A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized S epharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, du ring purification the binding activity of the protein was evaluated by moni toring the inhibition of lectin binding to the N-acetylglucosaminyl-transfe rase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc, The molecular mass of the purified protein mas found to b e 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electr ophoresis, By sequencing analysis, the isolated protein was identified as a nnexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that th e binding was not affected by the addition of phospholipids, Furthermore, s urface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K-d of 200 muM while esse ntially no binding was observed in the case of the corresponding non-bisect ed sample. These results suggest that annexin V has a novel carbohydrate bi nding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of bi ological functions.