Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.     

New cholesterol-specific antibodies remodel HIV-1 target cells�� surface and inhibit their in vitro virus production

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.     

Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase

Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of quartz crystal microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a K(D) below 1 nm being the highest affinity among HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential two-site model. Besides the one, which had been shown previously in course of x-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.     

Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine: IMPLICATIONS IN CALCIUM SIGNALING*

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca²⁺ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca²⁺ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca²⁺ signaling, we hypothesize that SPC could lead to Ca²⁺ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca²⁺ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca²⁺-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.     

GAP43 shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase

It has recently been proposed that prolyl oligopeptidase (POP), the cytosolic serine peptidase with neurological implications, binds GAP43 (Growth-Associated Protein 43) and is implicated in neuronal growth cone formation, axon guidance and synaptic plasticity. We investigated the interaction between GAP43 and POP with various biophysical and biochemical methods in vitro and studied the co-localisation of the two proteins in differentiated HeLa cells. GAP43 and POP showed partial co-localisation in the cell body as well as in the potential growth cone structures. We could not detect significant binding between the recombinantly expressed POP and GAP43 using gel filtration, CD, ITC and BIACORE studies, pull-down experiments, glutaraldehyde cross-linking and limited proteolysis. However, glutaraldehyde cross-linking suggested a weak and transient interaction between the proteins. Both POP and GAP43 interacted with artificial lipids in our in vitro model system, but the presence of lipids did not evoke binding between them. In native polyacrylamide gel electrophoresis, GAP43 interacted with one of the three forms of a polyhistidine-tagged prolyl oligopeptidase. The interaction of the two proteins was also evident in ELISA and we have observed co-precipitation of the two proteins during co-incubation at higher concentrations. Our results indicate that there is no strong and direct interaction between POP and GAP43 at physiological conditions.     

Neonatal FcR overexpression boosts humoral immune response in transgenic mice

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.     

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.     

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.