Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (K_D = ∼1.8 × 10⁻⁸ m, two-state model). Two arginine residues (Arg²⁷ and Arg¹³⁹) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (K_D = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg²⁷ and Arg¹³⁹) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15).     

The First Draft of the Endostatin Interaction Network

Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds to heparin/heparan sulfate and to a number of proteins, but its molecular mechanisms of action are not fully elucidated. We have used surface plasmon resonance (SPR) arrays to identify new partners of endostatin, and to give further insights on its molecular mechanism of action. New partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV, and VI), the amyloid peptide Aβ-(1–42), and transglutaminase-2. The biological functions of the endostatin network involve a number of extracellular proteins containing epidermal growth factor and epidermal growth factor-like domains, and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases.Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It inhibits angiogenesis and tumor growth (1–3). The effect of endostatin depends on its concentration (4, 5), on the length of exposure (6), on the type of endothelial cells (7), and on the growth factor inducing cell proliferation (fibroblast growth factor 2 or VEGF)³ (8, 9).Endostatin binds to several membrane proteins including α5β1 and αvβ3 integrins (10, 11), heparan sulfate proteoglycans (glypican-1 and -4) (12), and KDR/Flk1/VEGFR2 (13). We have previously characterized the binding of endostatin to heparan sulfate chains (9), and of endostatin to integrins (11). Furthermore, we have shown that α5β1, αvβ3, and αvβ5 integrins bind to heparin/heparan sulfate (11).The broad molecular targets of endostatin suggest that multiple signaling systems are involved in mediation of its antiangiogenic action. Endostatin is a broad spectrum angiogenesis inhibitor that suppresses angiogenesis by blocking general mechanisms that govern endothelial cell growth (14), and initiates a complex network of signaling at the gene level (15). However, its molecular mechanism of action is still a matter of debate.An integrative view of the endostatin interaction network, including interactions between endostatin partners, is necessary to provide a clear understanding of how all these molecules work together to regulate angiogenesis, and tumor growth. This global approach places individual proteins into a functional context, and takes into account the fact that a single molecule such as endostatin can affect a wide range of other cell components. Indeed, most proteins and other components carry out their functions within a complex network of interactions and this approach based on protein-protein interaction networks has been developed for several years to give new clues on biological processes (16).This study was thus designed to identify additional extracellular partners of endostatin in an attempt to obtain new insights into its mechanisms of action, and the biological processes in which it participates. For this purpose, we have developed protein and glycosaminoglycan arrays using an automated surface plasmon resonance (SPR) platform. Proteins and glycosaminoglycans selected for SPR analysis were present in the same tissues or structures, such as basement membranes (17), brain (18), cartilage (19), or they were involved in the same physio-pathological processes (angiogenesis, neuro-degenerative diseases) as endostatin, and they were available as full-length molecules. Collagens I and VI, for example, have been selected because the α1 and α2 chains of collagen VI were determined to be potential pan-endothelial markers as was the α1 chain of collagen XVIII containing endostatin (20), and because the genes coding for the α1 and α2 chains of collagen I, and the α3 chain of collagen VI are up-regulated in angiogenic vessels and elevated in tumor endothelium (20). Some proteins and glycosaminoglycans were also included to serve as positive controls for well known interactions with the potential partners of endostatin. We report that endostatin binds to other endogenous angiogenesis inhibitor, the matricellular proteins thrombospondin-1 and SPARC, and to several collagens (I, IV, and VI). Other interacting partners of endostatin are transglutaminase-2, the amyloid peptide Aβ-(1–42), chondroitin, and dermatan sulfate.     

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.     

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER

Background  

In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.     

Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.     

Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM''s cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM''s whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.     

Microbial communities in sugarcane field soils with and without a sugarcane cropping history

Microbial communities in rhizosphere soil from sugarcane (Saccharum inter-specific hybrids) and bulk soil were compared at paired field sites with and without a sugarcane cropping history to determine whether monoculture affects soil microbial community composition. Differences were evaluated for culturable microorganisms and functional diversity indicated by community level physiological profiles (CLPP). Qualitative differences in rhizosphere bacterial communities were detected between sites with no sugarcane cropping history (N_site) and sites with a long-term sugarcane cropping history (L_site). More fluorescent pseudomonads were detected in N_site than L_site rhizosphere soil at two of three sites, and Actinobacteria were more numerous in N_site than L_site rhizosphere soil at one site. Fusarial fungi were more numerous in N_site than L_site rhizosphere soils. Bacteria were more numerous in rhizosphere soil compared to bulk soil. Total bacterial, pseudomonad, and Actinobacteria population densities were greater in bulk soil from an N_site compared to an L_site. CLPP distinguished bulk from rhizosphere soil at one of two sites and N_site and L_site rhizosphere soils at two of four sites. Site affected CLPP similarity more than cropping history. The results demonstrated that sugarcane monoculture can affect the composition of the microbial community in field soil. The findings have possible implications for reduced yields associated with sugarcane monoculture.     

The non-native turf-forming alga Caulacanthus ustulatus displaces space-occupants but increases diversity

Non-indigenous seaweeds can be found in coastal habitats worldwide yet the ecological effects of only ~6 % of macroalgal introductions have been studied. The turf-forming red alga Caulacanthus ustulatus, a putative introduction from Asia, was discovered in southern California in 1999, yet has received very little attention despite being common in rocky intertidal habitats in the region. The purpose of this study was to evaluate the potential effects of Caulacanthus on native invertebrate and seaweed community composition. Macrofaunal, meiofaunal, and macroalgal community structure and diversity were compared between patches with (non-native) and without Caulacanthus (native) in the upper intertidal zone at 5 locations in southern California. Caulacanthus appears to displace macro invertebrates, such as barnacles, limpets, and periwinkles, while facilitating a more diverse array of meiofauna and macroalgae. This is likely due to the formation of a novel turf habitat in the upper zone where turfs are uncommon in this region naturally; algal turfs can increase habitat complexity, trap sediment, and maintain moisture during low tide which likely benefits meiofauna and seaweeds by providing food, habitat, or refuge from desiccation stress. Subsequent comparisons of invertebrate and seaweed assemblages were conducted in native and non-native patches at one site in the upper intertidal zone as well as in the middle intertidal zone where a native turf zone exists. Despite differences in community composition in the upper intertidal zone, no differences were observed in the middle zone, providing support that the novel turf created by Caulacanthus in the upper zone drives community differences.     

Low Birth Weight in Perinatally HIV-Exposed Uninfected Infants: Observations in Urban Settings in Cameroon

Background

The consequences of maternal HIV infection for fetal growth are controversial. Here, we estimated the frequency of small for gestational age and gender (SGAG) among neonates born to HIV-infected or uninfected mothers and assessed the contribution, if any, of maternal HIV to the risk of SGAG.

Methods

The data used were obtained from the ANRS-Pediacam cohort in Cameroon. Pairs of newborns, one to a HIV-infected mother and the other to an uninfected mother, were identified during the first week of life, and matched on gender and recruitment site from 2007–2010. SGAG was defined in line with international recommendations as a birth weight Z-score adjusted for gestational age at delivery and gender more than two standard deviations below the mean (−2SD). Considering the matched design, logistic regression modeling was adjusted on site and gender to explore the effect of perinatal HIV exposure on SGAG.

Results

Among the 4104 mother-infant pairs originally enrolled, no data on birth weight and/or gestational age were available for 108; also, 259 were twins and were excluded. Of the remaining 3737 mother-infant pairs, the frequency of SGAG was 5.3% (95%CI: 4.6–6.0), and was significantly higher among HIV-infected infants (22.4% vs. 6.3%; p<.001) and lower among HIV-unexposed uninfected infants (3.5% vs. 6.3%; p<.001) than among HIV-exposed uninfected infants. Similarly, SGAG was significantly more frequent among HIV-infected infants (aOR: 4.1; 2.0–8.1) and less frequent among HIV-unexposed uninfected infants (aOR: 0.5; 0.4–0.8) than among HIV-exposed uninfected infants. Primiparity (aOR: 1.9; 1.3–2.7) and the presence of any disease during pregnancy (aOR: 1.4; 1.0–2.0) were identified as other contributors to SGAG.

Conclusion

Maternal HIV infection was independently associated with SGAG for HIV-exposed uninfected infants. This provides further evidence of the need for adapted monitoring of pregnancy in HIV-infected women, especially if they are symptomatic, to minimize additional risk factors for SGAG.