New Analogues of Proline-Rich Protein Fragments. Synthesis and

Polish J. Chem., 74, 979–984 (2000)
New Analogues of Proline-Rich Protein Fragments.
Synthesis and Their Effect on Resistance
of Murine Thymocytes to Hydrocortisone*
by I. Wirkus-Romanowska1, H. Miecznikowska1, M. Janusz2, S. Szymaniec2,
W. Fortuna2, R. Miêdzybrodzki2, A. Zab³ocka2,
J. Lisowski2 and G. Kupryszewski1,3
1
Faculty of Chemistry, University of Gdañsk, 80-952 Gdañsk, Sobieskiego 18, Poland
2
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,
53-114 Wroc³aw, Rudolfa Weigla 12, Poland
3
Institute of Oceanology, Polish Academy of Sciences, 81-712 Sopot, Powstañców Warszawy 55, Poland
(Received February 7th, 2000; revised manuscript March 9th, 2000)
New analogues of proline-rich protein (PRP) fragment were synthesized by the solid
phase method using Boc/Bzl procedure. Dimer of the nonapeptide as well as dimer,
trimer and tetramer of hexapeptide fragments of PRP possessing immunotropic activity
were obtained. Effect of the peptides on the resistance of murine thymocytes to hydrocortisone was the same as that of the reference compounds (hexapeptide and nonapeptide).
Key words: PRP fragments analogues, solid phase peptide synthesis, immunotropic
activity
A proline-rich protein (PRP) was isolated by Janusz et al. from ovine colostrum
[1,2,3]. The polypeptide induces maturation and differentiation of thymocytes and affects humoral and cellular immune reactions both in vivo and in vitro [3,4,5]. PRP is
not species-specific and is active in mice [4,5], humans [6] and rats [7]. The molecular weight of PRP is about 6 kD [2]. PRP contains a high proportion of proline residues (25%) and hydrophobic amino acids (50%) [2]. An active nonapeptide fragment
(NP): Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the products of tryptic digestion of PRP. In mice it showed biological activity similar to that of the
nondigested PRP [3, 8]. Studies on the structure-activity relationship showed that the
C-terminal hexapeptide (HP), Tyr-Val-Pro-Leu-Phe-Pro, of the nonapeptide also exhibited immunotropic activity similar to that of NP and PRP [9]. Although both NP
and HP showed biological activity similar to that of PRP, the same biological effect
was achieved at concentrations higher than those for PRP. The preparation was subsequently found to be rather a complex of proline-rich polypeptides.
The aim of the present paper was to obtain synthetic analogues of NP and HP,
which could replace PRP isolated from colostrum and might show the same or even
* Abbreviations: The symbols of amino acids, peptides and their derivatives are in accordance with the
1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature [Eur. J.
Biochem., 138, 9 (1984)].
980
I. Wirkus-Romanowska et al.
higher immunotropic activity than that of PRP. For this purpose, linear oligomers of
NP and HP, and NP and HP containing corresponding D-amino acid residues at the
N-terminus were synthesized:
H-Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro-OH
H-(Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro)2-OH
H-D-Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro-OH
H-Tyr-Val-Pro-Leu-Phe-Pro-OH
H-(Tyr-Val-Pro-Leu-Phe-Pro)2-OH
H-(Tyr-Val-Pro-Leu-Phe-Pro)3-OH
H-(Tyr-Val-Pro-Leu-Phe-Pro)4-OH
H-D-Tyr-Val-Pro-Leu-Phe-Pro-OH
(NP)
(NP)2
(NP-D)
(HP)
(HP)2
(HP)3
(HP)4
(HP-D)
The immunotropic activity of the peptides was determined by measuring a protective activity of the preparations against apoptosis induced by hydrocortisone (HC)
on murine thymocytes [10,11].
Table 1. Physico-chemical properties of proline-rich protein fragment analogues.
Peptide*
[ a ]20
D
TLC**
c = 1 (5% aq.
acetic acid)
RF
HPLC
RT
H-Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro-OH
NP
–93°
0.48
[20–80%B,
30 min.]
H-(Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro)2-OH
(NP)2
–60°
0.52
[40–90%B,
15 min.]
H-D-Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro-OH
(NP-D)
+44°
0.57
[30–70%B,
20 min.]
H-Tyr-Val-Pro-Leu-Phe-Pro-OH
HP
–70°
0.68
[30–70%B,
H-(Tyr-Val-Pro-Leu-Phe-Pro)2-OH
(HP)2
–88°
0.73
[30–100%B,
H-(Tyr-Val-Pro-Leu-Phe-Pro)3-OH
(HP)3
–100°
0.78
[50–100%B,
Molecular ion
k¢
Calc.
Found
17.5
4.8
1050.2 1051.4
1.6
2082.4 2084.6
2.5
1049.5 1050.8
5.7
10.4
11.7
20 min.]
2.9
734.4
735.8
12.9
20 min.]
4.6
1451.2 1453.0
4.0
2168.7 2168.6
15.1
20 min.]
New analogues of proline-rich protein fragments
981
Table 1 (continuation)
H-(Tyr-Val-Pro-Leu-Phe-Pro)4-OH
(HP)4
–90°
0.72
[isocratic
H-D-Tyr-Val-Pro-Leu-Phe-Pro-OH
(HP-D)
+78°
0.61
[30–70%B,
10.8
59%B]
4.1
2885.6 2887.2
12.6
20 min.]
3.2
734.4
735.6
* The amino acid composition of the peptides is consistent with expectations. ** Solvent system:
1-butanol:ethyl acetate:acetic acid:water (1:1:1:1 v/v).
EXPERIMENTAL
Reagents: A proline-rich polypeptide complex (PRP) was isolated from the ovine colostrum according to
the method of Janusz et al. [2]. RPMI medium was obtained from the Laboratory of Biopreparates of the
Institute of Immunology and Experimental Therapy, Wroc³aw, Poland. Tissue culture plates were from
Costar, USA. Trypan blue, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide), actinomycin D, hydrocortisone were from Sigma, USA. Inactivated fetal calf serum was purchased from
Boehringer Mannheim, Germany.
Polypeptides: NP, (NP)2, NP-D, HP, (HP)2, (HP)3, (HP)4, HP-D were synthesized by the solid-phase
method using the Boc/Bzl procedure. The experimental details are presented in our previous publications
[12,13,14]. HPLC analyses of the peptides were performed on a Beckman Gold System chromatograph
with a Kromasil C-8 column (5 mm particle size, 4.6 ´ 250 mm); (A) 0.1% TFA in water, (B) 80%
acetonitrile in A; flow rate 1 ml/min.; A226. Mass spectra (FAB MS) were measured with an AMD mass
spectrometer with BE geometry. The ion source was equipped with a Cs+gun. Energy of the Cs+ ion was 12
keV.
Mice: Six-week-old C57/BL/6 mices were purchased from the Animal Farm of the Institute of Immunology and Experimental Therapy, Wroc³aw, Poland.
Resistance to hydrocortisone: The assay was performed according to the procedure of Trainin et al.
[10]. Thymuses were removed from mice treated with nembutal and dispersed by pressure through a
stainless steel mesh to obtain cell suspensions. The cells were twice washed with RPMI (Roswell Park
Memorial Institute 1640 medium for cell cultures: 0.02 M bicarbonate buffer containing amino acids, vitamins, and sugars, pH 8.1) and suspended in RPMI containing 10% inactivated fetal calf serum (4 ´ 106
cells/ml). One ml samples of the cell suspensions were then treated for 1 h with the peptides studied (concentrations 0.01–20.0 mg/ml). Then, the samples were treated with hydrocortisone (final concentration 30
mg/ml) and incubated for 18 h at 37°C (5% CO2). After the incubation, viability of cells was determined
using Trypan blue. The results are presented as a relative survival of cells exposed to hydrocortisone treatment and calculated as follows: (number of cell survival with HC/number of cell survival without HC).
The increase in resistance to hydrocortisone in the presence of peptides, Wo, was calculated: Wo = ((Op –
Ok)/Ok) ´ 100, where Op is the resistance in the presence of peptides; Ok – the resistance in the absence of
peptides.
RESULTS AND DlSCUSSION
To evaluate the immunotropic activity of the peptides studied, the resistance of
murine thymocytes to hydrocortisone was determined (Table 2). It was shown that
maturation and differentiation of murine thymocytes was accompanied by an increase in the resistance to hydrocortisone and that the assay correlates well with the
upregulation of the T cell antigen receptor [11]. The assay is relatively simple and re-
982
I. Wirkus-Romanowska et al.
producible. As presented in Table 2, the highest positive protecting activity was
shown by PRP. All other peptides showed a significantly lower activity. Dimerization of the nonapeptide did not improve its protective activity and the dimer expressed even a lower activity. There is a marked difference in the activity of the
nonapeptide and the hexapeptide, the latter showing a lower protective activity. This
is in contrast to the Jerne assay, where both peptides expressed similar, comparable
with PRP, activity [15]. This indicates different effects of peptides on different cell
effector activities. The biphasic character of the relationship between the concentration of the peptides and their activity suggests regulatory activity of the peptides.
In contrast to NP, oligomerization of the hexapeptide strongly improved its protective activity with the trimer and tetramer of HP, surpassing the activity of monomeric and dimeric forms of NP and approaching the activity of PRP isolated from
colostrum.
Table 2. Effect of peptides on resistance of murine thymocytes to hydrocortisone.
Preparation
Concentration
(mg/ml)
Control
PRP
NP
(NP)2
NP-D
HP
Increase of
resistance to
hydrocortisone
Mean standard
error
(%)
Positive protective effect (%,
confidence > 95%)
0.0
20.00
10.00
1.00
0.10
0.01
43.1
47.0
36.3
43.5
43.5
1.6
> 39.7
20.00
10.00
1.00
0.10
0.01
37.8
32.2
26.4
22.6
36.4
1.0
> 24.1
20.00
10.00
1.00
0.10
0.01
23.8
27.1
26.4
18.6
14.7
0.9
> 21.7
20.00
10.00
1.00
0.10
0.01
52.5
40.9
22.5
42.8
46.0
1.1
> 27.2
20.00
10.00
1.00
0.10
0.01
28.2
22.7
16.5
24.1
15.6
0.8
> 19.6
New analogues of proline-rich protein fragments
983
Table 2 (continuation)
(HP)2
(HP)3
(HP)4
HP-D
20.00
10.00
1.00
0.10
0.01
39.7
38.2
40.1
30.2
14.5
1.2
> 28.2
20.00
10.00
1.00
0.10
0.01
33.1
29.1
22.8
27.9
14.8
1.2
> 30.2
20.00
10.00
1.00
0.10
0.01
34.1
33.2
36.0
18.9
9.2
1.2
> 30.2
20.00
10.00
1.00
0.10
0.01
39.1
42.2
30.8
38.8
45.3
1.1
> 27.2
Statistical analysis was tested using two-way between-group ANOVA design and contrast comparison.
Confidence level (p-value) was chosen below 0.05. The positive protecting effect (probability 95%) was
calculated using values of standard mean error in each of the experiments. All values, compared with controls, showed confidence level p < 0.05. All results are means from at least four determinations.
The results obtained show differential cell activating ability of the peptides. This
effect could be a result of various conformations acquired by various peptides affecting their bioavailability. The results obtained also show a possibility of replacing the
colostral PRP by synthetic peptides to induce particular cell effector activities.
Acknowledgments
This work was supported by the Polish State Committee for Scientific Research, grant No. 6PO4B-013-11.
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