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J. Biol. Chem., Vol. 282, Issue 45, 32802-32810, November 9, 2007

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Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation^*

Marta Busse¹², Almir Feta¹, Jenny Presto, Maria Wilén, Mona Grønning, Lena Kjellén, and Marion Kusche-Gullberg³

From the Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway and the Department of Medical Biochemistry and Microbiology, University of Uppsala, BMC Box 582, SE-751 23 Uppsala, Sweden

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.

Received for publication, April 30, 2007 , and in revised form, August 9, 2007.

^* This work was supported by grants from the Swedish Research Council (to M. K.-G. and L. K.), Polysackaridforskning AB (Uppsala, Sweden) Gustav V:s 80-årsfond, the Norwegian Cancer Society, and the Meltzer Foundation (Bergen, Norway). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: Vascular Biology Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, WC2A 3PX London, UK.

³ To whom correspondence should be addressed. Tel.: 47-55-58-66-90; Fax: 47-55-58-64-10; E-mail: Marion.Kusche{at}biomed.uib.no.

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				S. Nadanaka and H. Kitagawa Heparan Sulphate Biosynthesis and Disease J. Biochem., July 1, 2008; 144(1): 7 - 14. [Abstract] [Full Text] [PDF]

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