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. 1996 Jan 9;93(1):250–254. doi: 10.1073/pnas.93.1.250

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

C Tiruppathi 1, A Finnegan 1, A B Malik 1
PMCID: PMC40216  PMID: 8552615

Abstract

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process 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 cell membranes were isolated from cultured cells by differential centrifugation 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 column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

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