Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

Immunoglobulin G4-related Retroperitoneal Fibrosis of the Pelvis

Retroperitoneal fibrosis (RPF) is a rare disease characterized by the replacement of normal tissue with fibrosis and/or inflammation. In this case, a 68-year-old man presented with RPF in the pelvis, a rare location for this disease. Biopsies were performed, which showed elevated levels of C-reactive protein, erythrocyte sedimentation rate, and, most importantly, immunoglobulin G4 (IgG4). It has been postulated that IgG4-related sclerosing disease is a systemic disease. Treatment has been successful with systemic corticosteroids.Key words: Retroperitoneal fibrosis, Serum immunoglobulin G4Retroperitoneal fibrosis (RPF) is characterized by the replacement of the normal retroperitoneal tissue with fibrosis and/or chronic nonspecific inflammation.¹ Pelvic retroperitoneal fibrosis is a rare location for the primary presentation of this disease, with only a few documented cases in the literature.² Identification of immunoglobulin G4 (IgG4) autoimmune activity among these cases has begun to improve our understanding of the inflammatory nature of this rare disease process.³ This case demonstrates the involvement of immune-mediated IgG4 within a case of pelvic retroperitoneal fibrosis.     

A1H NMR investigation of the hydrolysis of a synthetic substrate by KDN-sialidase fromCrassostrea virginica

The mechanism of hydrolysis of 4-methylumbelliferyl 3-deoxy-d-glycero--d-galacto-2-nonulopyranosidonic acid (KDN2MeUmb,4) by KDN-sialidase isolated from the hepatopancreas of the oysterCrassostrea virginica has been monitored by¹H NMR spectroscopy. The results of these experiments reveal that KDN-sialidase catalyses the hydrolysis of the synthetic substrate KDN2MeUmb, with initial release of -d-KDN. This is consistent with an overall mechanism for the hydrolysis which proceeds with retention of anomeric configuration. These results agree with earlier NMR studies of otherN-acetylneuraminic acid-recognising sialidases from both viral and bacterial sources.     

Applications of tritium NMR to macromolecules: A study of two nucleic acid molecules

Summary We have tritium labeled two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa hammerhead RNA for tritium NMR investigations. The DNA sequence studied has been previously used in homonuclear studies of DNA-bound water molecules and tritium NMR was expected to facilitate these investigations by eliminating the need to suppress the water resonance in tritium-detected ³H-¹H NOESY experiments. We observed the anticipated through-space interactions found in B-form DNA in the NOESY experiments and an unexpected antiphase cross-peak at the water frequency. T₁ measurements on the tritiated DNA molecule indicated that relaxation rates were also accelerated for tritium and protons. Tritium NMR spectra of the hammerhead RNA molecule indicated conformational dynamics in the conserved region of the molecule in the absence of Mg²⁺ and spermine, two components necessary for cleavage. The dynamics were also investigated by ¹⁵N-correlated ¹H spectroscopy and persisted after the addition of Mg²⁺ and spermine.     

Correlation of Immunoglobulin G Expression and Histological Subtype and Stage in Breast Cancer

Introduction

Recently, growing evidence indicates that immunoglobulins (Igs) are not only produced by mature B lymphocytes or plasma cells, but also by various normal cells types at immune privileged sites and neoplasm, including breast cancer. However, the association of breast cancer derived IgG with genesis and development of the disease has not yet been established.

Methods

In this study we examined the expression of IgG in 186 breast cancers, 20 benign breast lesions and 30 normal breast tissues. Both immunohistochemistry with antibodies to Igκ (immunoglobulin G κ light chain) and Igγ (immunoglobulin G heavy chain) and in situ hybridization with an antisense probe to IgG1 heavy chain constant region gene were performed. Various clinicopathological features were also analyzed.

Results

We found that IgG is specifically expressed in human breast cancer cells. Both infiltrating ductal carcinoma and infiltrating lobular carcinoma had significantly greater numbers of Igκ and Igγ positive cancer cells as compared with medullary carcinoma, carcinoma in situ, and benign lesions (all p<0.05). In addition, IgG expression was correlated with breast cancer histological subtypes (p<0.01) and AJCC stages (p<0.05), with more abundance of IgG expression in more malignant histological subtypes or in more advanced stage of the disease.

Conclusions

IgG expression in breast cancer cells is correlated with malignancy and AJCC stages of the cancers. This suggests that breast cancer derived IgG may be associated with genesis, development and prognosis of the cancer.     

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing ¹H–¹⁵N heteronuclear three-dimensional NMR experiments on uniformly ¹⁵N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with ³J_NH coupling constants and ¹H_N exchange data, indicate that the secondary structure consists of a five-stranded parallel -sheet and four -helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into ²H₂O, the ¹H^N3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.     

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: Structural refinement using heteronuclear multidimensional NMR spectroscopy

Summary The 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of ¹⁵N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131–1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for -angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 -angle restraints, and 32 -angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 ¹, 8 ², and 1 ³ angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44±0.12 (1 is 0.1 nm) for the backbone atoms C, C, and N in the four -helices A1, residues 4–15, A2, residues 21–36, A3, residues 51–62 and A4, residues 65–84. The loop-region of residues Gly⁴⁵-Lys⁵⁰ could not be defined by the restraints obtained by NMR.The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.     

A Monomeric Chicken IgY Receptor Binds IgY with 2:1 Stoichiometry

IgY is the principal serum antibody in birds and reptiles, and an IgY-like molecule was the evolutionary precursor of both mammalian IgG and IgE. A receptor for IgY on chicken monocytes, chicken leukocyte receptor AB1 (CHIR-AB1), lies in the avian leukocyte receptor cluster rather than the classical Fc receptor cluster where the genes for mammalian IgE and IgG receptors are found. IgG and IgE receptors bind to the lower hinge region of their respective antibodies with 1:1 stoichiometry, whereas the myeloid receptor for IgA, FcαRI, and the IgG homeostasis receptor, FcRn, which are found in the mammalian leukocyte receptor cluster, bind with 2:1 stoichiometry between the heavy chain constant domains 2 and 3 of each heavy chain. In this paper, the extracellular domain of CHIR-AB1 was expressed in a soluble form and shown to be a monomer that binds to IgY-Fc with 2:1 stoichiometry. The two binding sites have similar affinities: K_a1 = 7.22 ± 0.22 × 10⁵ m⁻¹ and K_a2 = 3.63 ± 1.03 × 10⁶ m⁻¹ (comparable with the values reported for IgA binding to its receptor). The affinity constants for IgY and IgY-Fc binding to immobilized CHIR-AB1 are 9.07 ± 0.07 × 10⁷ and 6.11 ± 0.02 × 10⁸ m⁻¹, respectively, in agreement with values obtained for IgY binding to chicken monocyte cells and comparable with reported values for human IgA binding to neutrophils. Although the binding site for CHIR-AB1 on IgY is not known, the data reported here with a monomeric receptor binding to IgY at two sites with low affinity suggest an IgA-like interaction.Fc receptors link the specificity of the adaptive immune system with the effector mechanisms of innate immune cells. In birds and reptiles, IgY is the principal serum antibody, and both mammalian IgG and IgE have evolved from an IgY-like ancestor, so studies of IgY offer insights into their origins (1). The historical contribution of chicken immunology to a wider understanding of the subject has been considerable (2), and recently several chicken IgY-Fc receptors have been identified. In this paper, the chicken antibody, IgY, is shown to bind to a chicken leukocyte receptor, CHIR-AB1,⁴ in a different manner from that of its mammalian orthologues, IgG and IgE, to their respective Fc receptors.Phagocytosis, mediated in mammals by IgG, and passive cutaneous anaphylaxis, mediated by both IgG and IgE in mammals, have been observed in chickens (3, 4), presumably both effected by IgY. In vitro, IgY binds to monocyte cell lines (5, 6), and an IgY receptor (CHIR-AB1) has been identified that is able to mediate the influx of calcium into cells (5).The genes for the mammalian high affinity IgE receptor, and several IgG receptors, are located in the classical Fc receptor cluster, whereas in chickens, this cluster is represented by a single gene, the product of which has been expressed and found not to bind IgY (7). Intriguingly, the first IgY leukocyte receptor, CHIR-AB1, was found to be a member of the chicken leukocyte receptor cluster (LRC) (5), adjacent to over 100 genes with high intersequence homology (8). This finding, together with phylogenetic analysis of the orthologous Fc receptor gene clusters (7, 9), implies that during the evolution of the IgY-like ancestor of both IgG and IgE, antibody-Fc binding function migrated from proteins expressed in the LRC to those in the classical Fc receptor cluster. The human LRC is the site of FcαRI, the leukocyte receptor for IgA (an antibody involved in mucosal immunity), the fetal IgG receptor (FcRn, involved in adult IgG homeostasis), and also a number of natural killer cell receptors including the HLA-G ligand, KIR2DL4 (10). A further leukocyte receptor for chicken IgY, also related to LRC receptors, was identified recently, on chromosome 20 (11), and remains to be characterized.Typically, the stoichiometry of the receptor-antibody complex differs for receptors located in the classical Fc receptor cluster and the LRC. Crystal structures of IgG complexes with FcγRIII and of IgE with FcϵRI show 1:1 receptor:antibody stoichiometry, with the receptor binding across both heavy chains in the lower hinge (12). In contrast, the crystal structure of FcαRI complexed with IgA shows 2:1 stoichiometry (13) as does that of FcRn with IgG (14), with the two receptors binding between the heavy chain constant domains 2 and 3 on each heavy chain. The IgY/receptor interaction could have either stoichiometry; on the one hand, IgY is an orthologue of IgG and IgE, which can both show 1:1 stoichiometry, but on the other hand, the location of the IgY receptor, CHIR-AB1, in the same gene cluster as the IgA and FcRn receptors suggests the possibility of a 2:1 stoichiometry. Consistent with either of these binding modes, the crystal structure of IgY-Fc reveals that many of the residues located in the receptor-binding sites in human IgE, IgG, and IgA are present and accessible in IgY (15).The single extracellular domain of the chicken leukocyte IgY receptor, CHIR-AB1, has been expressed in insect cells by Arnon et al. (16), who showed that this preparation consists of a mixture of soluble monomer and dimer. Because of the heterogeneity of the protein, it was not possible to ascertain whether the observed 2:1 stoichiometry of receptor binding to antibody involved two monomers or a single dimer binding to IgY. Thus, it was not possible to answer the question of whether the antibody-receptor complex most resembles that of human IgA or of IgG and IgE. We have expressed the extracellular domain of CHIR-AB1 in human HEK cells. It is a monomer, and we report here that it binds to IgY and IgY-Fc with 2:1 stoichiometry.     

Induction of Antiviral Antibodies by DNA Immunization Requires neither Perforin-Mediated nor CD8+-T-Cell-Mediated Lysis of Antigen-Expressing Cells

DNA immunization induces antibodies to the encoded protein, which indicates that the protein must gain access to the extracellular milieu, allowing it to interact with naïve B lymphocytes. It has been suggested that antigen release may be effected by cytotoxic-T-lymphocyte-mediated lysis of transfected antigen-expressing cells; this might be particularly important for the induction of responses to a noncytopathic, cytosolic protein. Here we show that the induction of antibody responses to one such DNA-encoded protein required neither perforin nor CD8⁺ T cells. In addition, there was no skewing of the immunoglobulin G isotypes in the absence of perforin.DNA immunization is unique in that antigenic proteins are synthesized within the transfected cell in the absence of any associated infectious agent, and the vaccine itself contains no soluble protein that could initiate humoral immune responses. Thus, if the immunizing plasmid expresses a cytoplasmic protein which is relatively noncytopathic and is unable to be effectively processed endogenously by the major histocompatibility complex (MHC) class II antigen presentation pathway, then it might be ineffective in inducing humoral immune responses. The nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) meets these criteria, yet intramuscular injection with a plasmid expressing the NP (pCMV-NP) has been shown by us and others to induce antibody responses in mice (13, 23, 24). Antibodies are also induced following DNA immunization with plasmids expressing other cytoplasmic antigens, such as the measles virus and influenza virus NPs (4, 18, 21). How might such antigens be released from the transfected cell? We hypothesized that the development of CD8⁺ antigen-specific cytotoxic T lymphocytes (CTL) could lead to the recognition and lysis of cells expressing plasmid-derived NP, resulting in the liberation of protein into the extracellular milieu, where it can then interact with B cells and antigen-presenting cells (APCs). CTL-mediated release of LCMV NP occurs during virus infection (12), and the possibility that it occurs following DNA immunization was strengthened by our observations of a profound myositis following intramuscular inoculation of pCMV-NP into LCMV-infected or -immune mice; the peak of the inflammatory infiltrate in acutely infected mice coincided with the development of anti-LCMV CTL, and destruction of muscle cells occurred (8, 22). Professional APCs, which are known to be a source of plasmid-expressed antigens (5) and which appear to be the cell type responsible for initiating immune responses following DNA immunization (6, 7), can also be recognized and lysed by CTL. Dendritic cells infected with human immunodeficiency virus are susceptible to lysis in vitro by CTL (11), and there is evidence to suggest that CD8⁺ T cells can limit the immune response by lysing APCs in vivo (1–3). Therefore, the possibility of lytic release of protein is not limited to NP-expressing myocytes but extends to most transfected somatic cells, including APCs. To assess what effect such lysis may have on the generation or maintenance of B-cell responses following DNA immunization, we analyzed humoral immune responses in DNA-immunized mice that lacked the cytolytic protein perforin (10, 20). Although antigen-specific CD8⁺-T-cell responses were induced in these mice by vaccination with pCMV-NP, they were unable to lyse NP-expressing cells in a perforin-dependent manner (data not shown).

Strong antibody responses are induced by DNA immunization of PKO mice.

To determine if a lack of perforin-mediated lysis by antigen-specific CTL resulted in an alteration in the temporal appearance or maintenance of antiviral serum antibodies, antibody levels were measured in PKO and C57BL/6 mice at 0, 2, 4, and 6 weeks after they received a single 50-μg intramuscular injection of pCMV-NP DNA. Serum immunoglobulin G (IgG) levels in individual mice were measured by enzyme-linked immunosorbent assay (ELISA), and the average for each group was calculated based upon the optical density measurement at a dilution of 1:200; the results are shown in Fig. ​Fig.1A.1A. Within 2 weeks of vaccination, anti-LCMV antibodies were demonstrable in both PKO and C57BL/6 mice, and the average levels in both groups were similar. The slight drop in antibody levels in the C57BL/6 mice at 4 weeks after DNA immunization is not statistically significant, but at 6 weeks, the difference is highly significant. In mice, the average half-life of an IgG molecule is approximately 6 to 10 days (17, 19). Therefore, the high level of antibodies present at 6 weeks postimmunization implies ongoing synthesis of NP-specific IgG in PKO mice, whereas the drop in antibody levels indicates decreased IgG synthesis in C57BL/6 mice. A representative analysis of the antibody response at 6 weeks postimmunization is presented in Fig. ​Fig.1B.1B. Anti-LCMV serum antibody levels were measured by ELISA in individual perforin-positive (C57BL/6, n = 6) or perforin-negative (PKO, n = 8) animals 6 weeks postvaccination. LCMV DNA-vaccinated C57BL/6 and PKO mice both produced anti-LCMV IgG. Together, these data clearly show that perforin-mediated release of plasmid-expressed LCMV NP is not required for the induction of humoral responses following intramuscular DNA injection. Open in a separate windowFIG. 1PKO mice mount strong antibody responses. (A) PKO and C57BL/6 mice (n = 4/group) were immunized with pCMV-NP and bled biweekly. Anti-LCMV serum antibodies were measured by ELISA. The mean optical density readings at 492 nm (OD₄₉₂) are shown for serum dilutions of 1:200, with standard errors. (B) Representative analyses of antibody responses at 6 weeks postimmunization. Anti-LCMV antibody levels in groups of DNA-immunized PKO (n = 8) and C57BL/6 (n = 6) mice were measured by ELISA. For controls, sera were derived from LCMV-immune or nonimmune C57BL/6 mice.

Isotype responses are unaltered in DNA-immunized perforin-deficient mice.

IgG responses to the LCMV NP require CD4⁺-T-cell help, and unlike the LCMV glycoprotein, endogenously synthesized NP cannot gain access to the MHC class II antigen presentation pathway (15). Thus, the CD4⁺ T cells required for immunoglobulin isotype class switching must be primed by APCs which have acquired extracellular NP. To determine if the isotype pattern was skewed in the absence of perforin, titers of LCMV-specific IgG, IgG1, IgG2a, and IgG2b were determined by ELISA in individual PKO and C57BL/6 mice 6 weeks after DNA immunization (Fig. ​(Fig.2).2). At this time point, all of the pCMV-NP DNA-immunized mice contained virus-specific immunoglobulin in their serum. Anti-LCMV IgG endpoint titers among PKO mice ranged from 1:4,000 to 1:15,000, with a geometric mean titer (Fig. ​(Fig.2)2) of 1:6,825. In contrast, among the C57BL/6 vaccines, the total IgG titers ranged from 1:1,700 to 1:3,400, with a geometric mean of 1:2,455. Detectable IgG1 and IgG2a were present in the sera of all mice of both strains with the exception of a single PKO mouse which lacked demonstrable levels of anti-LCMV IgG1. Therefore, the vaccinated PKO mice showed no skewing of their isotype classes, suggesting that CD4⁺-T-cell responses are appropriately induced in these mice and providing further evidence that perforin is not required for the release of NP from transfected cells. Open in a separate windowFIG. 2No skewing of antibody isotypes in PKO mice. IgG isotype titers were measured in individual PKO and C57BL/6 mice at 6 weeks after immunization with pCMV-NP. Triangles, individual mice; circles, geometric mean titers for each group. The LCMV-immune sample represents pooled sera from virus-immune C57BL/6 mice.

CD8⁺ T cells play no part in controlling humoral responses to DNA-encoded antigens.

Our data show that perforin is not required to prime B-cell responses against a plasmid-expressed, cytoplasmic antigen. Perforin is expressed by CD8⁺ CTL, some CD4⁺ T cells, and natural killer (NK) cells; we can therefore conclude that perforin-mediated lysis by any of these cell populations is not required for DNA-mediated antibody induction. However, other CD8⁺-T-cell functions, such as the Fas pathway (16) or the secretion of cytotoxic cytokines, can mediate target cell death and thus could liberate intracellular antigens (9). Our observation that PKO mice had elevated antibody titers compared to those of C57BL/6 mice (Fig. ​(Fig.11 and ​and2)2) also raised the possibility that perforin-mediated lysis might actually depress humoral responses to DNA immunization by killing cells expressing foreign plasmid-encoded antigens. Consistent with this idea, CD8⁺-T-cell-mediated lysis of APCs has been shown to suppress immune responses (1–3), and perforin has been shown to play a role in regulating immunity (14). If this hypothesis is correct, the absence of perforin might result in elevated and/or prolonged responses to DNA-encoded immunogens. To determine whether CD8⁺ T cells might play a perforin-independent role in antigen release and antibody induction, CD8⁺ T cells were removed from C57BL/6 mice by immunodepletion prior to and throughout the course of a DNA immunization experiment. C57BL/6 mice were treated with either an anti-mouse CD8⁺-T-cell monoclonal antibody or saline daily for 3 days prior to immunization with pCMV-NP and weekly thereafter. Nondepleted PKO mice served as controls. Mice were bled biweekly, and the average IgG response for each group is shown in Fig. ​Fig.3A.3A. Antibody responses were easily detected in CD8-depleted mice, showing that CD8⁺-T-cell functions are not required to release soluble NP for the initiation of humoral immune responses. Furthermore, at all time points, the antibody responses in CD8-depleted and nondepleted C57BL/6 mice were indistinguishable; therefore, it appears to be unlikely that CD8⁺ T cells play a role in down-regulating DNA-induced antibody responses. To confirm that the CD8⁺ cells had been successfully ablated by the monoclonal antibody treatment, splenocytes were obtained from mice immediately before DNA immunization (data not shown) and from three mice (two depleted and one sham-depleted) at the conclusion of the experiment and were analyzed for CD8 expression by flow cytometry (Fig. ​(Fig.3B).3B). The administration of anti-CD8 monoclonal antibody maintained CD8⁺-T-cell populations at almost undetectable levels throughout the course of the experiment. Thus, we conclude that the development of fully functional CD8⁺ CTL following DNA immunization neither inhibits nor enhances humoral immune responses. Open in a separate windowFIG. 3Depletion of CD8 cells has no effect on antibody induction by plasmid DNA. (A) Anti-LCMV antibody titers were measured over time in C57BL/6 mice depleted of CD8⁺ T cells (C57+anti CD8), nondepleted C57BL/6 mice (C57+saline), and nondepleted PKO mice (PKO+saline). Each group consisted of three animals, and the average antibody endpoint titer for each group at each time point is shown. (B) To confirm successful depletion of CD8⁺ cells, splenocytes were harvested from two mice treated with anti-CD8 antibody and from one control mouse. These cells were evaluated for CD8 expression by flow cytometry.These data argue against the hypothesis that lytic-antigen-specific CD8⁺ CTL have a profound effect on the generation of humoral immunity to plasmid-encoded antigens. Perforin is normally expressed not only in CD8⁺ T cells but also in some CD4⁺ T cells and in NK cells. Our observations therefore are not restricted to perforin-mediated CD8⁺-T-cell lysis but also show that perforin-mediated lysis by CD4⁺ T cells or NK cells plays no essential role in antigen release. Furthermore, the ratios of IgG1 to IgG2a were similar regardless of the perforin status of the mouse (Fig. ​(Fig.2).2). Thus, not only is perforin expendable in IgG induction but also its absence has no effect on the antibody classes—and, presumably, the ratio of Th1 to Th2 cells—induced.In conclusion, it is clear that plasmids encoding proteins which are relatively noncytotoxic and incapable of endogenous MHC class II antigen presentation can elicit strong IgG responses in the absence of CD8⁺ T cells and perforin. Thus, the mechanism of antigen release and B-cell stimulation remains to be determined. It is important that we identify the underlying mechanisms to permit the rational optimization of DNA vaccines.     

Determination of internal dynamics of deoxyriboses in the DNA hexamer d(CGTACG)2 by 1H NMR

The conformations and internal dynamics of the deoxyriboses of d(CGTACG)₂ have been determined by NMR measurements at 15°C. The conformations of the sugars were determined using coupling constants and time-dependent NOE measurements. The J-splitting patterns of the H1, H2 and H2 resonances show that the sugars exist as mixtures of conformations near C2 endo (south) and C3 endo (north). The population of the south conformation was larger for the purines than for the pyrimidines. The overall tumbling time of the molecule in ²H₂O was determined from measurements of the cross relaxation rate constant for the H6-H5 vectors of the two cytosine residues. Order parameters were determined for the H1-H2, H2-H2 and H2-H3 vectors from measurements of cross relaxation rate constants, making use of multi-spin analysis of the NOE build up rates. These order parameters are weakly dependent of the base sequence, and except for the terminal Cyt 1 residue, the H2-H2 and H2-H3 vectors are near unity, indicating the absence of rapid pseudorotation on the nanosecond time scale. However, the order parameter for the H1-H2 vector is significantly smaller than expected for rapid pseudorotation indicating the presence of other motions of the sugars. This motion must be about an effective axis parallel to the H2-H vector, and to occur with an angular fluctuation of about 30°.The results show that to obtain highly refined structures for nucleic acids by NMR the effects of spin diffusion and motional averaging cannot be ignored.Some of this work was presented as a poster at the 30^th Experimental NMR Conference at Asilomar, California 1989     

Insights from Fc receptor biology: A route to improved antibody reagents

Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year’s meeting will be no exception.From a therapeutic viewpoint, there is much to be gained from a detailed understanding of the biology of effector molecules such as Fc receptors and complement. Indeed, knowledge of the interaction of IgG with such molecules has been central to the development of improved mAbs with altered functions and transformed half-lives, tailored for particular therapeutic applications. Examples include mAbs designed to maximise complement recruitment¹ or to enhance Fc receptor engagement and triggering of ADCC,^2-5 or conversely, variants engineered to be unable to engage complement⁶ or Fc receptors.⁷ Glycoengineering of IgG Fc offers an alternative means to modify effector function capabilities,⁸ while development of IgG mutants that display extended or altered serum half-lives has been driven through exhaustive analysis of the interaction with FcRn.^9,10Despite the appreciable advances that have been made in unravelling the various facets of Fc receptor biology, new information pertinent to mAb engineering continues to emerge. A flavour of some of these new advances will be given below. They span novel receptors and receptor roles, structure-function relationships, the molecular architecture of signaling complexes, the influence of the membrane lipid environment and scaffolding interactions, isotype considerations, through to technical innovations likely to inform the field.Remarkably, new receptors that have previously eluded characterization are now being described. These include the IgM receptor, which evidence indicates is a molecule also known as TOPO/Fas apoptotic inhibitory molecule 3 whose gene lies close to other known immunoglobulin receptors on chromosome 1,¹¹ and a receptor for IgD recently documented on basophils.¹² Moreover, we are seeing an appreciation of new roles for existing Fc receptors. An example is the demonstration in a transgenic study that human FcγRIIa can trigger active and passive anaphylaxis and airway inflammation. Moreover, human mast cells, monocytes and neutrophils were shown to produce anaphylactogenic mediators when FcγRIIA was engaged.¹³ Hence IgG may contribute to allergic and anaphylactic reactions in humans by engaging FcγRIIa.Exciting new structural information on Fc receptors and their ligands is emerging. An important example is the solving of the X-ray crystal structure for human FcγRI.¹⁴ While the structural information supports a ligand binding mode similar to those of FcγRII or FcγRIII, the FG-loop in domain 2 of FcγRI with its conserved one-residue deletion appears critical for high affinity IgG binding. A second example concerns the high responder/low responder (HR/LR) polymorphisms of FcγRIIa, which are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. New insights into these differences have been provided by the recent solving of the structure for the complex of the HR allele with IgG Fc.¹⁵ Third, understanding of the human IgE-FcεRI interaction has moved forward significantly through the solving of the X-ray crystal structure of the complex of FcεRI and the entire Fc region of IgE (comprising domains Cε2, Cε3 and Cε4).¹⁶ In a final example, the structural basis for the improved efficacy of nonfucosylated mAbs has been investigated.¹⁷ The X-ray crystal structure of the complex between nonfucosylated IgG Fc and a soluble form of FcγRIIIa carrying two N-linked glycans showed that one of two receptor glycans interacts with nonfucosylated Fc to stabilize the complex. It is proposed that when the Fc glycan is fucosylated this interaction is inhibited due to steric hindrance and, together with the negative effects of Fc fucosylation on the dynamics of the receptor binding site, this provides a rationale for the improved ADCC displayed by nonfucosylated IgG.A question of interest is precisely how Fc receptors bound to antibody ligands organize themselves within signaling complexes in the cell membrane. Some intriguing clues to this conundrum of molecular architecture are now surfacing. In mast cells, FcεRI molecules loaded with IgE form a synapse when presented with antigen that is mobile within a lipid bilayer, via coalescence into large cholesterol-rich clusters.¹⁸ Of particular relevance to the therapeutic setting, clustering of receptors into immune synapses is also seen with FcγR. For instance, during in vivo ADCC mediated by tumor-specific mAb, clustering of FcγR, actin and phosphotyrosines has been noted at contact zones between tumor cells and macrophages or neutrophils.¹⁹ The theme of the influence of the membrane lipid domain environment on Fc receptor function is taken up elsewhere. It has been shown, for example, that serine phosphorylation of FcγRI influences membrane mobility and function. The cytoplasmic tail of FcγRI interacts with protein 4.1G,²⁰ and it is proposed that this is mediated via a phosphoserine-dependent mechanism critical for localization of the receptor to lipid rafts.²¹ With regard to FcγRIIa, a major role for lipid rafts in the regulation of IgG binding to FcγRIIa has been revealed.²² Notably, exclusion of FcγRIIa from lipid raft membrane microdomains is able to suppress IgG binding in myeloid cells.Increased knowledge of the capabilities of Fc receptors specific for other antibody classes is opening up new options for therapy. For example, IgA antibodies may offer a highly useful and efficacious alternative approach of particular relevance to treatment at mucosal sites. Human IgA mAbs have been demonstrated to mediate efficient tumor cell killing^23,24 and to have the capability to control certain infectious diseases.^25,26 The detailed understanding of functional sites in IgA that has resulted from numerous mutagenesis studies,²⁷ coupled with improved ways to produce and isolate recombinant IgA mAbs²⁸ should facilitate developments toward therapeutics based on this immunoglobulin class. Similarly, recent studies indicate that IgE may serve as an alternative to the classic IgG backbone for therapeutic antibodies.²⁹Finally, technical innovations seem poised to further inform the field and advances are arriving or may be anticipated from techniques such as solution nuclear magnetic resonance (NMR) spectroscopy,³⁰ cryo-electron tomography,³¹ single particle tracking,³² and ultrasensitive force techniques such as adhesion frequency assays.^33,34Interest in Fc receptors continues unabated, and the contribution that the field can make to mAb development and optimisation is unquestionable. The FASEB SRC on Immunoreceptors will serve as a forum for discourse on the above issues and much more, providing invaluable information and networking opportunities for all those interested in ways to maximise the efficacy of mAbs and mAb-based reagents. Registration is open until 24 June 2012.     

Surface charge and calcium binding in sarcoplasmic reticulum membranes

Vesicles of fragmented sarcoplasmic reticulum membranes have been adsorbed on to 2.68 latex spheres. Observation of these vesicle containing spheres in the presence of an electric field allows a calculation of the electrophoretic mobility of the vesicles Following this determination, the net membrane surface charge has been estimated. The mobility of sarcoplasmic reticulum membranes exhibited a dependency on pH. At an ionic strength of 0.10 a mobility (pH=7.0) of –0.67±0.10/sec/volt/cm was observed. At pH=7.0 and /2=0.150 the net excess negative charge density was 2.0×10^–2 coul/m². This is equivalent to one charge per 10³ A² (assuming a uniform charge distribution). With an average vesicle volume of 2.8×10⁸ A³ and a surface area of 2×10⁶ A² the surface of one vesicle would contain a net of approximately 2×10³ negative charges. While the mobility did not change during uptake of calcium by the vesicles, both glutaraldehyde fixation and lecithin extraction by phospholipase C greatly altered the mobility of the vesicle membrane. Calcium binding and uptake both exhibited a dependence on pH.     

Cell wall anionic polymers and peptidoglycan of Actinoplanes philippinensis VKM Ac-647

The cell wall of Actinoplanes philippinesis VKM Ac-647 harbours several carbohydrate-containing anionic polymers. (1) The main polymer of the wall is of a poly(glycosylglycerol phosphate) nature. Its monomeric units — O--d-mannopyranosyl-(14)--d-galactopyranosyl-(11)-glycerol monophosphates — are connected by phosphodiester bonds involving the hydroxyl groups at glycerol C3 and galactose C6. There also are chains without mannosyl substitutents. The teichoic acid structure has been established by chemical analysis and with ¹H and ¹³C NMR spectroscopy. This is the first finding of a teichoic acid with mannosyl residues in a bacterial cell wall. (2) The phosphorylated mannan contains mannose and 2-O-methylmannose. Its core chain has -1,2; -1,3; and -1,6 substitutions as revealed by ¹³C NMR spectroscopy.The peptide unit of the peptidoglycan contains no l-alanine, instead of which position 1 is occupied by glycine; and diaminopimelic acid is represented, besides its meso- (or DD) form, by small amounts of its LL isomer.Abbreviations Gro glycerol - Gro2P glycerol-2 phosphate - APT attached-proton-test - P_tot total content of phosphorus - P_lab phosphorus mineralized in 7 min at 100°C - P_NA phosphorus of nucleic acids - P_stab stable phosphorus - T trace amounts     

Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

Backbone dynamics of uniformly ¹⁵N-labeled free barnase and its complex with unlabelled barstar have been studied at 40°C, pH 6.6, using ¹⁵N relaxation data obtained from proton-detected 2D {¹H}-¹⁵N NMR spectroscopy. ¹⁵N spin-lattice relaxation rate constants (R₁), spin-spin relaxation rate constants (R₂), and steady-state heteronuclear {¹H}-¹⁵N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide ¹⁵N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (_m) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action.     

Lateral diffusion of plasma membrane receptors labelled with either platelet-derived growth factor (PDGF) or wheat germ agglutinin (WGA) in human polymorphonuclear leukocytes and fibroblasts

The aims of the present investigation were (a) to compare the lateral mobility of membrane receptors of human fibroblasts and polymorphonuclear leukocytes (PMNL) labelled with either platelet-derived growth factor (PDGF), or the lectin wheat germ agglutinin (WGA), and (b) to study effects of serum or PDGF on the mobility of these receptor molecules in human fibroblasts. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard (10%) or under serum-free conditions yielding normal and starved cells, respectively. The receptor mobility was studied in response to exposure to PDGF, or serum, in short time or prolonged incubations. Human polymorphonuclear leukocytes (PMNL) were adhered to microscope slides by clotting drops of blood. They were stained with rhodaminated PDGF or fluoresceinated WGA. The diffusion of labelled receptors was assessed with fluorescence recovery after photobleaching (FRAP). It was found that (a) fibroblasts grown at normal serum concentration had a lower diffusion coefficient (D=3×10^–10 cm² s^–1) for the PDGF-receptor and a slightly lower mobile fraction (R=60%) than starved cells (D=5×10^–10 cm²s^–1 and R=73%), (b) addition of serum to starved cells increased both D and R for the PDGF receptor to 12×10^–10 cm² s^–1 and 96%, respectively, (c) a similar pattern was obtained for WGA-labelled glycoconjugates indicating general membrane effects of serum-induced cell stimulation, and (d) in PMNL the PDGF receptor displayed motility characteristics (D=3–4×10^–10 cm² s^–1 and R=59%) similar to those in fibroblasts, possibly suggesting equivalent anchorage mechanisms in the membrane.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

The difference between protein structures obtained by x-ray analysis and nuclear magnetic resonance

We have analyzed the pairs of protein structures obtained by X-ray diffraction analysis and nuclear magnetic resonance (X-ray and NMR structures) that display no major differences when superimposed on one another (61 pairs). Analyzing atom-to-atom contacts (contact distances 2–8 Å), it has been found that the NMR structures (compared to the X-ray structures) have more contacts at distances below 3.5 Å and above 5.5 Å. In the case of residue-to-residue contacts, the NMR structures have more contacts at distances below 3 Å and between 4.5 and 6.5 Å. At other distances analyzed, the X-ray structures have more contacts. The difference in the numbers of atom-to-atom and residue-to-residue contacts is greater for buried residues inaccessible to water than for surface residues. Another important difference is related to the number of hydrogen bonds in the main chain: this number is greater in the X-ray structures. The coefficient of correlation between the numbers of hydrogen bonds identified in the structures obtained by both methods is only 32%. If a complete set of NMR models of protein structure is considered, the total number of hydrogen bonds proves to be 1.2 times greater than in the X-ray structures, whereas the correlation coefficient increases to only 65%. We have also demon-strated that -helices in the NMR structures are more distorted (compared to the ideal -helix) than those in the X-ray structures.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 129–138.Original Russian Text Copyright © 2005 by Melnik, Garbuzynskiy, Lobanov, Galzitskaya.     

Cystic Fibrosis: A Brief Look at Some Highlights of a Decade of Research Focused on Elucidating and Correcting the Molecular Basis of the Disease

The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter–like protein found in the plasma membrane of animal cells. CFTR is believed to function primarily as a Cl^– channel, but evidence is mounting that this protein has other roles as well. Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains. These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process. Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1. The most common mutation causing CF is the deletion () of a single phenylalanine (F) in position 508 within a putative helix located in NBF1. CF patients bearing this F508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30. Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the F508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect. In general, this pioneering or ground roots research has succeeded according to plan. This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory. For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and cost effective strategies to identify agents that correct the structural defect in the F508 CFTR protein causing most cases of CF.