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Lysosomal storage diseases result in various developmental and physiological complications, including cachexia. To study the causes for the negative energy balance associated with cachexia, we assessed the impact of sulfamidase deficiency and heparan sulfate storage on energy homeostasis and metabolism in a mouse model of type IIIa mucopolysaccharidosis (MPS IIIa, Sanfilippo A syndrome). At 12-weeks of age, MPS IIIa mice exhibited fasting and postprandial hypertriglyceridemia compared with wildtype mice, with a reduction of white and brown adipose tissues. Partitioning of dietary [3H]triolein showed a marked increase in intestinal uptake and secretion, whereas hepatic production and clearance of triglyceride-rich lipoproteins did not differ from wildtype controls. Uptake of dietary triolein was also elevated in brown adipose tissue (BAT), and notable increases in beige adipose tissue occurred, resulting in hyperthermia, hyperphagia, hyperdipsia, and increased energy expenditure. Furthermore, fasted MPS IIIa mice remained hyperthermic when subjected to low temperature but became cachexic and profoundly hypothermic when treated with a lipolytic inhibitor. We demonstrated that the reliance on increased lipid fueling of BAT was driven by a reduced ability to generate energy from stored lipids within the depot. These alterations arose from impaired autophagosome-lysosome fusion, resulting in increased mitochondria content in beige and BAT. Finally, we show that increased mitochondria content in BAT and postprandial dyslipidemia was partially reversed upon 5-week treatment with recombinant sulfamidase. We hypothesize that increased BAT activity and persistent increases in energy demand in MPS IIIa mice contribute to the negative energy balance observed in patients with MPS IIIa.

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy forMucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

International audience; Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the effect of D2 mutations conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors on the structure of the recombinant D2 Con1 mutants and their binding to CypA. Although resistance mutations D320E and R318W could induce some local and/or global folding perturbation, which could thus affect the kinetics of conformer interconversions, they do not significantly affect the kinetics of CypA/D2 interaction measured by surface plasmon resonance (SPR). The combination of all our data led us to build a model of the overall structure of NS5A, which provides a useful template for further investigations of the structural and functional features of this enigmatic protein.

Inherited defects in the ability to catabolize glycosaminoglycans result in lysosomal storage disorders known as mucopolysaccharidoses (MPS), causing severe pathology, particularly in the brain. Enzyme replacement therapy has been used to treat mucopolysaccharidoses; however, neuropathology has remained refractory to this approach. To test directly whether substrate reduction might be feasible for treating MPS disease, we developed a genetic model for substrate reduction therapy by crossing MPS IIIa mice with animals partially deficient in heparan sulfate biosynthesis due to heterozygosity in Ext1 and Ext2, genes that encode the copolymerase required for heparan sulfate chain assembly. Reduction of heparan sulfate by 30-50% using this genetic strategy ameliorated the amount of disease-specific biomarker and pathology in multiple tissues, including the brain. In addition, we were able to demonstrate that substrate reduction therapy can improve the efficacy of enzyme replacement therapy in cell culture and in mice. These results provide proof of principle that targeted inhibition of heparan sulfate biosynthetic enzymes together with enzyme replacement might prove beneficial for treating mucopolysaccharidoses.

Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

Glioblastomas (GBMs) are highly malignant tumors characterized by microvascular proliferation and the pseudopalisading pattern of necrosis. Investigations have, therefore, focused on vascular and endothelial cell biology in GBM. Endocan, also called endothelial cell-specific molecule-1, is a proteoglycan that is secreted by endothelial cells and upregulated by proangiogenic factors. We found that endocan is not only expressed in vitro by endothelial cells but also in the T98G and U118MG human GBM cell lines. In U118MG cells, tumor necrosis factor and fibroblast growth factor 2 upregulated endocan production, whereas exposure to hypoxia or cobalt chloride, an inducer of hypoxia inducible factor 1, increased endocan release without affecting cell viability. Endocan expression in 82 brain tumors was studied by immunohistochemistry. Endocan immunoreactivity was detected in hyperplastic endothelial cells in high-grade gliomas, mostly at the tumor margins; endothelial cells were mostly endocan negative in low-grade gliomas, and it was never detected in the cerebral cortex distant from the tumors. Tumor cells in high-grade but not low-grade gliomas also expressed endocan, and it was detected in palisading cells surrounding areas of necrosis in GBM. Endothelial cell endocan immunoreactivity also correlated with shorter survival in glioma patients. Taken together, these results suggest that endocan is associated with abnormal vasculature in high-grade gliomas.

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-gamma (IFNgamma), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNgamma monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNgamma. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Angstroms-long spacer termed 2O(10), displays high affinity for the cytokine and inhibits IFNgamma-HS binding with an IC(50) of 35-40 nm. Interestingly, this molecule also inhibits the binding of IFNgamma to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNgamma activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNgamma recognition, providing a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create"tailor-made"sequences based on the HS template to isolate therapeutic activities.

In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

International audience; The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein-heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.

Mucopolysaccharidoses are a group of genetically inherited disorders that result from the defective activity of lysosomal enzymes involved in glycosaminoglycan catabolism, causing their intralysosomal accumulation. Sanfilippo disease describes a subset of mucopolysaccharidoses resulting from defects in heparan sulfate catabolism. Sanfilippo disorders cause severe neuropathology in affected children. The reason for such extensive central nervous system dysfunction is unresolved, but it may be associated with the secondary accumulation of metabolites such as gangliosides. In this article, we describe the accumulation of dermatan sulfate as a novel secondary metabolite in Sanfilippo. Based on chondroitinase ABC digestion, chondroitin/dermatan sulfate levels in fibroblasts from Sanfilippo patients were elevated 2–5-fold above wild-type dermal fibroblasts. Lysosomal turnover of chondroitin/dermatan sulfate in these cell lines was significantly impaired but could be normalized by reducing heparan sulfate storage using enzyme replacement therapy. Examination of chondroitin/dermatan sulfate catabolic enzymes showed that heparan sulfate and heparin can inhibit iduronate 2-sulfatase. Analysis of the chondroitin/dermatan sulfate fraction by chondroitinase ACII digestion showed dermatan sulfate storage, consistent with inhibition of iduronate 2-sulfatase. The discovery of a novel storage metabolite in Sanfilippo patients may have important implications for diagnosis and understanding disease pathology.

International audience; Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.

Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40 kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and are mostly clustered in tetra- (35%), hexa- (12%), and octa- (5%) saccharide domains. Highly sulfated [up triangle, open]D, [up triangle, open]E, and [up triangle, open]B disaccharide units ([up triangle, open]HexA2S-GalNAc6S, [up triangle, open]HexA-GalNAc4S6S, and [up triangle, open]HexA2S-GalNAc4S) were also detected in significant amounts in both chains and may account for the HGF/SF-binding activity of the CS/DS. This work establishes that HEK-293 cells can be engineered to provide a valuable source of Endocan with authentic CS/DS chains, enabling the purification of sufficient amounts for structural and/or binding analysis and providing a possible model of Endocan CS/DS chain organization.

Standard culture systems of eukaryotic cells generally failed to deliver sufficient amounts of recombinant proteins without increasing the costs of production. We here showed that membrane-based bioreactors, initially developed for the production of monoclonal antibodies, can be very useful for the production using engineered HEK293 cells, of a recombinant proteoglycan called endocan, with achievement of high level expression and efficient long-term production. When compared to standard procedures, the growth in suspension and at high density of these cells in one bioreactor promoted a 60-fold increase of the concentration of the soluble recombinant endocan. These culture conditions did not affect cell viability, stable expression, recognition by specific monoclonal antibodies or electrophoretic profile of the recombinant endocan. Such an easy to scale up system to produce recombinant protein should open soon new opportunities to study structure and functions of endocan or any other glycosylated cell products newly investigated.

International audience; Endocan, previously called endothelial cell specific molecule-1, is a soluble proteoglycan of 50 kDa, constituted of a mature polypeptide of 165 amino acids and a single dermatan sulphate chain covalently linked to the serine residue at position 137. This dermatan sulphate proteoglycan, which is expressed by the vascular endothelium, has been found freely circulating in the bloodstream of healthy subjects. Experimental evidence is accumulating that implicates endocan as a key player in the regulation of major processes such as cell adhesion, in inflammatory disorders and tumor progression. Inflammatory cytokines such as TNF-alpha, and pro-angiogenic growth factors such as VEGF, FGF-2 and HGF/SF, strongly increased the expression, synthesis or the secretion of endocan by human endothelial cells. Endocan is clearly overexpressed in human tumors, with elevated serum levels being observed in late-stage lung cancer patients, as measured by enzyme-linked immunoassay, and with its overexpression in experimental tumors being evident by immunohistochemistry. Recently, the mRNA levels of endocan have also been recognized as being one of the most significant molecular signatures of a bad prognosis in several types of cancer including lung cancer. Overexpression of this dermatan sulphate proteoglycan has also been shown to be directly involved in tumor progression as observed in mouse models of human tumor xenografts. Collectively, these results suggest that endocan could be a biomarker for both inflammatory disorders and tumor progression as well as a validated therapeutic target in cancer. On the basis of the recent successes of immunotherapeutic approaches in cancer, the preclinical data on endocan suggests that an antibody raised against the protein core of endocan could be a promising cancer therapy.

International audience; We have recently described the preparation of three building blocks for the combinatorial synthesis of heparan sulfate (HS) fragments. Herein we show that one of these building blocks (disaccharide 4) allows the preparation, in high yields and with total alpha stereoselectivity, of tetra-, hexa- and octasaccharides from the heparin (HP) regular region, by using 2+2, 2+4 and 4+4 glycosylation strategies, respectively. These oligosaccharides were processed into sulfated derivatives bearing an allyl moiety in the anomeric position. The UV-promoted conjugation of these compounds with alpha,omega-bis(thio)poly(ethylene glycol) spacers of three different lengths allowed us to prepare nine benzylated glycoconjugates. After final deprotection, the glycoconjugates 1 a-c, 2 a-c and 3 a-c were obtained and their ability to inhibit the interaction between IFN-gamma and HP was tested by using surface plasmon resonance detection. Compound 3 b, containing two HP octasaccharides linked by a 50-A linker was able to inhibit the IFN-gamma/HP interaction with an IC(50) value of approximately 35 nM. In addition, the nine glycoconjugates were perfect tools in the study to ascertain the topology of the IFN-gamma binding site on HS. Compounds 1 a-c, 2 a-c and 3 a-c, by mimicking the alternating sulfated and nonsulfated regions found in HS, thus comprise the first example of a library of synthetic HS mimetics giving access to the "second level of molecular diversity" found in HS.

Introduction Endocan est un proteoglycane secrete specifiquement par la cellule endotheliale pulmonaire. Il est detecte au niveau serique chez des individus sains et retrouve surexprime chez des patients atteints de sepsis ou de cancer bronchique. En plus d’etre correlee au mauvais pronostic de ces deux maladies, la surexpression d’endocan est impliquee dans leur physiopathologie. Ainsi, pour pouvoir explorer endocan comme cible therapeutique potentielle, il apparait interessant de controler son expression, notamment en jouant sur son catabolisme. Cependant, le mecanisme de degradation d’endocan est encore a ce jour totalement inconnu. Endocan etant retrouve pour l’essentiel dans le sang, interagissant avec les leucocytes, nous nous sommes orientes sur l’etude de la degradation d’endocan par les leucocytes et plus precisement par les polynucleaires neutrophiles (PNNs). Methodes Des PNNs sont isoles a partir de poches de sang et resuspendus dans un milieu HBSS a 10 6 cellules/ml pour etre stimules par 10 nM de Phorbol Myristate Acetate, pendant 3 heures a 37°C. Le surnageant de PNNs, contenant les proteines secretees, est ensuite recueilli et teste pour son activite de degradation vis-a-vis d’endocan en le mettant en contact avec de l’endocan recombinant pendant 2 a 72 heures a 37 °C. La degradation d’endocan est ensuite evaluee par dosage ELISA et visualisee par Western blot. L’activite proteasique du surnageant de PNNs sur endocan est determinee par utilisation d’un cocktail inhibiteur de proteases. Resultats Les resultats montrent que les PNNs secretent des proteases clivant endocan en differents sites. Si l’extremite N-termi-nale d’endocan n’est pas degradee, les proteases coupent le corps proteique entre les acides amines 107 et 136 pour former des fragments de 15 et 14 kDa, et entre les acides amines 81 et 106 pour donner un peptide de 10 kDa. Un point important concerne le fragment de 14 kDa, qui constitue le metabolite majoritaire. Les donnees cinetiques montrent qu’il apparait tres precocement et persiste au moins pendant 3 jours en presence de proteases neutrophiles. Conclusion On peut penser qu’au cours d’etats infectieux severes, les PNNs actives sont capables de degrader plus rapidement endocan et modifient ainsi les activites biologiques de cette molecule.

Introduction By virtue of the multiplicity of their protein-binding partners (e.g. growth factors, cytokines/chemokines), proteoglycans have been shown to be involved in the regulation of a large number of pathophysiological processes including cancer and inflammatory diseases. We have studied and characterized endocan, also called endothelial cell-specific molecule-1 (ESM-1), which represents a new group of circulating proteoglycans. Endocan is mainly expressed by endothelial cells but also by epithelial cells from lung, gut and kidney. Structurally, endocan is constituted of a mature polypeptide of 165 amino acids with a single glycosaminoglycan chain covalently linked to the serine at position 137 (Bechard et al. 2001). Methods and results We showed that human umbilical vein endothelial cells expressed endocan specifically with a single chain of dermatan sulfate (DS) as glycosaminoglycan moiety. As shown by surface plasmon resonance, the DS chain directly interacts with cytokines and growth factors including hepatocyte growth factor/scatter factor and could be responsible for endocan's biological activities. Human embryonic kidney 293 cells, which have been genetically engineered to overexpress endocan, induce tumour growth when injected subcutaneously in SCID mice. Moreover, inflammatory cytokines such as TNF-a and IL-1 have been shown to increase the synthesis and the secretion of endocan from human umbilical vein endothelial cells. Conclusion These results suggest that circulating levels of endocan may represent a novel marker for cancer and inflammatory diseases. Further studies on its GAG structure could help us to better understand the biological activities of endocan and to design future glycomic-based therapies.

The migration of polymorphonuclear neutrophils (PMN) into inflamed tissue requires fine interactions with the endothelial cell surface. The PMN serine proteases cathepsin G (CG), neutrophil elastase (NE) and proteinase 3 (PR3) were originally thought to play a role by the cleavage of endothelial cell proteins that control the PMN firm adhesion and the transendothelial cell migration. However, how these proteases participate in leukocyte adhesion and transmigration remains controversial. Vascular endocan, also called esm1, is a restricted endothelial cell-secreted proteoglycan constituted by a protein core of 20 kDa and by a unique glycosaminoglycan chain of dermatan sulphate (DS). Endocan is preferentially expressed in lung and kidney tissues. Endocan binds to its leukocytic receptor, the LFA-1 integrin, with an affinity of 18 nM, and inhibits the LFA-1-ICAM-1 interactions. Its expression is upregulated by the proinflammatory mediators TNF, IL-1, and lipopolysaccharide (LPS). In human sepsis, the serum endocan increases from fivefold to 30-fold the normal value and correlates with bad prognosis. Here, we examined the role of PMN-derived proteases in the degradation of endocan.