Glycobiology, 2000, Vol. 10, No. 11 1209-1216
© 2000 Oxford University Press
A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine
2Department of Biochemistry, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan, 3Department of Surgical Oncology, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan, and 4Peptide Institute, Protein Research Foundation, 4–1–2 Ina, Minoh, Osaka 562–0015, Japan
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 Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a Kd of 200 µM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.
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