Abstract
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 (125)I-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.
Highlights
The majority of mammalian cell types express heparan sulfate proteoglycans (HSPGs),2 which are comprised of a protein core to which is attached linear chains of heparan sulfate (HS)
Glomerular Epithelial Cells Synthesize anticoagulant HSPGs (aHSPGs)—To address whether glomerular cells can synthesize aHSPGs, we evaluated primary glomerular epithelial cells (GECs) from human and rat, as well as an immortalized line that we generated from mouse
The probing of rat kidney cryosections with 125I-AT revealed that aHSPGs are not restricted to blood vessels and occur in the glomerulus and on the basolateral surface of the tubular epithelium
Summary
Mesangial Cell Culture—Mesangial cells were prepared from male Sprague-Dawley rat kidneys. GECs from these mice are able to grow when cultured under permissive conditions, at 33 °C in the presence of interferon-␥ By contrast if they are grown at 37 °C in the absence of interferon-␥, cell division does not occur and they resume a differentiated phenotype. AHS was eluted from the gel by dissociation of the aHS1⁄7AT complexes in buffer containing 1 M NaCl. AT was removed by phenol extraction and NaCl by dialysis. The 35S-aHS and 35S-iHS purified from conditioned medium or trypsinate (30,000 cpm for each sample) were electrophoresed through the AT-containing wells at 60 V and 200 mA for 5 h. AT (Cutter) was initially repurified over heparin-Sepharose, with washing at 350 mM NaCl and elution at 1 M NaCl. The AT flow-through fraction, which lacks heparin affinity, was used as a negative control for nonspecific binding.
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