http://www.abbs.info e-mail:[email protected] ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(2): 177-181                                    CN 31-1300/Q

　

Synthesis of Osteogenic Growth Peptide and Its Synergetic Effect with Granulocyte-Colony Stimulating Factor

 

LIU Zhi-Hui, SHAO Yun-Chao¹, CHEN Hong-Hong², SHI De-Yuan¹, CHEN Tong-Yi¹, CUI Da-Fu*

( Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031; ¹Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai 200030; ²Shanghai Yi Zhong Biological Technique Co., Ltd.., Shanghai 201203 )

 

Abstract    Osteogenic growth peptide (OGP) has been synthesized through Fmoc solid phase synthesis procedure. The purity of synthetic OGP (sOGP) is over 98.6% identified by HPLC, the amino acid sequence and electro-spray mass spectroscopy are consistent with theoretical values. The synergetic effect of sOGP with recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the hematopoiesis was investigated in normal mice. To assess the synergy of sOGP with rhG-CSF, two schemes were designed. In one scheme rhG-CSF was used at the last 8 days of a 13-day treatment with sOGP, while in the other one both cytokines were given concurrently for 10 days [sOGP, 0.5 nmol/day (mouse); rhG-CSF, 2 µg/day (mouse)]. Both schemes showed that sOGP remarkably synergized with rhG-CSF on increment of white blood cell number and lymphocyte number in peripheral blood without any change of red blood cell and platelet counts. Quantitative differential analysis of bone marrow and histological examination of the spleen and sternum showed that sOGP plus rhG-CSF did not cause abnormal hyperplasia, so sOGP is a very hopeful new drug to improve the effectiveness of clinical used rhG-CSF.

 

Key words    OGP; G-CSF; synergy; hematopoiesis; solid phase synthesis

 

Now a newly found peptide, named osteogenic growth peptide (OGP), working like a hematopoietic modulator, increases blood and bone marrow (BM) cellularity ^[1]. The OGP, a 14-amino acid, highly conserved, H4 histone-related peptide, is characterized in regenerating BM and normal serum. The sequence of OGP is ALKRQGRTLYGFGG. OGP was abundant in human and other mammalian blood, mainly in the form of OGP-OGP-binding protein complex ^[1-3]. Meanwhile, its C-terminal pentapeptide was also found at a high level in mammalian serum and osteogenic cultures^[4]. In vivo OGP increased osteogenesis^{[2, 5, 6]} and blood granulocyte count, enhanced engraftment of bone marrow transplants of mice^[1]. The C-terminal pentapeptide had also some effects on peripheral neutrophil recovery after cyclophosphamide-induced aplasia, and stem/progenitor cell mobilization from BM into peripheral blood^[7]. On human CD34⁺ enriched cells, the activity of stem cell factor (SCF) and granulocyte-megakaryocyte colony-stimulating factor is increased by the pentapeptide^[8].

It is suggested that OGP may enhance some cytokines and/or receptor synthesis, and synergize with some other cytokines, such as rhG-CSF, which is a popular clinically used hematopoietic cytokine, at granulocytosis. The detailed mechanism of its action on hematopoiesis is not clear yet.

In this paper, OGP was synthesized (sOGP, which has the same sequence and the same function of natural OGP ^[1]) by Fmoc system and the synergetic effect of sOGP with rhG-CSF was studied.

 

1  Materials and Methods

1.1  Materials

Male BALB/C weighing 16~18 g were purchased from Shanghai Laboratory Animal Center, the Chinese Academy of Sciences. Fmoc-amino acids were purchased from ABI Company. P-alkoxybenzyl alcoholic (HMP)-resin was purchased from Sigma Company. Human recombinant G-CSF (rhG-CSF) was purchased from Kirin Brewery Co., Ltd. (China). Human albumin was purchased from Shanghai RAAS Blood Products Co., Ltd..

1.2  Synthesis of OGP

Fmoc solid phase synthesis (SPS) was performed manually starting from a P-alkoxybenzyl alcoholic (HMP)-resin, N-terminal and side chain protected amino acids were connected step by step as the sequence of natural OGP by Fmoc system. These side-chain protected amino acids are: Fmoc-Arg (Pmc), Fmoc-Lys (Boc), Fmoc-Thr (tBu) and Fmoc-Tyr (tBu). After the final cycle, the resin was cleavaged by TFA containing 5% p-cresol and 100 mg phenol for 1 h at room temperature. The other side-chain deprotection and resin cleavage conditions were also tried, 2 h reaction, 3 h reaction, or reactive solvent containing 2% water. The crude peptide was desalted on a Sephadex G10 and purified by FPLC (ACTA purifier, Amersham Pharmacia). The column used was SP Sepharose XL (column volume: 1 mL; dimension: 0.7 cm × 2.5 cm). The purity of sOGP was identified by rpHPLC (Hewlett Packard series 1050, HP Company; column: Beckman C8 4.6 mm × 250 mm), the exactness and sequence of sOGP was identified by electro-spray mass spectroscopy (MS) and protein sequence analyzer (ABI 491A protein sequence analyzer).

1.3  Cytokines and Treatment Schemes

Mice were injected subcutaneously with sOGP, or rhG-CSF, or a combination of these two growth factors. The carrier consists of 10 g/L human albumin in sterile saline. As shown in Table 1, two schemes were applied. Scheme 1, the mice received either sOGP 0.5 nmol /day from day 0 to 12, or rhG-CSF 2 µg/day from day 5 to 12 (from day 0 to day 4, carrier was injected), and a combination group with only sOGP at first 5 days and both at the last 8 days of the 13-day treatment. Scheme 2, the mice received either sOGP 0.5 nmol /day from day 0 to 9, or rhG-CSF 2 µg /day from day 0 to 9, or a combination of sOGP and rhG-CSF concurrently for 10 days. In both schemes, the control mice were injected with carrier only.

 

Table 1 Normal mice treated with sOGP plus rhG-CSF, sOGP alone and rhG-CSF alone (Days of treatment)

 

1.4  Quantitative differential analysis for peripheral blood (PB) and BM

In scheme 1, PB was collected on days: 0, 4, 7, 9, 11, and 13; while in scheme 2, PB was collected on days: 0, 2, 4, 6, 8 and 10. The mice were bled retro-orbitally, and the samples were analyzed with differential cell count with Coulter counter (MEK-5108-K, Japan). In Scheme 1, BM nucleated cells were also counted with hemocytometer and the stained BM smears were then differentially numerated.

1.5  Histological examination of the spleen and sternum

In scheme 1, histological examination of the spleen and sternum was performed with Bouin’s-fixed or formaldehyde-fixed paraffin-embedded sections stained with hematoxylin and eosin and by the Giemsa method, observed under optic microscope.

1.6  Statistical analysis

The statistical analysis of the results was done using the student t-test, results are given as mean±SEM.

 

2  Results

2.1  The purity and exactness of sOGP

The purity of sOGP after purification is over 98.6% identified by HPLC; the molecular weight of sOGP is 1523.5 kD identified by electro-spray mass spectroscopy, which is consistent with the theoretical value (1523.75 kD); the result of whole sequence analysis of sOGP by N-terminal protein sequence analyzer is consistent with the theoretical sequence of OGP. The results of side-chain deprotection and resin cleavage under different conditions indicated that 1 h reaction time without water is the best.

2.2  The synergetic effect between sOGP and rhG-CSF on blood cellularity of mice

In scheme 1 that rhG-CSF was used for the last 8 days of a 13-day treatment with sOGP, there was synergistic increase in circulating WBC as compared with sOGP alone or rhG-CSF alone at day 11 and 13 [Fig.1(A)]. At day 11, a marked increase of circulating WBC after the coinjection of sOGP plus rhG-CSF to (39.7±4.5)×10⁹ cells/L was observed as compared with (16.1±1.1)×10⁹ cells/L in rhG-CSF treated mice and (6.7±0.5)×10⁹ cells/L in sOGP treated mice. At day 13, a marked increase in the number of circulating WBC after the coinjection of sOGP plus rhG-CSF to (35.1±5.67) × 10⁹ cells/L was observed as compared with (17.6±1.6)×10⁹ cells/L in rhG-CSF treated mice and (6.6±0.4)×10⁹ cells/L in sOGP treated mice [Fig.1(A)]. A marked increase of circulating lymphocyte could also be seen at day 11 and day 13 [Fig.1(B)]. In scheme 2, coinjection of sOGP and rhG-CSF for 10 days causes a higher synergistic increase in circulating WBC than sOGP alone or rhG-CSF alone at day 10, with increases of (53.1±2.7)×10⁹ cells/L, (22.7±1.9)×10⁹ cells/L and (6.3±1.2)×10⁹ cells/L respectively [Fig.2(A)]. What’s more, a marked increase of the circulating lymphocyte was also caused by the coinjection of these two factors at day 10 [Fig.2(B)].

 

 

Fig. 1 G-CSF used for the last 8 days of a 13-day treatment with OGP causes a synergistic increase of WBC (A) and a synergistic increase of LY (B)

n = 7; *P < 0.0005， ^#P < 0.05 compared with the group that injected with rhG-CSF alone

 

Fig. 2 Daily coinjection of sOGP and rhG-CSF for ten days causes a synergistic increase of WBC (A) and a synergistic increase of LY (B)

n = 10; *P < 0.0005 compared with the group that injected with rhG-CSF alone.

 

The proportion of the granulocyte and lymphocyte was altered slightly in sOGP plus rhG-CSF as compared with rhG-CSF or OGP alone. The red blood cells (RBC) and platelets (PLT) counts kept unchanged in different groups (Table 2), showing that the synergistic role mainly acts on neutrophils or perhaps lymphocytes in blood. The BM cellularity was not altered by the treatment of different cytokines. An increase in myelocytes/metamyelocytes was seen in sOGP and sOGP plus rhG-CSF compared with the carrier controls. An increase in neutrophils cells was seen in sOGP, sOGP plus rhG-CSF and rhG-CSF 16 hours later after the cytokine injection (Table 3). No significant increase was noted in sOGP plus rhG-CSF treated mice as compared with sOGP or rhG-CSF alone.

 

Table 2 Differential counts in blood of normal mice treated with OGP, OGP+G-CSF and G-CSF
(G-CSF used for the last 8 days of a 13-days treatment with OGP)

 

2.3  The influence of sOGP plus rhG-CSF at sternum BM and spleen of mice

Histological examination of sternum BM shows that the percentages of erythroid line, myeloid line and megakaryocyte were normal in sOGP, sOGP plus rhG-CSF and rhG-CSF treated mice. The spleens of sOGP plus rhG-CSF treated mice and rhG-CSF treated mice show increased myelopoiesis, erythropoiesis, and megakaryocytopoiesis as compared with the spleens of the carrier control and sOGP treated mice. Although the average weight of spleen of the sOGP plus rhG-CSF treated mice was larger than that in the rhG-CSF treated mice, yet there was no significant difference.

 

Table 3 The influence of OGP and G-CSF on BM total nucleated cell number and the percent of differential cells when G-CSF was used for the last 8 days of a 13-days treatment together with OGP