A NOVEL SOLUBLE FORM OF MOUSE VCAM-1 IS GENERATED FROM A GLYCOLIPID-ANCHORED SPLICING VARIANT

VCAM-1 is a cytokine-induced endothelial adhesion molecule which belon gs to the immunoglobulin (Ig) superfamily and mediates the binding of various leukocytes. In addition to the 110-kDa form of VCAM-1, we have found four additional glycoproteins on mouse brain-derived endothelio ma cells after stimulation with tumor necrosis factor-alpha (TNF-alpha ), which are recognized by several monoclonal antibodies against VCAM- 1. Biochemical analysis revealed that the two smaller proteins (35 kDa and 37 kDa) are intracellular precursors of the two larger forms (44 kDa and 35 kDa), that the 44 kDa and 45 kDa proteins are glycolipid-an chored at the cell surface and that they differ in their N-glycosylati on. Most likely they are identical to the:recently identified glycolip id-anchored splice variant of VCAM-1, since they are recognized by the M3 antiserum which we raised against a peptide from the unique protei n domain of this splicing variant. With the help of this antiserum we could show by immunohistology that the corresponding VCAM-1 protein va riant is induced in vivo by lipopolysaccharide (LPS) on endothelium of the mouse. In addition, we found a 42-kDa soluble form of VCAM-1 in t he serum of LPS-stimulated mice, which was recognized by the M3 antise rum. This soluble form was undetectable in the serum of unstimulated m ice in contrast to the soluble 100-kDa form of VCAM-1 which was clearl y detected in serum of unstimulated mice and only increased 2-3-fold u pon stimulation with LPS. Thus, only the expression of the 42-kDa shed ded form and not of the 100-kDa soluble form of VCAM-1 is strictly dep endent on stimulation by LPS.