The Role of Sialylated Glycans in Human Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)-mediated Trans Homophilic Interactions and Endothelial Cell Barrier Function.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) is a major component of the endothelial cell intercellular junction. Previous studies have shown that PECAM-1 homophilic interactions, mediated by amino-terminal immunoglobulin homology domain 1, contribute to maintenance of the vascular permeability barrier and to its re-establishment following inflammatory or thrombotic insult. PECAM-1 glycans account for ∼30% of its molecular mass, and the newly solved crystal structure of human PECAM-1 immunoglobulin homology domain 1 reveals that a glycan emanating from the asparagine residue at position 25 (Asn-25) is located within the trans homophilic-binding interface, suggesting a role for an Asn-25-associated glycan in PECAM-1 homophilic interactions. In support of this possibility, unbiased molecular docking studies revealed that negatively charged α2,3 sialic acid moieties bind tightly to a groove within the PECAM-1 homophilic interface in an orientation that favors the formation of an electrostatic bridge with positively charged Lys-89, mutation of which has been shown previously to disrupt PECAM-1-mediated homophilic binding. To verify the contribution of the Asn-25 glycan to endothelial barrier function, we generated an N25Q mutant form of PECAM-1 that is not glycosylated at this position and examined its ability to contribute to vascular integrity in endothelial cell-like REN cells. Confocal microscopy showed that although N25Q PECAM-1 concentrates normally at cell-cell junctions, the ability of this mutant form of PECAM-1 to support re-establishment of a permeability barrier following disruption with thrombin was significantly compromised. Taken together, these data suggest that a sialic acid-containing glycan emanating from Asn-25 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface.

Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31).

PURPOSE OF REVIEW: The purpose of this article is to describe the function of the vascular cell adhesion and signaling molecule, platelet/endothelial cell adhesion molecule-1 (PECAM-1), in endothelial cells, with special emphasis on its role in maintaining and restoring the vascular permeability barrier following disruption of the endothelial cell junction. RECENT FINDINGS: In addition to its role as an inhibitory receptor in circulating platelets and leukocytes, PECAM-1 is highly expressed at endothelial cell-cell junctions, where it functions as an adhesive stress-response protein to both maintain endothelial cell junctional integrity and speed restoration of the vascular permeability barrier following inflammatory or thrombotic challenge. SUMMARY: Owing to the unique ability of antibodies that bind the membrane proximal region of the extracellular domain to trigger conformational changes leading to affinity modulation and homophilic adhesion strengthening, PECAM-1 might be an attractive target for treating vascular permeability disorders.

Structural basis for PECAM-1 homophilic binding.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a ß-sandwich topology formed by 2 sheets of antiparallel ß strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å(2). These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.

Regulation of endothelial cell barrier function by antibody-driven affinity modulation of platelet endothelial cell adhesion molecule-1 (PECAM-1).

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

Distal effect of amino acid substitutions in CYP2C9 polymorphic variants causes differences in interatomic interactions against (S)-warfarin.

Cytochrome P450 2C9 (CYP2C9) is crucial in excretion of commonly prescribed drugs. However, changes in metabolic activity caused by CYP2C9 polymorphisms inevitably result in adverse drug effects. CYP2C9*2 and *3 are prevalent in Caucasian populations whereas CYP2C9*13 is remarkable in Asian populations. Single amino acid substitutions caused by these mutations are located outside catalytic cavity but affect kinetic activities of mutants compared to wild-type enzyme. To relate distal effects of these mutations and defective drug metabolisms, simulations of CYP2C9 binding to anti-coagulant (S)-warfarin were performed as a system model. Representative (S)-warfarin-bound forms of wild-type and mutants were sorted and assessed through knowledge-based scoring function. Interatomic interactions towards (S)-warfarin were predicted to be less favorable in mutant structures in correlation with larger distance between hydroxylation site of (S)-warfarin and reactive oxyferryl heme than wild-type structure. Using computational approach could delineate complication of CYP polymorphism in management of drug therapy.

Modeling of Anopheles minimus Mosquito NADPH-cytochrome P450 oxidoreductase (CYPOR) and mutagenesis analysis.

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two- to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs.

Homology modeling of mosquito cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in substrate selectivity.

BACKGROUND: Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds. FINDINGS: Homology models of the three An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center. CONCLUSIONS: Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes.

Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure.

Aedes aegypti mosquitoes are resistant to various insecticides, including pyrethroids, throughout Thailand. We previously reported that Ae. aegypti from Mae Wong district, Nakhon Sawan Province in north-central Thailand, were resistant to insecticides, including pyrethroids (deltamethrin and permethrin), organophosphates and carbamates, and that high levels of detoxification enzymes were present. In the present study we used the method of suppression by subtractive hybridization to determine differential expression of genes in Mae Wong Ae. aegypti that survived the exposure to increasing doses (approximately 1.5-2x10(-5) M) of deltamethrin beyond the diagnostic dose compared to unexposed mosquitoes. Screening of 350 cDNA clones from the suppression subtractive library by cDNA array hybridization revealed that 58 clones were over-expressed in the mosquito that survived high dose deltamethrin. The over-expressed cDNA insert sequences corresponded to 11 functional genes, five hypothetical protein genes, and five sequences of unknown function that could be located on the supercontig of the Ae. aegypti genome. The functional genes are those coding for cuticular proteins, muscle proteins, proteins related to controlling the release of synaptic vesicles, and other genes such as heat shock protein and small subunit ribosomal RNA. Over-expression of tomosyn and myosin light chain kinase genes was verified using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), confirming their increased expression in response to deltamethrin exposure in insecticide-resistant Ae. aegypti.