A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria.
The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.
Ploug
M
M
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Eriksen
J
J
Plesner
T
T
Hansen
N E
NE
Danø
K
K
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D006017:Glycolipids / Q000097:blood*; D017261:Glycosylphosphatidylinositols; D006457:Hemoglobinuria, Paroxysmal / Q000097:blood*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000097:blood; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D012333:RNA, Messenger / Q000097:blood; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; C524898:PLAUR protein, human; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D043265:Phosphatidylinositol Diacylglycerol-Lyase