Characterization of a hydrophilic form of Thy-1 purified from human cerebrospinal fluid.
Thy-1 is a developmentally regulated cell surface glycoprotein in nervous tissue. An inositol-containing glycolipid structure is covalently attached to its carboxyl terminus, which anchors the protein to the cell membrane. In the present paper we report the characterization of a water-soluble form of Thy-1, purified from human cerebrospinal fluid (CSF). In contrast to the membrane-bound form of Thy-1 (M-Thy-1) isolated from human brain cerebral cortex, CSF-Thy-1 behaved like a completely hydrophilic glycoprotein, as analyzed by charge-shift electrophoresis in the presence of detergents and by liposome incorporation experiments. CSF-Thy-1 displayed a slightly higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than M-Thy-1. Digestions with endoglycosidases demonstrated that this difference in size was correlated to different processing of the three N-linked oligosaccharides, and the mobilities of the deglycosylated molecules were indistinguishable in sodium dodecyl sulfate gels. A Pronase-resistant carboxyl-terminal fragment was isolated from the CSF-Thy-1 after trypsin digestion and compared with the corresponding structure of M-Thy-1, obtained by treatment either with bacterial phosphatidylinositol-specific phospholipase C or with human serum (as a source of phosphatidylinositol-specific phospholipase D). The major fragment from CSF-Thy-1 behaved identically, with respect to size and charge, to the carboxyl-terminal fragment from M-Thy-1 solubilized by phospholipase D. These findings suggest an in vivo release of phosphatidylinositol-anchored Thy-1 glycoprotein from brain cells by the action of an endogenous phospholipase D.
D000954:Antigens, Surface / Q000134:cerebrospinal fluid*; D002462:Cell Membrane / Q000276:immunology; D002540:Cerebral Cortex / Q000276:immunology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004563:Electrochemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D012995:Solubility; D018800:Thy-1 Antigens; D010738:Type C Phospholipases / Q000378:metabolism; D014867:Water
D000954:Antigens, Surface; D003902:Detergents; D008081:Liposomes; D010446:Peptide Fragments; D018800:Thy-1 Antigens; D014867:Water; D010738:Type C Phospholipases; D010739:Phospholipase D; D006026:Glycoside Hydrolases; D011402:Pronase