(Received for publication, February 27, 1995; and in revised form, June 23, 1995)
From the
The human megakaryocyte potentiating factor (hMPF) has been previously purified from a culture supernatant of human pancreatic cancer cells HPC-Y5 (Yamaguchi, N., Hattori, K., Oh-eda, M., Kojima, T., Imai, N., and Ochi, N.(1994) J. Biol. Chem. 269, 805-808). We have now isolated hMPF cDNA from a HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The hMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular mass of 68 kDa, although HPC-Y5 cells secrete a 33-kDa form of hMPF. Human MPF does not show any significant homology with other previously described sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and megakaryocyte potentiating activity was detected in their culture supernatant. The COS-7 cells secreted only a 33-kDa recombinant hMPF, whereas an additional 30-kDa form was detected in the culture medium of CHO cells. The 33-kDa rhMPF purified from CHO cells showed megakaryocyte potentiating activity, but not the purified 30-kDa rhMPF. The difference in structure and activity between the 33- and 30-kDa forms of hMPF was ascribed to the existence in the 33-kDa form of the C-terminal 25 amino acid residues.
Megakaryocytes originate from pluripotent hematopoietic stem
cells through a complex process involving commitment of the pluripotent
hematopoietic progenitor to megakaryocytic precursor cells and their
mitotic amplification. The regulatory system governing
megakaryocytopoiesis and platelet production is thought to take place
in at least two stages, 1) during proliferation and differentiation of
megakaryocytic progenitor cells, leading to the production of
megakaryocytes, and 2) during maturation of megakaryocytes which leads
to the production of platelets. Megakaryocyte proliferation is thought
to be dependent on an essential megakaryocyte colony-stimulating
factor, and this proliferation can be potentiated in vitro by
ancillary megakaryocyte potentiators
(Meg-POT)()(1, 2, 3) , which also
stimulate the maturation of the megakaryocytes. We have recently
identified, in the culture supernatant of the human pancreatic cancer
cells HPC-Y5, a novel megakaryocyte potentiating factor (hMPF), which
stimulates the megakaryocyte colony forming activity of murine
interleukin-3 in mouse bone marrow cell culture(4) . The factor
was found to consist of a single polypeptide of about 32 kDa with at
least one N-linked sugar chain. In this paper, we describe the
molecular cloning and characterization of the cDNA encoding human MPF.
In order to isolate a full-length hMPF
cDNA, approximately 1.7 10
clones were screened
with Q197A(7) , a 197-base pair MPF cDNA fragment generated by
PCR. This fragment was labeled by
P using a Random Primer
Labeling kit (TAKARA) in the presence of QS and AA primers instead of
the kit's random primers, and used as hybridization probe.
Filters (Hybond N
, Amersham) were hybridized with the
P-labeled Q197A probe. Isolated clones were subjected to in vivo excision of pBluescript SK(-) phagemid
(Stratagene), and plasmid DNA was prepared by the standard method.
For 30-kDa MPF purification, the
cell culture supernatant was filtrated, concentrated, and MPF was
precipitated at 4 °C with ammonium sulfate at 50% saturation. The
precipitate was dissolved in 10 mM Tris-HCl (pH 7.4)
containing 1 M ammonium sulfate, and applied to a
Phenyl-Sepharose Fast Flow column (5 15 cm) equilibrated with
the same buffer. MPF was eluted with 10 mM Tris-HCl (pH 8.5)
containing 0.1% Tween 20. The 30-kDa MPF was further purified by a
combination of reverse-phase HPLC and TSK G3000SW gel permeation
chromatography as described above. Fractions containing MPF were
identified by Western blotting at each step of the purification.
Figure 1: A, strategy for PCR. The cDNA insertion is represented by a rectangle. T3 and T7, promoters for T3 and T7 polymerases, respectively. ZAP phage sequence is shown as solid bars. Primers used for amplification are denoted by arrows. Four inner oligonucleotide primers (KS1, KS2, KA1, and KA2) derived from this DNA fragment were then synthesized and two sets of primers, T7 promoter sequence primer/KS1 and T3 promoter sequence primer/KA1, were used to carry out PCR amplification using HPC-Y5 cDNA library as template. Aliquots of the PCR products were subjected to further amplification in the presence of either T7/KS2 or T3/KA2 primers. The PCR products (200 and 240 base pairs) were gel-purified, and their sequences were determined by direct sequencing. One more set of primers, sense primer QS and antisense primer AA, were synthesized based on this sequence information, and used for the final PCR amplification. B, amino acid sequences and corresponding oligonucleotide sequences used as primers for PCR. Numbering of sequences 1-6 was taken from that of the corresponding amino acids in Fig. 2. The primers contain all the possible combinations of nucleotide sequences that correspond to a given amino acid. Code for nucleotides is as follow: R, either A or G; Y, either C or T; N, A, G, C, or T.
Figure 2: cDNA structure and deduced amino acid sequence of MPF precursor. The nucleotide sequence of pKPO27 cDNA and the deduced amino acid sequence are shown. The numbering of nucleotides in the cDNA is indicated at the end of each line. The predicted amino acid sequence of pre-proMPF is numbered by designating the first methionine as 1. The four potential N-linked glycosylation sites (Asn-X-Ser/Thr, X, any amino acid except for Pro) are indicated by N-CHO. A potential polyadenylation signal is dot underlined in the 3`-untranslated region. The bold underlined Arg-X-Arg-X-Arg-Arg precedes the potential proteolytic cleavage site.
Twenty-three positive clones were
isolated from the cDNA library (II) (1.7 10
) by
plaque hybridization using the Q197A fragment as a probe. One of them,
clone pKPO27, was subjected to further analysis, since it contained the
longest insert.
Figure 3: Detection of MPF in a culture supernatant of transfected COS cells. The culture supernatant of cDNA-transfected COS cells was concentrated by Centriprep-10 (Amicon), electrophoresed on a 12% gel (120 V, 2 h), and transferred on a nitrocellulose membrane. MPF was detected with rabbit anti-MPF antiplasma and alkaline phosphatase-conjugated antibody to rabbit IgG (Cappel). Lane 1, culture supernatant of mock transfected COS cells; lane 2, cDNA transfected COS cells.
Figure 4: SDS-PAGE analysis of the purified recombinant MPF. The two MPF forms (100 ng) were electrophoresed on a 12% gel (120 V, 2 h) and stained with Coomassie Brilliant Blue R-250. Apparent molecular weight was determined by using a molecular weight standard mixture (Bio-Rad). Lane 1, 33-kDa MPF; lane 2, 30-kDa MPF.
The C-terminal sequences were
identified by analyzing the C-terminal fragments obtained from the 33-
and 30-kDa MPF after cyanogen bromide cleavage and endoproteinase Asp-N
digestion. The amino acid sequence of the C-terminal peptide derived
from the 30-kDa MPF was determined as
Met-Asp-Ala-Leu-Arg-Gly-Leu-Leu-Pro-Val-Leu-Gly-Gln-Pro-Ile-Ile-Arg.
The C-terminal peptide obtained from the 33-kDa MPF by cyanogen
bromide cleavage was further digested with endoproteinase Asp-N, since
this peptide was too long to determine the complete amino acid
sequence. The peaks obtained by reverse phase HPLC were analyzed in a
protein sequencer, and their amino acid sequences,
Met-Asp-Ala-Leu-Arg-Gly-Leu-Leu-ProVal-Leu-Gly-Gln-Pro-Ile-Ile-Arg-Ser-Ile-Pro-Gln-Gly-Ile-ValAla-Ala-Trp-Arg-Gln-Arg-Ser-Ser-Arg and Asp-Pro-Ser-Trp-Arg-Gln-Pro-Glu-Arg
were
identified. These results demonstrated that the 30-kDa rMPF was
generated by truncation of the C-terminal 25 amino acid residues from
the 33-kDa rMPF.
Figure 5:
Megakaryocyte potentiating activities of
natural and recombinant MPFs. Bone marrow cells (2
10
) were cultured as described under ``Materials and
Methods.'' Megakaryocyte colonies were scored using dehydrated
cultures stained for acetylcholine esterase. Each point represents the
mean of duplicate cultures. Similar results were obtained with repeated
experiments, and a typical pattern is shown here.
, natural MPF;
, 33-kDa rMPF;
, rhIL-6;
, mouse IL-3
alone.
Figure 6: Megakaryocyte potentiating activities of 30- and 33-kDa rMPFs. The Meg-POT activities of two molecular species of MPF were determined as described under ``Materials and Methods.'' Each value represents the mean of duplicate cultures. Similar results were obtained with repeated experiments, and a typical pattern is shown here.
Using a sandwich enzyme-linked immunosorbent assay, as shown in Fig. 7, the antigenicity of 33-kDa rMPF was detected with the prepared anti-MPF monoclonal and polyclonal antibodies, whereas the 30-kDa rMPF displayed only a weak antigenicity, approximately 1/50 of 33-kDa rMPF, for any antibody. Thus, deletion of the C-terminal 25 amino acids from the 33-kDa rMPF decreased the antigenicity of the protein for these antibodies.
Figure 7: Immunological activities of 30- and 33-kDa rMPFs. Two molecular species of MPF samples were incubated in a 96-well plate coated with mouse anti-MPF monoclonal antibody against MPF. The detection was then carried out with rabbit anti-MPF antiplasma and anti-rabbit IgG-horseradish peroxidase-conjugated antibody, and absorbance was read at 490 nm.
Figure 8:
MPF mRNA distribution in human tissues. A, poly(A) RNA (2 µg) prepared from
HPC-Y5 was size-fractionated on formaldehyde agarose gels (1%), and
transferred to Hybond-N
. B, lane 1,
heart; lane 2, brain; lane 3, placenta; lane4, lung; lane5, liver; lane6, skeletal muscle; lane7, kidney; lane8, pancreas.
We have isolated a cDNA for hMPF, which exhibits a Meg-POT
activity in the presence of murine IL-3 in a colony-forming assay with
mouse bone marrow cells, by means of PCR and plaque hybridization
methods. By structural analysis of the cDNA, the primary translation
product of MPF was shown to consist of 622 amino acid residues. The
sequence of the mature MPF protein (33 kDa) starts at Ser and ends at Arg
in complete agreement with that of
natural MPF(4) , and the C-terminal Arg residue of the mature
33-kDa MPF is followed by a 336-amino acid polypeptide. Several
polypeptide hormones are synthesized as large precursor proteins, such
as human pro-parathyroid hormone related protein(12) , porcine
proendothelin-1(13) , human pro-transforming growth factor
1(14) , mouse pro-nerve growth factor(15) , and
human pro-platelet-derived growth factor(16) . In each of these
cases, active polypeptide sequences are usually bonded by pairs of
basic amino acid residues such as Arg-Arg or Lys-Arg, and it is at
these sites that proteolytic processing occurs. Watanabe et al.(17, 18) have demonstrated that the sequence
Arg-X-Arg/Lys-X-Lys/Arg-Arg is a signal for precursor
cleavage catalyzed with furin, a mammalian homologue of the yeast
precursor-processing endoprotease Kex2, within the constitutive
secretory pathway in nonendocrine cells. The sequence
Arg
-Pro-Arg-Phe-Arg-Arg
deduced from the
MPF cDNA matches this cleavage signal model. The C-terminal Arg
residues of the 33- and 30-kDa rMPFs, Arg
, and
Arg
, are located 9 and 34 amino acid residues upstream of
this cleavage signal, respectively. Moreover, the hydrophobicity
profile predicts that the signal is located in a hydrophilic region
between Trp
and Glu
. This suggests that the
MPF precursor would fold and then, be successively processed with
furin-like and trypsin-like proteases. A similar processing has been
found in leukocyte-derived natural interferon-
(19) and
interferon-
(20, 21) . Although cleavage of the
precursor gives rise to the mature MPF, it is not known if other
peptides released from the C-terminal region of the MPF precursor are
also biologically active.
The 33-kDa rMPF displayed a Meg-POT
activity, whereas the 30-kDa rMPF, which was generated by truncation of
the C-terminal 25 amino acid residues from the 33-kDa rMPF, possessed
no Meg-POT activity equivalent to that of natural hMPF and IL-6.
Furthermore, the 30-kDa rMPF was not antigenic for any of the
antibodies raised against the rMPF precursor protein produced in E.
coli, although the 30- and 33-kDa forms of rMPF are detectable in
Western blotting analysis with these antibodies. Therefore, the
C-terminal 25 amino acid residues are not epitopes. Circular dichroism
(CD) analysis showed no influence of C-terminal truncation on the
secondary structure, that is, there are no significant differences
between the CD spectra of the 33- and 30-kDa rMPFs. ()However, Hattori et al. (22) have
previously shown that monoclonal antibodies can detect subtle
conformational changes in local areas within a protein molecule during
denaturation, by determining affinity changes. Shortle and Meeker (23) have also found that removal of the C-terminal 13 amino
acids of staphylococcal nuclease is sufficient to destabilize the
protein native state or stabilize it in the denatured state. Therefore,
loss of the C-terminal 25 amino acids might give rise to a local
conformational change, altering the antigenicity of MPF.
Slodowski et al. (24) have previously examined the biological
function of the C-terminal 20 amino acid residues of IFN-. The
antiviral activities of the INF-
analogue proteins lacking 14
(C-14) or more amino acids from the C-terminal are less than 2% of the
native protein activity. In contrast, a variant, shortened by 11 only
amino acids (C-11), showed a higher biological activity than the wild
type INF-
. CD analysis showed no influence of these C-terminal
truncations on the secondary structure. In the case of 33-kDa MPF,
however, removal of the Arg
-Arg
region
leads to a drastic loss in biological activity and antigenicity, while
truncation at Arg
, excising the 33-kDa rMPF from its
precursor, may result in a significant increase in activity. The loss
of biological activity of the 33-kDa rMPF due to the absence of its
C-terminal peptide may be linked to the delicate regulation of
megakaryocytopoiesis in vivo. It remains to be determined
whether MPF exists in vivo as a precursor of the shorter
species, 33- and 30-kDa MPF.
MPF was originally isolated from a pancreatic cancer cell line, HPC-Y5, however, Northern blot analysis shows that the MPF gene is expressed in the lung, not in the pancreas. Since the structure of the lung consist of aloveoli and abundant capillaries, and is fraught with the danger of bleeding, this would suggest that MPF might play a role in the growth of megakaryocytes and/or the regulation of platelet production. But since MPF is weakly expressed in heart, placenta, and kidney, MPF may exhibit other biological activities.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49441 [GenBank]for the human mRNA for megakaryocyte potentiating factor.