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dc.contributor.authorPrice, Laura King Hannan
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 53-04, Section: B, page: 1680.;Advisors: E. Richard Stanley.
dc.description.abstractColony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375-kDa and 250-dKa with a smaller amount of a 100-kDa species. Analysis by gel-filtration in 4M guanidine HCl buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The 3 predominant CSF-1 species were shown to be an 80-kDa/80-kDa homodimer, an 80-kDa/50-kDa heterodimer and a 50-kDa/50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80-kDa and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase and endo-{dollar}\alpha{dollar}-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 kDa and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyterminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid precipitable counts released from {dollar}\sp{lcub}35{rcub}{dollar}SO{dollar}\sb4\sp{lcub}2-{rcub}{dollar}-labeled L cells, has important implications for regulation by this growth factor. In addition to soluble CSF-1, a plasma membrane-associated form of CSF-1 was demonstrated by trypsin release and immunofluorescent localization. SDS-PAGE analysis of lysates of L cells surface-labeled with lactoperoxidase demonstrated a major 80-kDa CSF-1 species and trace amounts of the 375- and 250-kDa CSF-1 species. The 80-kDa cell surface CSF-1 species is released slowly compared with the 375-, 250- and 100-kDa secreted species. In vivo evidence of comparable high molecular weight, diffuse CSF-1 species in a variety of murine tissue homogenates and serum was provided by western analysis. It would therefore appear that the biosynthesis of soluble proteoglycan and membrane bound glycoprotein species of CSF-1 renders the growth factor available for intercellular and extracellular matrix interactions as well as humoral regulation.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.titleBiosynthetic studies of membrane-associated and secreted L cell CSF-1

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