Ploug M, Eriksen J, Plesner T, Hansen N E, Danø K
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Eur J Biochem. 1992 Sep 1;208(2):397-404. doi: 10.1111/j.1432-1033.1992.tb17200.x.
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.
细胞型尿激酶型纤溶酶原激活物（uPA）受体（uPAR）是一种糖脂锚定膜蛋白，被认为在细胞迁移和肿瘤侵袭过程中的细胞周围蛋白水解中发挥作用。在本研究中，我们鉴定并表征了两种保留其配体结合能力的可溶性uPAR形式。一种变体是通过用磷脂酰肌醇特异性磷脂酶C（PLC）或天冬氨酸蛋白酶-N处理完整的正常细胞在体外产生的。另一种可溶性uPAR变体是在体内由受干细胞疾病阵发性夜间血红蛋白尿（PNH）影响的外周血白细胞分泌的，并且在这些PNH患者的血浆以及培养的PNH白细胞的条件培养基中被发现。在正常条件下，我们没有发现血浆中存在与人类uPAR结合的可溶性对应物的任何脱落或分泌的证据。与正常白细胞不同，受PNH影响的细胞在细胞表面不表达uPAR，尽管它们确实含有明显正常水平的uPAR特异性mRNA。源自PNH细胞的分泌型uPAR在SDS/PAGE中的迁移率略高于通过磷脂酰肌醇特异性PLC或去污剂溶解的uPAR，但类似于截短的重组uPAR变体，其C末端靠近拟议的糖脂附着位点，这表明分泌蛋白已被蛋白水解加工以进行糖脂附着。PNH患者血浆中存在这种分泌的亲水性uPAR形式，支持了以下假设：PNH疾病的潜在病变要么存在于糖脂生物合成中，要么存在于一种尚未鉴定的转酰胺酶的功能中，该酶假定可切割并将截短的uPAR与预先形成的糖脂部分组装在一起。
