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