Molecular cloning and expression of megakaryocyte potentiating factor cDNA.
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.
Kojima
T
T
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan.
Oh-eda
M
M
Hattori
K
K
Taniguchi
Y
Y
Tamura
M
M
Ochi
N
N
Yamaguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D016466:CHO Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D058851:GPI-Linked Proteins; D006801:Humans; D015151:Immunoblotting; D008533:Megakaryocytes / Q000187:drug effects / Q000502:physiology*; D008562:Membrane Glycoproteins; D000090204:Mesothelin; D051379:Mice / Q000276:immunology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
D000906:Antibodies; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D058851:GPI-Linked Proteins; C000715527:MSLN protein, human; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000090204:Mesothelin