Bisecting GlcNAc mediates the binding of annexin V to Hsp47.
The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.
Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka, Japan.
Cong-Xiao
CX
Gao
Eiji
E
Miyoshi
Naofumi
N
Uozumi
Rina
R
Takamiya
Xiangchun
X
Wang
Katsuhisa
K
Noda
Jianguo
J
Gu
Koichi
K
Honke
Yoshinao
Y
Wada
Naoyuki
N
Taniguchi
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D045744:Cell Line, Tumor; D050958:HSP47 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods; D013997:Time Factors
D017304:Annexin A5; D050958:HSP47 Heat-Shock Proteins; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine