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Yamada, J., Marshak, D., Basinger, S., Walsh, J., Mosley, J., & Stell, W. (1980) Proc. Natl. Acad. Sci. U.S.A.77, 1691-1695. Role of Carbohydrate in the Function of Human Granulocyte-Macrophage Colony-Stimulating Factort Kenneth Kaushansky* Division of Hematology, University zyxwvu of Washington,Seattle, Washington 981 95 Patrick J. O’Hara, Charles E. Hart, John W. Forstrom, and Frederick S. Hagen ZymoGenetics, Inc., Seattle, Washington 981 03 Received August 12, 1986: Revised Manuscript Received February 17, 1987 ABSTRACT: cDNA clones for the human hematopoietic regulator granulocytemacrophage colony-stimulating factor (hGM-CSF) were isolated from a Xgtl 1 cDNA library prepared from RNA of COS cells transiently expressing the gene for hGM-CSF. As the RNA was a rich source of hGM-CSF mRNA, approximately 0.1% of the clones of this library contained hGM-CSF sequences. All of the clones analyzed were full length and were correctly processed. When subcloned into an expression vector and transfected into COS cells, the cDNA clones direct the synthesis of higher levels of the growth factor than the gene from which they were derived. The cDNA for native hGM-CSF was used to generate structural mutants which lack N-linked carbohydrate, 0-linked carbohydrate, or both. Although the mutant proteins had differing specific activities, the nonglycosylated forms reproduce many, if not all, of the physiologic functions of authentic hGM-CSF. The role of carbohydrate in the secretion and function of hGM-CSF is discussed. Colony -stimulating factors (CSFs) are acidic glycoproteins required for the survival, proliferation, and differentiation of hematopoietic progenitor cells in culture (Burgess & Metcalf, 1980). Functionally, the various CSFs are defined by the type of hematopoietic colony produced in semisolid culture. Hence, granulocytemacrophage colony-stimulating factor (GM-CSF) stimulates the growth of progenitors which give rise to colonies containing granulocytes, macrophages, or a combination of both cell types (Wong et al., 1985). In addition to GM-CSF, granulocyte CSF (G-CSF or CSFP), macrophage CSF (M- CSF or CSF-l), and multi-CSF (or IL-3) have been char- acterized and cloned from human sources (Souza et al., 1986; Kawasaki et al., 1985; Yang et al., 1986). Recently, we obtained a genomic clone for GM-CSF from a human library in X Charon 4A (Kaushansky et al., 1986). The gene is divided into four exons and three introns and contains the transcription- and translation-controlling elements typical of a eukaryotic structural gene. It is present in a single copy in the human genome and encodes a mature polypeptide +This work was supported in part by NIH Grants CA-31615 and AI22112. * Address correspondence to this author. Recipient of Physician- Scientist Award AM-01304 from the National Institutes of Health. 0006-2960/87/0426-4861$01.50/0 of 127 amino acids. There are two N-linked glycosylation sites, and there are reported to be three 0-linked glycosylation sites (S. C. Clark, personal communication) which account for approximately 6 kilodaltons (kDa) of the estimated 22 kDa of the mature growth factor. Colony-stimulating factors are proteins of diverse physiologic function. We (Kaushansky et al., 1986) and others (Emerson et al., 1985) have found that recombinant hGM-CSF expressed in COS cells stimulates not only neutrophilic, eosinophilic, and monocytemacrophage progenitor cells but also megakaryocyte colony-forming cells and, in the presence of erythropoietin, erythroid and mixed erythroid-nonerythroid colony-forming cells. Further, hGM-CSF has been shown to stimulate mature neutrophils to localize at sites of inflammation (Weisbart et al., 1985), mature eosinophils and monocytes to become ac- tivated and to enhance their killing of helminths (Handman & Burgess, 1979; Vadas et al., 1983), and mature monocytes and macrophages to enhance phagocytosis and tumor cell killing (Grabstein et al., 1986). In addition to these in vitro activities, recombinant hGM-CSF was recently demonstrated in primates to stimulate in vivo hematopoiesis (Donahue et al., 1986). Despite the growing body of knowledge surrounding the in vitro and now in vivo physiology of hGM-CSF, little is known zyxwvutsrqpo 0 1987 American Chemical Society