Bisecting GlcNAc mediates the binding of annexin V to Hsp47

Glycobiology. 2005 Nov;15(11):1067-75. doi: 10.1093/glycob/cwj005. Epub 2005 Jul 6.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / chemistry
  • Acetylglucosamine / metabolism*
  • Amino Acid Sequence
  • Animals
  • Annexin A5 / chemistry
  • Annexin A5 / isolation & purification
  • Annexin A5 / metabolism*
  • Binding Sites
  • Carbohydrate Metabolism
  • Cell Line, Tumor
  • HSP47 Heat-Shock Proteins / chemistry
  • HSP47 Heat-Shock Proteins / isolation & purification
  • HSP47 Heat-Shock Proteins / metabolism*
  • Humans
  • Molecular Sequence Data
  • N-Acetylglucosaminyltransferases / chemistry
  • N-Acetylglucosaminyltransferases / metabolism
  • Protein Binding / physiology
  • Rats
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Surface Plasmon Resonance / methods
  • Time Factors


  • Annexin A5
  • HSP47 Heat-Shock Proteins
  • N-Acetylglucosaminyltransferases
  • beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
  • Acetylglucosamine