days, the animals were killed and the spleens were isolated. Spleen cell suspensions were prepared and PFC-19s were determined in the same way as described for the in vitro assay. In control experiments, neither BSA nor poly-LPro showed effects on the immune response.
RESULTS
s 6
V c 0; IOOCO-
SOOO-
7coO“0
6Oco-
T z1 a
5000-
4000
-
3m2OOo-
lw-tl
6
V
c?
; Fig. 2. Effect of the synthetic nonapeptide: Val-Glu-Ser-TyrVal-Pro-Leu-Phe-Pro and its C-terminal hexapeptide fragment: Tyr-Val-Pro-Leu-Phe-Pro on the primary humoral immune response in uiuo measured by determination of PFC-198 Mice were treated intraperitoneally with 1.0 or 10pg of the hexapeptide (2 and 3) or with 1.0 or 10pg of the nonapeptide (4 and 5) or with 1.0 or 10pg of PRP (6 and 7) 3 hr before immunization with SRBC. (1) Control mice. The values presented are averages of five determinations. Bars denote standard error.
s
11 1 0
-
0
AND DISCUSSION
The synthetic nonapeptide: Val-Glu-Ser-Tyr-ValPro-Leu-Phe-Pro and its C-terminal hexapeptide: Tyr-Val-Pro-Leu-Phe-Pro showed similar stimulation of the immune response as PRP, both in vitro and in vivo (Fig. 1 and Fig. 2). The immunoregulatory activity of the synthetic nonapeptide is the same as
9000
et al.
!I
I 4
Fig. 1. Effect of the synthetic nonapeptide: Val-Glu-Ser-TyrVal-Pro-Leu-Phe-Pro, its C-terminal hexapeptide fragment: Tyr-Val-Pro-Leu-Phe-Pro, and PRP on the primary hu&oral immune response in vitro measured by determination of PFC-19s. Mice were mimed with SRBC. (1) Control mice; (2, 3 and 4) spleen- cells cultured with 0: i, 1.O and 10 pg of the hexapeptide, respectively; (5, 6 and 7) spleen cells cultured with 0.1, 1.0, and 10 pg of the nonapeptide, respectively; (8 and 9) spleen cells cultured with 1.0 and 1Opg of PRP, respectively. The results are the mean of four determinations f standard error. The experiments were performed twice.
that of the nonapeptide obtained by chymotryptic digestion of PRP (StaroScik et al., 1983). This confirms the correctness of amino acid sequence of the nonapeptide given by Staroscik et al. (1983). Control experiments excluded the possibility of contamination of PRP with endotoxin. There also was a possibility that the biological effects of PRP were due to a contamination of PRP with a very low concentration of a substance with a very high biological activity. However, the experiments showing that the synthetic nonapeptide has similar immunoregulatory activity to PRP and the nonapeptide obtained from the chymotryptic digest of PRP presented evidence that the observed effects on the immune response are caused by PRP and not by contaminations. It has recently been found that N-terminal tripeptide, Val-Glu-Ser, was inactive whereas the Cterminal pentapeptide fragment, Val-Pro-Leu-PhePro, showed immunoregulatory activity (Kubik et al., 1984). This confirms our results with the C-terminal hexapeptide that C-terminal region of the nonapeptide is responsible for its immunoregulatory activity. Preparation of biologically active fragments of PRP with lower molecular weight will be of greater help in studies of the mechanism of action of PRP and also the mechanism of the immune response.
Synthetic immunoregulatory REFERENCES
Chan E. L., Mishell B. B. and Shiigi S. M. (1980) Secondary immunization to heterologous erythrocytes. In Selected Methorls in Cellular Immunology (Edited by Mishell B. B. and Shiigi S. M.), pp. 43-45. Freeman and Company, San Francisco. Janusz M., Lisowski J. and Fransk F. (1974) Isolation and characterization of a proline-rich polypeptide from ovine colostrum. FEBS Lett. 49, 276-279. Janusz M., Staroscik K., Zimecki M., Wieczorek Z. and Lisowski J. (1981) Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum. Biothem. J. 199, 9-15. Janusz M., StaroScik K., Zimecki M., Wieczorek Z. and Lisowski J. (1986) A proline-rich polypeptide (PRP) with immunoregulatory properties isolated from ovine colostrum. Mu&e thymocytes have on their surface a receptor soecific for PRP. Arch. Immun. Ther. Exe. 34. 427-436. K&k A., KliH W. A., Szewczuk Z., Siemion I. Z., Janusz M., StaroScik K., Zimecki M., Lisowski J. and Wieczorek Z. (1984) Proline-rich polypeptide (PRPt-_A new peptide immunoregulator and its partial sequences. In Peptides 1984. Proceedings of the 18th European Peptide Symposium (Edited by Ragnarsson U.), pp. 457460.
Almqvist and Wigzell, Stockholm. Mishell R. J. and Dutton R. W. (1967) Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med. 126, 423-442.
fragments of PRP
1031
Staroscik K., Janusz M., Zimecki M., Wieczorek Z. and Lisowski J. (1983) Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory prop_erties. Molec. Immuk 20, 1277-1282. Wieczorek 2.. Zimecki M.. Janusz M.. StaroHcik K. and Lisowski J.’ (1979) Proline-rich polypeptide from ovine colostrum: its effect on skin permeability and on the immune response. Immunology 36, 875-88 1. Zimecki M., Janusz M., Staroscik K., Lisowski J. and Wieczorek Z. (1982) Effect of a proline-rich polypeptide on donor cells in graft-versus-host reaction. Immunology 47, 141-147.
Zimecki M., Lisowski J., Hraba T., Wieczorek Z., Janusz M. and Staroscik K. (1984a) The effect of a proline-rich polypeptide (PRP) on the humoral immune response I. Distinct effect of PRP on the T-cell properties of mouse glass-nonadherent (NAT) and glass-adherent (GAT) thymocytes in thymectomized mice. Arch. Immun. Ther. Exp. 32, 191-196.
Zimecki M., Lisowski J., Hraba T., Wieczorek Z., Janusz M. and StaroScik K. (19846) The effect of a proline-rich polypeptide (PRP) on the humoral immune response II. PRP induces differentiation of helper cells from glassnonadherent thymocytes (NAT) and suppressor cells from glass-adherent thymocytes (GAT). Arch. Immun. Ther. Exp. 32, 197-201.