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J. Biol. Chem., Vol. 280, Issue 45, 38059-38070, November 11, 2005

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Synthesis of Anticoagulantly Active Heparan Sulfate Proteoglycans by Glomerular Epithelial Cells Involves Multiple 3-O-Sulfotransferase Isoforms and a Limiting Precursor Pool^*

Eric P. Girardin1, Sassan HajMohammadi, Béatrice Birmele, Armin Helisch, Nicholas W. Shworak, and Ariane I. de Agostini¶

From the Department of Pediatrics, and the ^¶Department of Obstetric and Gynecology, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland and the Section of Cardiology, Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03756

Endothelial and other select cell types synthesize a subpopulation of heparan sulfate (HS) proteoglycans (HSPGs), anticoagulant HSPGs (aHSPGs) that bear aHS-HS chains with the cognate 3-O-sulfated pentasaccharide motif that can bind and activate anti-thrombin (AT). Endothelial cells regulate aHSPG production by limiting levels of HS 3-O-sulfotransferase-1 (3-OST-1), which modifies a non-limiting pool of aHS-precursors. By probing kidney cryosections with ¹²⁵I-AT and fluorescently tagged AT we found that the glomerular basement membrane contains aHSPGs, with the staining pattern implicating synthesis by glomerular epithelial cells (GECs). Indeed, cultured GECs synthesized aHS with high AT affinity that was comparable with the endothelial product. Disaccharide analyses of human GEC (hGEC) HS in conjunction with transcript analyses revealed that hGECs express predominantly 3-OST-1 and 3-OST-3_A. aHS production has not been previously examined in cells expressing multiple 3-OST isoforms. This unusual situation appears to involve novel mechanisms to regulate aHS production, as HS structural analyses suggest hGECs exhibit excess levels of 3-OST-1 and an extremely limiting pool of aHS-precursor. A limiting aHS-precursor pool may serve to minimize aHS synthesis by non-3-OST-1 isoforms. Indeed, we show that high in vitro levels of 3-OST-3_A can efficiently generate aHS. Non-3-OST-1 isoforms can generate aHS in vivo, as the probing of kidney sections from 3-OST-1-deficient mice revealed GEC synthesis of aHSPGs. Surprisingly, Hs3st1^-/- kidney only expresses 3-OST isoforms having a low specificity for aHS synthesis. Thus, our analyses reveal a cell type that expresses multiple 3-OST isoforms and produces minimal amounts of aHS-precursor. In part, this mechanism should prevent aHS overproduction by non-3-OST-1 isoforms. Such a role may be essential, as 3-OST isoforms that have a low specificity for aHS synthesis can generate substantial levels of aHSPGs in vivo.

Received for publication, July 22, 2005

^* This work was supported in part by Swiss National Foundation Grants 32-51032.97 (to E. P. G.), 32-59030.99 (to A. I. A.), 32-61807.00 (to E. P. G.), 3200B0-10148/I (to A. I. A.), Human Frontier Science Program Grant RG0304 (to N. W. S.), and by grants from the Fondation C. and E. De Reuter and from the Fonds Caroline Rigaud (to E. P. G.). Deconvolution confocal microscopy was conducted in The Herbert C. Englert Cell Analysis Laboratory (Dartmouth Medical School), which is supported in part by grants from the Fannie E. Rippel Foundation, the National Institutes of Health Shared Instrument Program, and Norris Cotton Cancer Center Core Grant CA 23108. 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 To whom correspondence should be addressed. Tel.: 22-382-46-00; Fax: 22-382-45-05; E-Mail: Eric.girardin{at}hcuge.ch.

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