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Glycobiology Advance Access published online on July 6, 2005
Glycobiology, doi:10.1093/glycob/cwj005
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received December 6, 2004
Revised June 25, 2005
Accepted June 26, 2005

Article

Bisecting GlcNAc Mediates the Binding of Annexin V to Hsp47

Cong-xiao Gao 1, Eiji Miyoshi 2*, Naofumi Uozumi 3, Rina Takamiya 1, Xiangchun Wang 1, Katsuhisa Noda 1, Jianguo Gu 1, Koichi Honke 4, Yoshinao Wada 5, and Naoyuki Taniguchi 1

1 Department of Biochemistry, Osaka University Graduate School of Medical, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Department of Biochemistry, Osaka University Graduate School of Medical, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; JST, Japan Science and Technology Agency
3 Department of Surgical Oncology, Osaka University Graduate School of Medical, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; JST, Japan Science and Technology Agency
4 Department of Molecular Medicine, Kochi University Medical School
5 Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan

* To whom correspondence should be addressed.
Eiji Miyoshi, E-mail: miyoshi34{at}biochem.med.osaka-u.ac.jp


  Abstract

The bisecting GlcNAc structure, formed via catalysis by N-acetylglucosaminyltransferase 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 the present 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 47kDa 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 pre-incubation 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 E4-PHA and MALDI-TOF MS analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5µM, while no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the co-localization of annexin V, Hsp47 and a 4 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.

Keywords: annexin V/ Hsp47/bisecting GlcNAc.
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[Abstract] [Full Text] [PDF]


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