Glycobiology Advance Access originally published online on July 6, 2005
Glycobiology 2005 15(11):1067-1075; doi:10.1093/glycob/cwj005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Bisecting GlcNAc mediates the binding of annexin V to Hsp47
2 Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 3 JST, Japan Science and Technology Agency, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan; 4 Department of Surgical Oncology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 5 Department of Molecular Medicine, Kochi University Medical School, 2-5-1 Akebono-cho, Kochi 780-8520, Japan; and 6 Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan
1 To whom correspondence should be addressed; e-mail: miyoshi34{at}biochem.med.osaka-u.ac.jp
Received on December 6, 2004; revised on June 25, 2005; accepted on June 26, 2005
The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : ß-D-mannoside ß-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 µM, 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.
Key words: annexin V / bisecting GlcNAc / Hsp47
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Nishioka, J.-i. Aikawa, M. Ida, I. Matsumoto, M. Street, M. Tsujimoto, and K. Kojima-Aikawa Ligand-binding Activity and Expression Profile of Annexins in Caenorhabditis elegans J. Biochem., January 1, 2007; 141(1): 47 - 55. [Abstract] [Full Text] [PDF]
|
|