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Molecular cloning and expression of megakaryocyte potentiating factor cDNA
T Kojima1, M Oh-eda, K Hattori
1Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
Abstract:
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.
Insights
Researchers isolated the human megakaryocyte potentiating factor (hMPF) cDNA, revealing a 622-amino acid protein. The 33-kDa secreted form exhibits megakaryocyte potentiating activity, unlike the 30-kDa form.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Human megakaryocyte potentiating factor (hMPF) was previously purified from human pancreatic cancer cells (HPC-Y5).
- The exact molecular basis and functional characteristics of hMPF require further elucidation.
Purpose of the Study:
- To isolate and characterize the cDNA encoding human megakaryocyte potentiating factor (hMPF).
- To investigate the expression and functional activity of recombinant hMPF in different cell systems.
Main Methods:
- hMPF cDNA isolation from HPC-Y5 library using PCR and plaque hybridization.
- Expression of hMPF cDNA in COS-7 and Chinese hamster ovary (CHO) cells.
- Purification and activity assays of recombinant hMPF (rhMPF) from cell culture supernatants.
Main Results:
- hMPF cDNA encodes a 622-amino acid polypeptide (68 kDa theoretical mass), with HPC-Y5 cells secreting a 33-kDa form.
- Expressed hMPF cDNA yielded a 33-kDa active form in COS-7 cells and both 33-kDa and 30-kDa forms in CHO cells.
- The 33-kDa rhMPF demonstrated megakaryocyte potentiating activity, while the 30-kDa form did not, attributed to the absence of C-terminal residues.
Conclusions:
- The isolated hMPF cDNA provides a molecular basis for the protein.
- Differential processing or post-translational modification in CHO cells leads to distinct hMPF isoforms.
- The C-terminal 25 amino acids are crucial for the megakaryocyte potentiating activity of hMPF.