Avidity confers FcγR binding and immune effector function to aglycosylated immunoglobulin G1

Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc-mediated immune effector functions. Human IgGs have an N-glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N-linked glycosylation site has been reported to "silence" FcγR-binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen-engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes.     

A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A

Peptidyl prolyl cis/trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca(2+)-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.     

Chemiluminescent acridinium-9-carboxamide boronic acid probes: application to a homogeneous glycated hemoglobin assay

Chemiluminescent acridinium-9-carboxamide probes containing 1, 3, 9, and 27 phenylboronic acids were prepared and their chemiluminescent properties evaluated. The relative chemiluminescent signal from the probes varied from 4 to 0.83 x 10(19)counts/mol across the series, while the apparent affinity of the probes for the diabetes marker glycated hemoglobin increased from 211 to 0.43 microM. The dose-dependent modulation of the chemiluminescent intensity of the probes upon binding was used to demonstrate a homogeneous assay for glycated hemoglobin.     

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.     

Dimer dissociation is essential for interleukin-8 (IL-8) binding to CXCR1 receptor

Chemokines play a fundamental role in trafficking of immune cells and in host defense against infection. The role of chemokines in the recruitment process is highly regulated spatially and temporally and involves interactions with G protein-coupled receptors and cell surface glycosaminoglycans. The dynamic equilibrium between chemokine monomers and dimers, both free in solution and in cell surface-bound forms, regulates different components of recruitment such as chemotaxis and receptor signaling. The binding and activity of the chemokine interleukin-8 (IL-8) for its receptors, previously studied using "trapped" non-associating monomers and non-dissociating dimers, show that the monomer has a native-like function but support conflicting roles for the dimer. We have measured the binding of native IL-8 to the CXCR1 N-domain, using isothermal titration calorimetry and sedimentation equilibrium techniques. The N-domain constitutes a critical binding site, and IL-8 binding affinity to the receptor N-domain is in the same concentration range as the IL-8 monomerdimer equilibrium. We observed that only the IL-8 monomer, and not the dimer, is competent in binding the receptor N-domain. Based on our results, we propose that IL-8 dimerization functions as a negative regulator for the receptor function and as a positive regulator for binding to glycosaminoglycans and that both play a role in the neutrophil recruitment process.     

Evaluation of healing following frenectomy

Aberrant frenum attachment would cause plaque accumulation and malalignment of teeth. It can be managed by frenotomy or frenectomy methods, through a conventional surgical technique or laser technique. Therefore, it is of interest to compare frenectomy healing surgical and laser techniques. Data from 51 outpatients and post-operative healing of frenectomy was assessed by Landry’s healing score index using 3 weeks postoperative photographs followed by statistical analysis. Based on the healing score index, the laser technique showed better outcomes than the surgical technique. Moreover, the association between the management of high frenal attachment and the healing score index was found to be statistically significant.     

Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding

Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.     

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D ¹⁵N-edited, ¹³C-edited HSQC-NOESY and 2D homonuclear ¹H-¹H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 Å for single spectral data, of 1 Å to 1.5 Å for double spectral data, and of 0.6 Å for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 Å and 1.3 Å for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 Å rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.     

Heterochromatin,HP1 and methylation at lysine 9 of histone H3 in animals

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.     

Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N‐terminal domain

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N‐terminal domain (Site‐I) and the extracellular/transmembrane domain (Site‐II). Therefore, higher monomer affinity could be due to stronger binding at Site‐I or Site‐II or both. We have now characterized the binding of a human CXCR1 N‐terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N‐domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ～10‐ to 100‐fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site‐I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.