Functional roles of Phe12 of deacetyl-thymosin β4 in the impaired blastogenic response of uraemic T-lymphocytes

Phe12 of deacetyl-thymosin β4 is one of the structural essentials for restorative effect on the impaired blastogenic response of uraemic T-lymphocytes. In order to evaluate the functional roles of this phenyl group in the restorative effect on impaired T-lymphocytes, two analogues, [1- Nal12]deacetyl-thymosin β4 and [Cha12]deacetyl4 thymosin β4, were synthesized by a solid-phase method and evaluated for restorative effect on the impaired blastogenic response of uraemic T-lymphocytes. The results indicated that [1-Nal12]deacetyl-thymosin β4 which had a bulky naphthyl ring showed a stronger restorative effect than that of deacetyl-thymosin β4, but it was slightly weaker than that of [Phe(4F)12]deacetyl-thymosin β4. However, [Cha12]deacetyl-thymosin β4 showed no restorative effect on the impaired blastogenic response of uraemic T-lymphocytes.


Introduction
The impairment of immunological responsiveness in uraemic patients is well known. All aspects of the immune response appear to be affected by the uraemic state. The numbers, subpopulations and reactivities of circulating lymphocytes may be altered by uraemia. 1,2 This impairment has been implicated in easy susceptibility to infections and increased incidence of malignancy.
Thymosin b 4 , an N-terminal acetylated peptide containing 43 amino acid residues, was rst isolated from calf thymus by Low et al. 3 and has the following amino acid sequence: Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Leu-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH. This peptide exhibits several biological activities that are important for maturation and functioning of the immune systems. 4 Previously 5 7 we reported syntheses of deacetyl-thymosin b 4 and its fragments and that some of the fragments could have a restorative effect on the impaired cell-mediated immunological functions. We also noticed that the acetyl group at the N-terminal serine residue of thymosin b 4 , is not required for the restorative effect on the impaired cell-mediated immunological functions. 5 In an earlier paper, 8 we reported that the synthetic [Phe(4F) 12 ]deacetyl-thymosin b 4 exhibited stronger restorative effect on the impaired blastogenic response of T-lymphocytes isolated from uraemic patients than that of our synthetic deacetyl-thymosin b 4 . In this study, the strong electron-withdrawing uoride atom on the para-position of the aromatic ring results in an analogue that possesses stronger activity than that of the parent molecule. 8 This result seems to suggest that modi cation of the Phe residue of thymosin b 4 could produce more potent analogues capable of a restorative effect on impaired blastogenic response of T-lymphocytes.
The purpose of the present study was to synthesize two thymosin b 4 analogues, [1-Nal 12 ]deacetyl-thymosin b 4 and [Cha 12 ]deacetylthymosin b 4 by the solid-phase method and to compare the restorative effect of these two analogues on the impaired blastogenic response of uraemic T-lymphocytes.
After the deprotection, the resin was removed by ltration and the ltrate was evaporated under reduced pressure and the residue was solidi ed by addition of anhydrous peroxide free ether to give a crude peptide. The resulting powder was dissolved in H 2 O (6 ml). The solution was treated with Amberlite CG-4B (acetate form, approximately 3 g) for 30 min, and ltered by suction and evaporated in v acuo. The residue was dissolved in H 2 O (10 ml). The solution, after addition of dithiothreitol (20 mg), was incubated at 608 C under N 2 gas for 36 h. The solvent was evaporated off in v acuo and the residue was dissolved in a small amount of 1%AcOHand then applied to a column of Sephadex G-25 (2.3 3 95 cm), which was eluted with the same solvent. Individual fractions (5 ml each) were collected and absorbancy at 230 nm was determined for each fraction. The fractions corresponding to the front main peak were combined and the solvent was removed by lyophilization. The peptide was further puri ed by semi-preparative PR-HPLC. The semi-preparative PR-HPLC was performed on a Nucleosil C18 column (250 3 10 mm I.D.; 7 mm particle size; Macherey Nagel). Solvent A was 0.05% TFA in water and solvent B was 60% acetonitrile in solvent A. A linear gradient was applied from 10 to 50% B during 50 min, at a ow rate of 3.0 ml/min. Detection of the peptide was set at 230 nm. The major peak was lyophilized to give the puri ed product. [1-Nal 12 ]deacetyl-thymosin b 4 ; 20.3 mg (20%, calculated from the starting Cterminal amino acid). [Cha 12 ]deacetyl-thymosin b 4 ; 22.6 mg (23%, calculated from the starting C-terminal amino acid) (Fig. 1).

Patient selection
Three uraemic patients who needed dialysis treatment three times a week and were suffering from recurrent infectious diseases (pneumonia and tuberculosis) were selected. Examination of cellular immunocompetence of these patients revealed a signi cant decrease in blastformation by PHA. 3 H-thymidine incorporation values of these patients were 11 826, 12 042 and 12 153 cpm respectively (normal values: 41 195± 42 659 cpm).
Venous blood was obtained from these uraemic patients for the uorometric blast-formation test. Venous blood samples from three healthy donors were used as a control. The uorescence excitation spectrum was measured with an Oyo-Bunko ULOG-FLOUSPEC 11A uorometer. Kits for the uorometric blast-formation test were purchased from Japan Immunoresearch Laboratories Co. Ltd (Japan).

Fluorometric blast-formation test
A 3 ml aliquot of venous blood was drawn into a syringe containing 25 U/ml of heparin and then mixed with 3 ml of PBS. Lymphocytes were isolated in a Hypaque-Ficoll gradient. Isolated lymphocytes were adjusted to 1 0 3 10 6 /ml with PBS. The lymphocytes were cultured in 0.5 ml of RPMI 1640 (Gibco) with 10% FCS (Dainippon Pharmaceutical Co.) in microplates. Cultures of each combination were incubated at 378 C in the presence of one of the peptides in a humidi ed atmosphere of 5%CO 2 in air or 12 h and then PHA (0.125%) was added to each well and incubation was continued under the same conditions for 60 h. Lymphocytes in each well were transferred into a test tube and centrifuged for 10 min at 240 g, then the supernatant was removed. A 2 ml aliquot of 0.125% SDS was added to the residue and stirred for 20 min at room temperature; lymphocytes were completely destroyed and solubilized by this procedure. Ethidium bromide solution (2 ml) was added to the above solution and the mixture was stirred for 15 min at room temperature. The uorescence excitation spectrum was measured as previously described. 7

Results
In order to construct the peptide chain, the Fmoc-based solid-phase method was employed. Fmoc-Ser(tBu)-Pam-resin was placed in the reaction vessel and the combination of piperidine treatment and DCC plus HOBT procedure served to elongate the peptide chain manually according to the usual method.
We encountered no serious dif culties during the elongation of the entire sequences of the two analogues, although the double coupling procedure was employed when the resin became positive to the ninhydrin test, after a single coupling. The coupling cycle included a capping step with acetic anhydride (5 min) to prevent the formation of deleted sequences. The amino acid compositions of the protected peptide resins thus assembled were in good agreement with those predicted by theory after acid hydrolysis with 12 N HCl-proprionic acid (1:1). The protected peptide resins thus obtained were then treated with 2 M HBF 4 -thioanisole in TFA at 48 C for 90 min to cleave the peptide chain from the resin and at the same time to remove all side-chain protecting groups employed. The Met(O) residue was reduced back to Met in two steps, rstly with 2 M HBF 4thioanisole in TFA during the above acid treatment, and secondly with dithiothreitol during incubation of the unprotected peptide.
The crude peptides thus obtained were then successively puri ed by gel-ltration on Sephadex G-25 and semi-preparative HPLC. The two puri ed peptides exhibited single peaks on analytical HPLC. The two products possessed amino acids in ratios consistent with those predicted from the sequences of the two analogues after acid hydrolysis. The homogeneity of the peptides was checked by TLC, HPLC, amino acid analysis after 6 N HCl hydrolysis, and FAB-MS spectrometry. Physicochemical data for the synthetic analogues are shown in Tables  1 and 2.  The immunological effects of the synthetic deacetyl-thymosin b 4 , [Phe(4F) 12 ]deacetyl-thymosin b 4 , [1-Nal 12 ]deacetyl-thymosin b 4 and [Cha 12 ]deacetyl-thymosin b 4 were examined by the JIMRO (Japan Immunoresearch Laboratories Ltd) uorometric blast-formation test. Responses of T-lymphocytes to mitogenic stimulation were signi cantly lower in uraemic patients that were those of normal persons. The in vitro effect of the synthetic peptides on the impaired PHA response of T-lymphocytes from uraemic patients is shown in Table 3.

Discussion
Comparison of the stimulation index (SI) values of the blastogenic transformation of T-lymphocytes into lymphoblasts with mitotic activity upon PHA stimulation shows that in the case of the uraemic patients investigated, the synthetic analogue, [1-Nal 12 ]deacetyl-thymosin b 4 which had a bulky naphthyl ring exhibited stronger restorative activity than that of our synthetic deacetyl-thymosin b 4 , but it was a little bit weaker than that of [Phe(4F) 12 ]deacetyl-thymosin b 4 . However, the synthetic [Cha 12 ]deacetyl-thymosin b 4 had no restorative effect even at a much higher concentration (Table 4).
Those results exhibited that not only 4-uorophenyl ring of deacetyl-thymosin b 4 but also more bulky naphthyl ring of deacetyl-thymosin b 4 could bind with receptors of T-lymphocytes more strongly than a phenyl ring of deacetylthymosin b 4 . On the contrary, another analogue, [Cha 12 ]deacetyl-thymosin b 4 which contains an aliphatic ring at position of 12 instead of an aromatic ring showed no restorative effect. This result seems to suggest that aromaticity at position of 12 of thymosin b 4 plays signi cant roles for restorative activity on impaired blastogenic response of T-lymphocytes.