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J. Biol. Chem., Vol. 283, Issue 41, 27724-27735, October 10, 2008

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Sulf Loss Influences N-, 2-O-, and 6-O-Sulfation of Multiple Heparan Sulfate Proteoglycans and Modulates Fibroblast Growth Factor Signaling^*

William C. Lamanna, Marc-André Frese, Martina Balleininger, and Thomas Dierks¹

From the Fakultät für Chemie, Biochemie I, Universität Bielefeld, 33615 Bielefeld, Germany and Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, 37073 Göttingen, Germany

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.

Received for publication, March 17, 2008 , and in revised form, August 6, 2008.

^* This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and Shire Human Genetic Therapies Inc. (Cambridge, MA). 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 Tables S1–S6 and Figs. S1 and S2.

¹ To whom correspondence should be addressed: Fakultät für Chemie, Biochemie I, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany. Tel.: 49-521-106-2092; Fax: 49-521-106-6014; E-mail: thomas.dierks{at}uni-bielefeld.de.

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