Arivalagan Muthusamy

Plasmodium falciparum-Infected Erythrocytes Adhere Both in the Intervillous Space and on the Villous Surface of Human Placenta by Binding to the Low-Sulfated Chondroitin Sulfate Proteoglycan Receptor

In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies.

Characterization of chondroitin sulfate and dermatan sulfate proteoglycans of extracellular matrices of human umbilical cord blood vessels and Wharton's jelly.

The chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) of the human umbilical cord vein, arteries and Whartons jelly matrices were characterized and localized by immunohistochemical analysis. The CS/DSPGs were found to be decorins and biglycans with 43-48 kDa core proteins and are distributed throughout the umbilical cord. A truncated form of decorin having only the ∼14 kDa NH2-terminal portion of the core protein was found exclusively in the vein. The proteoglycans, regardless of their locations, have two types of CS/DS chains, one with ∼90% CS and ∼10% DS and the other with ∼65% CS and ∼35% DS. The glycosaminoglycan (GAG) chains of the truncated decorin consist of ∼53% CS and ∼47% DS. Both decorin and biglycan including the truncated form of decorin could efficiently bind collagen I and fibronectin. The decorin and biglycan with ∼10% DS and ∼90% CS were loosely bound in the extracellular matrices, whereas those with ∼35% DS bound strongly. Together, these data demonstrate that, the GAG chains with 35-47% DS but not those with 10% DS, interact strongly with the matrix. Our data also show that the GAG chain composition is a significant factor in binding of the decorin and biglycan to matrix proteins. The expression of decorin and biglycan with distinctively different CS/DS proportions implies specific biological functions for these PGs in the umbilical cord. The occurrence of the truncated form of decorin exclusively in the umbilical vein suggests a specific functional role. Published in 2004.

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

BackgroundImmobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro.ResultsIn solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2.ConclusionsOur observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

Targeted Disruption of a Ring-infected Erythrocyte Surface Antigen (RESA)-like Export Protein Gene in Plasmodium falciparum Confers Stable Chondroitin 4-Sulfate Cytoadherence Capacity

Background: Factors that influence the cytoadherence property of P. falciparum are not completely understood. Results: Targeted disruption of the gene that encodes a RESA-like protein in P. falciparum results in stable and increased chondroitin 4-sulfate-dependent cytoadherence capacity. Conclusion: A RESA-like protein gene regulates PfEMP1 expression and cytoadherence of P. falciparum. Significance: Our findings provide a significant insight into the regulation of P. falciparum cytoadherence. The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasites cytoadherence.

Binding affinity of Plasmodium falciparum-infected erythrocytes from infected placentas and laboratory selected strains to chondroitin 4-sulfate

The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta is mediated by chondroitin 4-sulfate (C4S). The C4S-adherent parasites selected from laboratory strains have been widely used for determining the C4S structural elements involved in IRBC binding and for the identification of parasite adhesive protein(s). However, as far as we know, the relative binding strength of the placental versus laboratory-selected parasites has not been reported. In this study, we show that IRBCs from the infected placentas bind to C4S about 3-fold higher than those selected for C4S adherence from laboratory strains. Although adherent parasites selected from several laboratory strains have comparable binding strengths, the one obtained from 3D7 parasites designated as 3D7N61 used for malaria genome sequencing, exhibits markedly lower binding strength. Furthermore, 3D7N61-CSA parasites lose most of the binding capacity by tenth generation in continuous culture.

Characterization of High Molecular Weight Mucins of Rabbit Bladder

Epithelial mucin glycoproteins of bladder act as an effective barrier against invasion by pathogenic microor- ganisms and injury by toxic substances in urine. Although these glycoconjugates play important roles in the pathophysiol- ogy of bladder disorders such as intestinal cystisis, cancer, and urinary tract infections, they have not been characterized in detail either in humans or in animals. Rabbits could be useful for developing models for studying bladder disorders. In this study, we purified and partially characterized two major high molecular weight rabbit bladder mucin glycoproteins, desig- nated RBM1 and RBM2, found in urine. Consistent with their mucin characteristics, amino acid compositions showed have high levels of serine, glutamic acid, proline, glycine and alanine, which together comprise 34% and 42% of the total amino acids in RBM1 and RBM2, respectively. Carbohydrate compositional analysis indicated that RBM1 and RBM2 con- sist of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), N-acetylneuraminic acid (NeuAc) and fucose (Fuc) in the molar ratio of 1.0: 0.82: 0.12: 0.30: 0.02 and 1.0: 1.03: 0.46: 0.16: 0.05, respectively; mannose (Man) was not detected in either mucin. Both mucin fractions were strongly reactive to wheat germ agglutinin, but not to Ca2 antibody specific to a human tumor mucin antigen (asialylated carbohydrate linked to protein core), sug- gesting that most of the galactosyl residues of oligosaccharides are sialylated. Together, the data suggest that rabbit mucin glycoproteins characterized here are distinctively different from MUC1 mucin glycoprotein found in human urine.