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J. Biol. Chem., Vol. 282, Issue 23, 17014-17023, June 8, 2007

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O-Fucosylation Is Required for ADAMTS13 Secretion^*

Lindsay M. Ricketts, Malgosia Dlugosz, Kelvin B. Luther, Robert S. Haltiwanger, and Elaine M. Majerus¹

From the Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, St. Louis, Missouri, 63110 and the Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [³H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1–8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.

Received for publication, January 11, 2007 , and in revised form, March 29, 2007.

^* This work was supported by American Heart Association 0475035N and by the Edward Mallinckrodt, Jr. Foundation and by National Institutes of Health Grant GM61126 (to R. S. H.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text and a supplemental figure.

This article was selected as a paper of the week.

¹ To whom correspondence should be addressed: Washington University School of Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel.: 314-362-8866; Fax: 314-362-8826; E-mail: emajerus{at}im.wustl.edu.

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