Direct Binding Studies Research Articles

Asn792 Participates in the Hydrogen Bond Network Around the K+-binding Pocket of Gastric H,K-ATPase

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin

Structural data have indicated that the 60-loop of thrombin with 8-9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However,previous deletion of 3-4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired approximately 2-4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved approximately 2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.

The functional significance of the autolysis loop in protein C and activated protein C

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

The Critical Role of the 185–189-Loop in the Factor Xa Interaction with Na+ and Factor Va in the Prothrombinase Complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.

Analysis of calibration methodologies for solvent effects in drug discovery studies using evanescent wave biosensors

Recent improvements in sensitivity have enabled direct binding studies of small molecules with evanescent wave biosensors, which monitor binding by measuring refractive index changes close to the sensing surface. The universal solvent for small molecules, dimethylsulfoxide has a high refractive index; consequently, on ligate addition a large non-specific solvent effect is seen which can mask the specific signal. It has been previously noted that different sensor surfaces can respond differently to the same buffer change. The difference is proposed to arise from differences in buffer space and contraction and swelling of the surface hydrogel. Within this paper, a number of calibration approaches are investigated and tested using warfarin binding to human serum albumin as a model system. A number of recommendations are made for accurate referencing for non-specific effects. Changes to the ionic strength of the running buffer had little effect, whilst changes to the charge density of the carboxylmethyl dextran significantly affected how well the control surface reflects the non-specific signal. An amended 'calibration method' can be used, however, it is an additional complex step that was found to overcorrect in the presence of non-specific binding. Matching immobilisation levels between control and active surface significantly reduces solvent differences allowing accurate correction providing solvent compositional changes are minimised in experimental design. Under these circumstances, the traditional method of simple subtraction of the control from the active response is the most appropriate method of correction.

Light chains are a ligand for megalin.

Cubilin and megalin are giant glycoprotein receptors abundant on the luminal surface of proximal tubular cells of the kidney. We showed previously that light chains are a ligand for cubilin. As cubilin and megalin share a number of common ligands, we further investigated the ligand specificity of these receptors. Three lines of evidence suggest that light chains can also bind megalin: 1) anti-megalin antiserum largely displaces brush-border light chain binding and megalin-expressing BN-16 cell uptake more than anti-cubilin antiserum, 2) direct binding studies on isolated proteins using surface plasmon resonance techniques confirm that megalin binds light chains, and 3) light chains compete with known megalin ligands for brush-border membrane binding and BN-16 cell uptake. The megalin-light chain interaction is divalent ion dependent and similar for both kappa- and lambda-light chains. A fit of the data on light chain binding to megalin over a concentration range 0.078-2.5 mg/ml leads to an estimated dissociation constant of 6 x 10(-5) M, corresponding approximately to one light chain-binding site per megalin and in the same range for dissociation constants for cubilin binding. These data suggest that light chains bind the tandem megalin-cubilin complex. Megalin is the major mediator of light chain entry into megalin-expressing membrane such as the apical surface of proximal tubular epithelial cells.

Nucleic Acid Is a Novel Ligand for Innate, Immune Pattern Recognition Collectins Surfactant Proteins A and D and Mannose-binding Lectin

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)(8)(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)(8)(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues.

The adaptor disabled-2 binds to the third ΨxNPxY sequence on the cytoplasmic tail of megalin

The cytoplasmic tail (CT) of megalin possesses several functional motifs likely to participate in protein–protein interactions within the proximal tubular epithelial cell (PTEC) of the kidney. One such interaction is with the phosphotyrosine interaction domain (PID) of the adaptor protein disabled-2 (Dab2), a mitogen-responsive phosphoprotein, which interacts via its PID with ΨxNPxY (where Ψ represents a hydrophobic residue) motifs on its binding partners. Megalin CT has three such motifs; it has been established that there is no interaction of Dab2 with the first (from N to C) (Biochem. J. 3 (2000) 613). Here, we analyse in real-time the binding of recombinant megalin CT, and of synthetic peptide sequences encompassing the second and third ΨxNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of KD = 2.6 × 10–7 ± 5.3 × 10–8. Direct binding and competition studies indicate that this interaction is with the third ΨxNPxY motif. The dissociation of Dab2 from the third ΨxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05). Synthetic peptide sequences encompassing the third ΨxNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third ΨxNPxY motif. Tyrosine phosphorylation of either motif did not exert a major effect upon competition efficacy. We further demonstrate for the first time the presence of Dab2 expression in primary human PTEC.

The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

Effect of HLA-DR-based T cell peptides derived from phospholipase A2 on late-phase skin reactions and T cell function in subjects with mild honeybee allergy

10 Effect of HLA-DR-BasedT Cell Peptides Derived from Phospholipase A2 on Late-Phase Skin Reactions and T Cell Function in Subjects with Mild Honeybee Allergy M. TarzU, C. Texier 2, B. Maill~re 2, M. Larch6 l, A. B. Kayl; 1Allergy and Clinical Immunology, NHLI Division, Imperial College London, London, UNITED KINGDOM, 2Ing6nierie et d'Etudes des Prot6ines, CEASaclay, Gif sur Yvette, FRANCE. RATIONALE: Whole venom immunotherapy for bee sting allergy is highly efficacious, but IgE-mediated systemic reactions frequently complicate treatment. Allergen-derived T cell peptides may negate this problem, however the diversity of epitopes conferred by HLA polymorphism makes peptide selection difficult. Texier et al (Journal of Immunology 2000; 164:3177-84) characterized T cell peptide epitopes of phospholipase A 2 (PLA2, the major allergen of honeybee venom) by direct binding studies with commonly expressed HLA-DRBI molecules. In this study, we assessed the effect on various immunological parameters of repeated injections of an HLA-DR based PLA 2 peptide mixture. METHODS: Eight volunteers allergic to honeybee venom received 9 injections of PLA 2 peptides. At baseline and 3 months the late-phase cutaneous reaction to bee venom was measured and peripheral blood mononuclear cells (PBMCs) were extracted for proliferation and cytokine production assays. RESULTS: Peptides were not associated with any adverse reactions. There was a significant decrease in the size of late-phase cutaneous reactions to bee venom (baseline 54.0 ram, outcome 38.8 mm). The proliferation of PBMCs to bee venom decreased significantly (baseline stimulation index 20.5, outcome 6.8). The production of IL-13 by PBMCs decreased (baseline 400.3 pg/ml, outcome 115.8 pg/ml), as did the production of IFN7 (baseline 100.9 pg/ml, outcome 47.6 pg/ml) whilst there was a trend for an increase in IL-10 (baseline 28.3 pg/ml outcome 53.4 pg/ml). CONCLUSIONS: The selection of peptides by HLA binding affinity may be a useful method for selection of immunodominant T cell epitopes. This study supports the potential of HLA-DR based T cell peptides in the treatment of human allergic disease. Funding: Imperial College l~ndon 20 The Kinetics of Eotaxin-2 Release in Nasal Secretions Following Nasal Allergen Challenge: Its Inhibition by a Topical Steroid

Neoglycolipid Technology: Deciphering Information Content of Glycome

Publisher Summary Neoglycolipid technology was introduced in 1985 to enable direct binding studies to be conveniently performed with oligosaccharides of glycoproteins. At that time, there was a lack of microscale methods for examining the carbohydrate–protein interactions, after release of the oligosaccharides from the carrier proteins. Most carbohydrate–protein interactions are of such low affinities that di- or multivalence of both oligosaccharide and recognition protein is required for detection by precipitation, radioimmunoassay, or enzyme-linked immunosorbent assay experiments. It was clear that a method was required to examine the released oligosaccharides in a multivalent state. The existing methods for examining the recognition of specific oligosaccharides by antibodies and other carbohydrate-recognizing proteins involved purification of the oligosaccharides, often from highly heterogeneous mixtures, and testing them individually as inhibitors of the binding of the recognition proteins to macromolecules or cells. The amounts of oligosaccharides required for inhibition of binding were frequently prohibitive. The approach chosen to address the problem was chemical conjugation of oligosaccharides to lipid. The artificial glycolipids, neoglycolipids, formed would enable the presentation of the oligosaccharides in the clustered state. The conjugation principle selected was reductive amination to a phosphatidylethanolaminetype aminolipid. For O-glycans released by reductive alkali treatment, a mild periodate oxidation procedure was included to generate reactive aldehydes for the conjugation. A major advantage of conjugating each oligosaccharide in a mixture to a lipid molecule rather than to a protein, such as bovine serum albumin, is that each component remains discrete and can be isolated.

Kininogen domain 3 contains regions recognized by antiphosphatidylethanolamine antibodies

Antiphosphatidylethanolamine antibodies (APE) have been described in patients with thrombotic diseases and recurrent pregnancy loss (RPL). It has been reported that certain APE are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, called kininogens. Our recent in vitro data suggest that APE may recognize the domain 3 (D3) region of kininogens. In this study, we have used synthetic peptides that span the D3 of kininogens in inhibition and direct binding studies to identify epitopes that are sites for binding APE. Our present data demonstrate that among 24 RPL patients who were positive for kininogen-dependent immunoglobulin (IgG) APE, 17 patients (70.8%) recognized the LDC27 peptide. We mapped the APE-binding region on D3 using plasma from a RPL patient (X) who had a high titer of IgG APE that recognized LDC27. APE of patient X recognized a 13-residue segment in LDC27, named CNA13. Leu331-Met357 (LDC27) and Cys333-Lys345 (CNA13) are located on the carboxyl-terminal portion of kininogen D3, which is known as the major kininogen heavy chain cell attachment site where it overlaps its cysteine protease inhibitory region. Because APE interferes with the balance of hemostasis in vitro, APE may therefore induce a similar condition in patients thereby causing thrombosis and RPL.

Localization of the Heparin Binding Exosite of Factor IXa

Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.

Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav.

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cgamma1 (PLCgamma1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y(132), Y(171), Y(191), and Y(226) have been shown previously to be critical for binding to Gads, Grb2, and PLCgamma1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y(171) and potentially Y(226) are docking sites for the Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

Functional Consequences of the Loss of High Affinity Agonist Binding to γ-Aminobutyric Acid Type A Receptors: IMPLICATIONS FOR RECEPTOR DESENSITIZATION

We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.

Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic.

We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds >30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins.

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.

Heparan sulfate-growth factor interactions

Publisher Summary This chapter examines the techniques that are typically used to demonstrate the dependence of receptor–growth factor interactions on heparan sulfate (HS), focusing on the fibroblast growth factors (FGFs). It describe methods for blocking the binding of growth factor to cell surface HS, and a novel method in which FGF and a soluble form of FGF receptor are used to detect endogenous HS that is capable of mediating their assembly into a signaling complex. Binding of growth factors to HS is dependent on the sulfation of the HS chain, a process that occurs in the Golgi apparatus through the action of specialized sulfotransferases. These enzymes utilize the high-energy sulfate donor phosphoadenosine phosphosulfate (PAPS), transferring sulfate from PAPS to the growing HS chain. A useful method for examining the binding specificity of a growth factor-receptor pair is to use the isolated proteins for direct binding studies in vitro . For the FGFs, this has been used extensively to determine the general sulfation requirements for binding. In addition, as the assembly of the FGF with the receptor is also dependent on HS, this assembly can be examined in vitro using FGF and soluble FR constructs as probes.

Is the class A macrophage scavenger receptor (SR-A) multifunctional? - The mouse's tale.

The class A macrophage scavenger receptor (SR-A) is the prototypic member of an expanding family of membrane receptors collectively termed scavenger receptors (SRs) (1–3). Receptors of this group recognize a number of ligands, including chemically modified or altered molecules and, in particular, the modified lipoproteins that are pertinent to the development of vascular disease. As shown in Table ​Table1,1, all characterized SR-A ligands are polyanionic, although many polyanions fail to bind SR-A. Here, we wish to summarize and comment upon the evidence for SR-A being both a multiligand and multifunctional receptor, considering both the available in vitro data and the results of several studies of SR-A–deficient mice. Two other SRs, namely SR-B1 (Krieger, ref. 4) and LRP (Herz and Strickland, ref. 5), are the subject of separate reviews in this Perspective series. Table 1 SR-A ligands. Receptor recognition or nonrecognition has been shown for these molecules, either through direct binding studies or the inhibition of modified lipoprotein endocytosis. Two themes that can be drawn from studies of SRs as a whole are particularly relevant to this discussion. First, because these receptors display broad and seemingly overlapping ligand-binding properties, biological specificity is likely to be determined not only by ligand structure and the signal transduced following ligand binding, but also by other considerations. These include the distribution and availability of the various SRs, their ability to interact with other receptors, and their relative affinities for the various ligands. Second, the apparent redundancy of ligand binding is achieved despite the absence of conserved protein sequences among the distinct classes of SRs. A mechanistic understanding of individual SRs’ broad yet specific ligand recognition and of the features shared by these unrelated molecules will require the resolution of tertiary structures of multiple SRs.

Fibrin-mediated plasminogen activation.

Fibrin, but not fibrinogen, enhances the rate of activation of plasminogen by tissue type plasminogen activator (t-PA). Studies with enzymatic and chemical fragments of fibrinogen showed that several sites in fibrinogen are involved in this rate enhancement; these are, A alpha 148-160 (located in CNBr fragment FCB-2), and FCB-5 (a CNBr fragment comprising gamma 312-324), and recently discovered sites in the fibrinogen alpha C domains. All these sites are buried in fibrinogen, but exposed in fibrin and some fibrinogen fragments. For the first two of these, located in the D-domains, this was shown by the fact that monoclonal antibodies against A alpha 148-160 and gamma 312-324 bind to fibrin and rate enhancing fibrin(ogen) fragments, but not to fibrinogen. Direct binding studies indicate that at physiological concentrations plasminogen binds to FCB-2, and t-PA to FCB-5. More detailed studies have demonstrated the importance of residues A alpha-157 and A alpha-152, and that the minimum stretch with rate enhancing properties is A alpha 154-159. The sites in the alpha C domains await further identification. With the recently reported three-dimensional structure of fragments D and D-dimer it is now possible to explain these findings at the molecular level. Molecular calculations and experimental data show that the site A alpha 148-160 in fibrinogen is covered among others by a part of the A alpha chain (A alpha 166-195) that forms an alpha-helix, and by a globular domain formed by the beta-chain. On fibrin formation, the last two may move away, and give access to A alpha 148-160. It is conceivable that in the alpha C domain sites are involved in the early phases of fibrinolysis. The site A alpha 148-160 and that in FCB-5 may be more important at later stages. It is also clear that fibrin polymerization is important. This polymerization has probably several effects: exposure of the rate enhancing sites; mutual positioning of the t-PA and plasminogen binding sites; a concentrating effect of t-PA and plasminogen on the fibrin surface; effects on the kinetic properties of t-PA and plasminogen. These effects together explain the rate enhancement.