Characterization, Molecular Cloning and Expression of Megakaryocyte Potentiating Factor

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Abstract
We examined whether the conditioned media of 64 kinds of cell lines, which have been maintained by a protein-free culture system, could produce megakaryocyte potentiating (Meg-POT) activity. In these cell lines, HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from its conditioned medium by a combination of ion-exchange chromatography, gel filtration and reversed-phase HPLC. The purified MPF showed Meg-POT activity almost equal to human (Hu) interleukin 6 (IL-6) in the presence of murine IL-3 in a colony-forming assay with mouse bone marrow cells. The molecular weight of MPF was estimated to be 33 kDa by SDS-PAGE. Glycopeptidase F digestion and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF may be a novel cytokine which has Meg-POT activity. Then, we isolated HuMPF cDNA from an HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The HuMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular weight of 68 kDa, although HPC-Y5 cells secrete a 33 kDa form of HuMPF. HuMPF cDNA does not show any significant homology with other known sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and Meg-POT activity was detected in their culture supernatant. The COS-7 cells secreted only a 33 kDa recombinant (r)HuMPF, however, an additional 30 kDa form was detected in the culture medium of CHO cells. The 33 kDa rHuMPF from CHO cells showed Meg-POT activity, but not the purified 30 kDa rHuMPF. The difference in structure and activity between the 33 and 30 kDa forms of HuMPF was ascribed to the existence in the 33 kDa form of the C-terminal 25 amino acid residues. The expression of MPF mRNA was examined by Northern blot analysis using labeled MPF cDNA as a probe. MPF mRNA was detected in HPC-YS cells, with an approximate molecular size of 2.4 kb. We also examined the expression of the MPF gene in various human tissues, and the 2.4 kb band was detected only in lung. Then, the immunohistocytochemical analysis and in situ hybridization revealed that MPF-producing cells were identified as lung macrophages. MPF may exhibit other biological activities such as regeneration of the lung tissues.