Synthesis of anticoagulantly active heparan sulfate proteoglycans by glomerular epithelial cells involves multiple 3-O-sulfotransferase isoforms and a limiting precursor pool.

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

Structural Basis of Pattern Recognition by Innate Immune Molecules

The importance of the innate immune system as a first line defence against pathogenic challenge has long been recognised. Over the last decade the identity of many of the key molecules mediating innate host defence have been clarified and a model of self/nonself discrimination by families of pattern recognition receptors (PRRs) has emerged. Although a large amount of information is now available concerning the action of these innate immune molecules at the level of the cell and organism, little is known about the molecular interface between pathogens and innate immune recognition molecules. In this chapter the molecular basis for innate immune discrimination of a wide variety of pathogen derived molecules is discussed in the context of the emerging literature