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 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 K(d) of 200 microM 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.
Gao-Uozumi
C X
CX
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Uozumi
N
N
Miyoshi
E
E
Nagai
K
K
Ikeda
Y
Y
Teshima
T
T
Noda
K
K
Shiba
T
T
Honke
K
K
Taniguchi
N
N
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D007700:Kinetics; D037102:Lectins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism; D013552:Swine; D014162:Transfection
D017304:Annexin A5; D002241:Carbohydrates; D037102:Lectins; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine