T Kojima

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein carrying one O-linked sugar chain. To clarify the role of the oligosaccharide in hG-CSF, some biological and physicochemical properties of the deglycosylated hG-CSF and the intact factor were compared. Recombinant hG-CSF produced in transfected Chinese hamster ovary cells was sequentially digested with neuraminidase and endo-alpha-N-acetylgalactosaminidase. The deglycosylated hG-CSF was one-third as active as the intact form in the colony-forming assay, but it was almost as active as the intact hG-CSF in the cell proliferation assay using NFS-60 cells (NFS-60 bioassay). Inactivation of the deglycosylated hG-CSF was also found by NFS-60 bioassay after incubation for 2 days at pH values from 7 to 8 and at 37 degrees C. This inactivation was accompanied by polymerization of the factor which did not occur with the glycosylated factor. Circular dichroic and calorimetric analyses demonstrated that the deglycosylated hG-CSF is more sensitive to heat denaturation than the intact form and that the inactivation of both forms of hG-CSF was accompanied by conformational change of the proteins. From these results, it was concluded that the O-linked sugar chain of hG-CSF contributes to the stability of the factor by suppressing polymerization and/or its conformational changes.

Sixty-four kinds of cell lines were examined for their ability to produce megakaryocyte potentiating activity by means of conditioned media obtained from a protein-free culture system. Six human tumor cell lines were shown to produce this activity, and the cell line HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from an HPC-Y5 conditioned medium by a combination of ion-exchange chromatography, gel filtration and reverse-phase HPLC. The purified MPF showed a megakaryocyte potentiating activity almost equal to human interleukin-6 in the presence of murine interleukin-3 in a colony formation assay with mouse bone marrow cells. The apparent molecular weight of MPF is 32,000 when determined by SDS-polyacrylamide gel electrophoresis. 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 is a novel cytokine that has megakaryocyte potentiating activity in the murine assay system.