Multiple Variables at the Leukocyte Cell Surface Impact Fc γ Receptor-Dependent Mechanisms

Fc γ receptors (FcγR) expressed on the surface of human leukocytes bind clusters of immunoglobulin G (IgG) to induce a variety of responses. Many therapeutic antibodies and vaccine-elicited antibodies prevent or treat infectious diseases, cancers and autoimmune disorders by binding FcγRs, thus there is a need to fully define the variables that impact antibody-induced mechanisms to properly evaluate candidate therapies and design new intervention strategies. A multitude of factors influence the IgG-FcγR interaction; one well-described factor is the differential affinity of the six distinct FcγRs for the four human IgG subclasses. However, there are several other recently described factors that may prove more relevant for disease treatment. This review covers recent reports of several aspects found at the leukocyte membrane or outside the cell that contribute to the cell-based response to antibody-coated targets. One major focus is recent reports covering post-translational modification of the FcγRs, including asparagine-linked glycosylation. This review also covers the organization of FcγRs at the cell surface, and properties of the immune complex. Recent technical advances provide high-resolution measurements of these often-overlooked variables in leukocyte function and immune system activation

Direct Determination of the Site of Addition of Glucosyl Units to Maltooligosaccharide Acceptors Catalyzed by Maize Starch Synthase I

Starch synthase (SS) (ADP-glucose:1,4-α-D-glucan 4-α-D-glucosyltransferase) elongates α-(1→4)-linked linear glucans within plastids to generate the storage polymers that constitute starch granules. Multiple SS classes are conserved throughout the plant kingdom, indicating that each provides a unique function responsible for evolutionary selection. Evidence has been presented arguing for addition of glucosyl units from the ADPglucose donor to either the reducing end or the non-reducing end of the acceptor substrate, although until recently direct evidence addressing this question was not available. Characterization of newly incorporated glucosyl units determined that recombinant maize (Zea mays L.) SSIIa elongates its substrates at the non-reducing end. However, the possibility remained that other SSs might utilize distinct mechanisms, and that one or more of the conserved enzyme classes could elongate acceptors at the reducing end. This study characterized the reaction mechanism of recombinant maize SSI regarding its addition site. Newly incorporated residues were labeled with 13C, and reducing ends of the elongation products were labeled by chemical derivitization. Electrospray ionization-tandem mass spectroscopy traced the two parameters, i.e., the newly added residue and the reducing end. The results determined that SSI elongates glucans at the non-reducing end. The study also confirmed previous findings showing recombinant SSI can generate glucans of at least 25 units, that it is active using acceptors as short as maltotriose, that recombinant forms of the enzyme absolutely require an acceptor for activity, and that it is not saturable with maltooligosaccharide acceptor substrates

Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (Kd 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed. Published by Wiley-Blackwell. © 2010 The Protein Society

A single amino acid distorts the Fc γ receptor IIIb/CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a

Therapeutic mAbs engage Fc γ receptor III (CD16) to elicit a protective cell-mediated response and destroy the target tissue. Newer drugs designed to bind CD16a with increased affinity surprisingly also elicit protective CD16b-mediated responses. However, it is unclear why IgG binds CD16a with more than 10-fold higher affinity than CD16b even though these receptors share more than 97% identity. Here we identified one residue, Gly-129, that contributes to the greater IgG binding affinity of CD16a. The CD16b variant D129G bound IgG1 Fc with 2-fold higher affinity than CD16a and with 90-fold higher affinity than the WT. Conversely, the binding affinity of CD16a-G129D was decreased 128-fold relative to WT CD16a and comparably to that of WT CD16b. The interaction of IgG1 Fc with CD16a, but not with CD16b, is known to be sensitive to the composition of the asparagine-linked carbohydrates (N-glycans) attached to the receptor. CD16a and CD16b-D129G displaying minimally processed oligomannose N-glycans bound to IgG1 Fc with about 5.2-fold increased affinity compared with variants with highly processed complex-type N-glycans. CD16b and the CD16a-G129D variant exhibited a smaller 1.9-fold affinity increase with oligomannose N-glycans. A model of glycosylated CD16b bound to IgG1 Fc determined to 2.2 Å resolution combined with a 250-ns all-atom molecular dynamics simulation showed that the larger Asp-129 residue deformed the Fc-binding surface. These results reveal how Asp-129 in CD16b affects its binding affinity for IgG1 Fc and suggest that antibodies engineered to engage CD16b with high affinity must accommodate the Asp-129 side chain

Species-Specific and Inhibitor-Dependent Conformations of LpxC: Implications for Antibiotic Design

LpxC is an essential enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different Gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against Gram-negative infections

Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)

BACKGROUND: The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish

Mouse IgG2c and hIgG1 Fc show different strand and loop interactions.

<p><b>A</b>. Mouse and human Fc fragments show a high degree of global similarity with an rmsd of 0.7 Å. <b>B</b>. P291 of hIgG1 disrupts a beta sheet formation in the C' strand, and shifts the register by one residue that is relieved by a bulge at mIgG2c D295. Differences and similarities are identified in residues that mediate loop interactions in mIgG2c Fc (<b>C</b>) and hIgG1 Fc (<b>D</b>).</p