ISOLATION AND CHARACTERIZATION OF A CELL-SURFACE ALBUMIN-BINDING PROTEIN FROM VASCULAR ENDOTHELIAL-CELLS

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a proce ss involving transcytosis, in the present study; we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells, The endothelial c ell membranes were isolated from cultured cells by differential centri fugation and solubilized with sodium cholate and urea, The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this co lumn,were further separated using preparative sodium dodecyl sulfate/p olyacrylamide gel electrophoresis and used for immunizing rabbits. Flu orescence-activated cell sorter analysis using the anti-gp60 antibodie s demonstrated the expression of gp60 on the endothelial cell surface, Affinity-purified anti-gp60 antibodies inhibited approximate to 90% o f the specific binding of I-125-labeled albumin to bovine pulmonary mi crovessel endothelial cell surface. The anti-gp60 antibodies reacted w ith gp60 from bovine pulmonary artery, bovine pulmonary microvessel, h uman umbilical vein, and rat lung endothelial cell membranes, Bovine a nti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal seque nce (1-56 residues) antibodies did not react with gp60, indicating tha t the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC, We con clude that the endothelial cell-surface-associated gp60 mediates the s pecific binding of native albumin to endothelial cells and thus may re gulate the uptake of albumin and its transcytosis.