Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

Deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mice results in pulmonary alveolar proteinosis (PAP) from impaired surfactant catabolism by alveolar macrophages (AMs). Recently, we have shown that neutralizing anti-GM-CSF autoantibodies develop specifically in patients with idiopathic pulmonary alveolar proteinosis (iPAP). Analogous to murine PAP models, it is plausible that the autoantibodies reduce GM-CSF activity, resulting in AM dysfunction and surfactant accumulation. To examine this hypothesis, we estimated the neutralizing activity of the autoantibodies in the lungs of patients and characterized their biologic properties. GM-CSF bioactivity was completely abrogated in the bronchoalveolar lavage fluid (BALF) of patients with iPAP but not in healthy subjects. Autoantibodies were present in the alveoli in high concentrations and colocalized with GM-CSF. They recognized human GM-CSF with high avidity (K AV ‫؍‬ 20.0 ؎ 7.5 pM) and high specificity, reacting with its superstructure and neutralizing GM-CSF activity to a level 4000 to 58 000 times the levels of GM-CSF normally present in the lung. Although target epitopes varied among patients, GM-CSF amino acids 78 to 94 were consistently recognized. Thus, autoantibodies bind GM-CSF with high specificity and high affinity, exist abundantly in the lung, and effectively block GM-CSF binding to its receptor, inhibiting AM differentiation and function. Our data strengthen the evidence associating anti-GM-CSF autoantibodies with the pathogenesis of this disease.

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.

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 (1-3), 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. MATERIALS AND METHODSProtein and Amino Acid Sequence Determination -Human MPF was purified from HPC-Y5 culture supernatant as described previously (4) and partially digested with endoproteinase Glu-C (Boehringer-Mannheim) in the presence of 10 mM 2-mercaptoethanol and 2 M urea at 37 °C for 18 h. Peptide fragments were then separated on a Vydac C18 reverse-phase column, and subjected, with purified MPF, to sequence analysis in an Applied Biosystems model 473A o...

A colony‐stimulating factor (CSF) has been purified to homogeneity from the serum‐free medium conditioned by one of the human CSF‐producing tumor cell lines, CHU‐2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O‐linked glycosides. Amino acid sequence determination of the molecule gave a single NH2‐terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte‐lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non‐adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G‐CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.

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Two different cDNAs for human granulocyte colony‐stimulating factor (G‐CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G‐CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G‐CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G‐CSF cDNA and the DNA fragment containing the G‐CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G‐CSF gene is interrupted by four introns and a comparison of the structures of the two G‐CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5′ splice donor sequences in the second intron of the G‐CSF gene. When the G‐CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.