Heparan sulphate proteoglycans reside on the plasma membrane of all animal cells studied so far and are a major component of extracellular matrices. Studies of model organisms and human diseases have demonstrated their importance in development and normal physiology. A recurrent theme is the electrostatic interaction of the heparan sulphate chains with protein ligands, which affects metabolism, transport, information transfer, support and regulation in all organ systems. The importance of these interactions is exemplified by phenotypic studies of mice and humans bearing mutations in the core proteins or the biosynthetic enzymes responsible for assembling the heparan sulphate chains.

Malaria infection is initiated when Anopheles mosquitoes inject Plasmodium sporozoites into the skin. Sporozoites subsequently reach the liver, invading and developing within hepatocytes. Sporozoites contact and traverse many cell types as they migrate from skin to liver; however, the mechanism by which they switch from a migratory mode to an invasive mode is unclear. Here, we show that sporozoites of the rodent malaria parasite Plasmodium berghei use the sulfation level of host heparan sulfate proteoglycans (HSPGs) to navigate within the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype, a process accompanied by proteolytic cleavage of the sporozoite's major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry.

We examined the role of hepatic heparan sulfate in triglyceride-rich lipoprotein metabolism by inactivating the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) in hepatocytes using the Cre-loxP system, which resulted in an approximately 50% reduction in sulfation of liver heparan sulfate. Mice were viable and healthy, but they accumulated triglyceride-rich lipoprotein particles containing apoB-100, apoB-48, apoE, and apoCI-IV. Compounding the mutation with LDL receptor deficiency caused enhanced accumulation of both cholesterol-and triglyceride-rich particles compared with mice lacking only LDL receptors, suggesting that heparan sulfate participates in the clearance of cholesterol-rich lipoproteins as well. Mutant mice synthesized VLDL normally but showed reduced plasma clearance of human VLDL and a corresponding reduction in hepatic VLDL uptake. Retinyl ester excursion studies revealed that clearance of intestinally derived lipoproteins also depended on hepatocyte heparan sulfate. These findings show that under normal physiological conditions, hepatic heparan sulfate proteoglycans play a crucial role in the clearance of both intestinally derived and hepatic lipoprotein particles.

Elevated plasma triglyceride levels represent a risk factor for premature atherosclerosis. In mice, accumulation of triglyceride-rich lipoproteins can occur if sulfation of heparan sulfate in hepatocytes is diminished, as this alters hepatic lipoprotein clearance via heparan sulfate proteoglycans (HSPGs). However, the relevant HSPG has not been determined. In this study, we found by RT-PCR analysis that mouse hepatocytes expressed the membrane proteoglycans syndecan-1, -2, and -4 and glypican-1 and -4. Analysis of available proteoglycandeficient mice showed that only syndecan-1 mutants (Sdc1 -/-mice) accumulated plasma triglycerides. Sdc1 -/-mice also exhibited prolonged circulation of injected human VLDL and intestinally derived chylomicrons. We found that mice lacking both syndecan-1 and hepatocyte heparan sulfate did not display accentuated triglyceride accumulation compared with single mutants, suggesting that syndecan-1 is the primary HSPG mediating hepatic triglyceride clearance. Immunoelectron microscopy showed that syndecan-1 was expressed specifically on the microvilli of hepatocyte basal membranes, facing the space of Disse, where lipoprotein uptake occurs. Abundant syndecan-1 on wild-type murine hepatocytes exhibited saturable binding of VLDL and inhibition by heparin and facilitated degradation of VLDL. Furthermore, adenovirus-encoded syndecan-1 restored binding, uptake, and degradation of VLDL in isolated Sdc1 -/-hepatocytes and the lipoprotein clearance defect in Sdc1 -/-mice. These findings provide the first in vivo genetic evidence that syndecan-1 is the primary hepatocyte HSPG receptor mediating the clearance of both hepatic and intestinally derived triglyceride-rich lipoproteins.

Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection. High density lipoprotein (HDL)2 has several well established physiological activities. The best understood of these is the ability of HDL to protect against atherosclerotic cardiovascular disease by promoting the efflux of excess cholesterol from peripheral tissues and its subsequent transport to the liver for excretion. In addition, apolipoproteins and enzymes carried by HDL have antioxidant activities that inhibit the oxidative modification of low density lipoprotein (LDL), thereby reducing the atherogenicity of these lipoproteins. A less well known activity of HDL is an antimicrobial property that protects some primates from infection by certain eukaryotic pathogens. In these primate species, HDL apolipoproteins have been proposed to have novel innate immune protective functions that provide selective protection against infection.One such innate immune activity in the serum of humans, apes, and old world monkeys limits the host range of Trypanosoma brucei brucei to non-primate mammals (1). This activity fractionates with a minor subclass of human HDL termed trypanosome lytic factor 1 (TLF-1) that, like all HDLs, is composed of apolipoproteins, phospholipids, and neutral lipids (2, 3). Trypanosome killing by these cytotoxic HDLs requires high affinity binding to receptors on the trypanosome surface, endocytosis, and lysosomal localization (4 -8). Following lysosomal acidification, destabilization of the lysosome membrane is facilitated by either free radical-mediated lipid peroxidation or pore formation (9 -11). Two HDL-associated proteins have been implicated in the toxicity of TLF-1, haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I) (12, 13).Haptoglobin-related protein differs by only 27 amino acids from human haptoglobin, an acute phase serum protein that binds hemoglobin released from red blood cells during trauma or infection and transfers the hemoglobin to liver cells for detoxification (14,15). However, Hpr does not bind hemoglobin, and...

It has been known for almost a decade and a half that in trypanosomes all

Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis.

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.