Search

The development of methods for conjugation of DNA to proteins is of high relevance for the integration of protein function and DNA structures. Here, we demonstrate that protein-binding peptides can direct a DNA-templated reaction, selectively furnishing DNA-protein conjugates with one DNA label. Quantitative conversion of oligonucleotides is achieved at low stoichiometries and the reaction can be performed in complex biological matrixes, such as cell lysates. Further, we have used a star-like pentameric DNA nanostructure to assemble five DNA-Rituximab conjugates, made by our reported method, into a pseudo-IgM antibody structure that was subsequently characterized by negative-stain transmission electron microscopy (nsTEM) analysis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A gold nanoparticle-based colorimetric antibody structure and activity evaluation method is developed without using complicated and expensive instrumentation. In this assay, a minimum number of antibodies to stabilize nanoparticles are conjugated to gold nanoparticles to prepare minimally stable nanoparticle probes, and the addition of salt rapidly induced particle aggregation and a color change of the solution from red to blue (25-min assay time). It is found that the solution color change is affected by the degree of structural denaturation of antibodies, and the conformational change of antibodies affects the modification of antibodies to nanoparticles and particle stability. Importantly, the colorimetric method can be applied to different types of antibodies (IgG, IgA, and IgM) and it shows comparable or better structural sensitivity than conventional circular dichroism spectroscopy. Moreover, immunoassay results show that these structural changes of antibodies are highly correlated with their antigen-binding activities. Rapid particle aggregation and high structural sensitivity are achieved in this assay because particles are modified with a minimum number of antibodies to stabilize particles in solution. This nanoparticle-based colorimetric method could be useful in evaluating the structural and activity changes of an array of antibodies in an easy, rapid, and sensitive manner., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcalpha/mu receptor (hFcalpha/muR). Ligand polymerization status was crucial for the interaction, because hFcalpha/muR binding did not occur with monomeric Ab of either class. hFcalpha/muR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcalpha/muR binding. IgM binding required contributions from both Cmu3 and Cmu4 Fc domains, whereas for dIgA, an exposed loop in the Calpha3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcalphaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFcalpha/muR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFcalpha/muR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFcalpha/muR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.

Enhanced cell death and deficient clearance of cellular debris are thought to contribute to increased self-antigen exposure in systemic autoimmune disease. To investigate the characteristics of early humoral autoimmune responses, six monoclonal antibodies were generated from two autoimmune prone strains of mice. All antibodies specifically bound the surface of late-stage apoptotic cells. Similar antibody reactivities were present in the sera of patients with systemic lupus erythematosus. While IgM antibodies significantly reduced the phagocytic uptake of apoptotic thymocytes, IgG antibodies enhanced uptake. Poly-reactivity was demonstrated in the recognition of ribonucleoproteins and lipids. An antibody reactive towards lysophosphatidylcholine reversed lysophosphatidylcholine-mediated inhibition of LPS-induced TNF-alpha production and adversely affected the transmigration of phagocytes towards an apoptotic stimulus. In several instances, CDR were characterized by the accumulation of somatic mutations. Anti-idiotypic antibodies generated upon immunization bound distinct cellular moieties and self-antigens. Poly-specific, apoptotic cell-reactive autoantibodies can therefore directly impact upon the course of disease by influencing phagocytic uptake of apoptotic cells, by inducing a pro-inflammatory environment through neutralization of bioactive lipids, by blinding phagocytes to the presence of dying cells through the negation of lipidic chemotactic signals, and by mediating diversification of the humoral autoimmune response via the idiotypic network.

Polyvalent carbohydrate-protein interactions occur frequently in biology, particularly in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions, rendering it difficult to control them with individual small molecules. Artificial macromolecules have been used as polyvalent ligands to inhibit polyvalent processes; however, both reproducible synthesis and appropriate characterization of such complex entities is demanding. Herein, we present an alternative concept avoiding conventional macromolecules. Small glycodendrimers which fulfill single molecule entity criteria self-assemble to form non-covalent nanoparticles. These particles-not the individual molecules-function as polyvalent ligands, efficiently inhibiting polyvalent processes both in vitro and in vivo. The synthesis and characterization of these glycodendrimers is described in detail. Furthermore, we report on the characterization of the non-covalent nanoparticles formed and on their biological evaluation.

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.

Human and mouse IgM can be polymerized as a hexamer in addition to a pentamer. Our previous work with mouse IgM measured activation of guinea pig complement by highly enriched preparations of hexamer and pentamer and showed that hexamer is >100-fold more active than pentamer. In this report pentamer and hexamer were compared for their capacity to activate complement in a homogeneic system, i.e. chimeric mouse V/human Cmu IgM pentamer and hexamer were assayed separately for their capacity to activate human (and guinea pig) complement. In both the homogeneic and the xenogeneic systems hexamer was more active than pentamer, but the magnitude of the difference between hexamer and pentamer depended on the complement source. Whereas chimeric hexamer activated guinea pig complement >100-fold more efficiently than did chimeric pentamer, this hexamer was only 4-13-fold more active than pentamer when assayed with human complement. Similarly, mouse hexamer, which was >100-fold more active than mouse pentamer with guinea pig complement, was only approximately 2-fold more active than mouse pentamer with human complement. Mouse hexameric and pentameric IgM were each approximately 20-fold more active with human complement than were the corresponding chimeric isoforms of IgM.

In 1993, we reported the presence of an IgM-associated peptide (M(r) 44 kDa; pI 5.45) in all immunoglobulin M (IgM) fractions purified from plasma/serum by various methods. This peptide was absent in Ig fractions of non-IgM isotypes. The N-terminal sequence was determined as being APPSGVRLVGGLH. To gain insight into the nature of this peptide, we further analyzed, using modern proteomic tools, the IgM-associated peptide isolated from cryoglobulins. Mass spectrometry revealed three peptides of different masses: 2203.13 (ELGCGAASGTPSGILYEPPAEK), 1564.83 (KPIWLSQMSCSGR), and 1544.77 (EATLQDCPSGPWGK). Theses sequences together with the already known N-terminal sequence allowed us to identity the IgM-associated peptide as Sp alpha (O43866 in TrEMBL database; CD5 antigen-like). Sp alpha is a member of the scavenger receptor cysteine-rich superfamily of proteins. This family includes the T-and B-cell antigens CD5 and CD6, and several of its members influence immune cell fate. Our finding may have important implications in the understanding of the homeostasis of IgM antibodies.

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell-dependent antigen ph-Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ-line sequence of the prototype anti-ph-Ox heavy chain variable element V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild-type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgE(KVK Delta tail)). IgE(KVK Delta tail) mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high-affinity antibodies by measuring relative affinities of phage-expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen-receptor is responsible for the selection process and the expansion of antigen-specific cells, leading to an isotype-specific antibody repertoire.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self-antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre- and post-infected Xid mice. In particular, relatively increased natural (pre-existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivities to self and non-self proteins. These results suggest that self-reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self-aggressive components responsible for pathology.

Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodies have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high performance liquid chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. This procedure not only allowed measurement of the purity of the intact antibodies but also provided detailed analysis of the complex mixtures of oligosaccharides covalently attached to these glycoproteins. The methodology out-lined allows the simultaneous processing of a number of glycoproteins separated on one single gel. In contrast to the release of carbohydrate from glycoproteins in solution, this procedure can also be conveniently applied when only impure glycoprotein is available.

To characterize the variable heavy chain (VH)3 antibody response to HIV-1 gp120, we analyzed a panel of IgM and IgG1 Fab fragments from phage display isotype libraries from a long-term, non-progressor HIV-1-infected individual. The IgM Fab antibodies isolated had low affinity for gp120, were not restricted to a particular VH3 germ-line gene, and consisted mainly of unmutated VH genes. In contrast, IgG Fab fragments were gp120 specific, with high affinity and extensive somatic mutation; all were clonally related and were derived from a single VH3 germ-line gene (DP50). One IgG Fab (S8) has DP50 VH region nucleotide substitutions identical to those of IgM Fab M025 and uses similar DH and JH segments, suggesting that S8 arose from M025 by isotype switching. In addition, somatic mutation in the IgG heavy chain third complementarity-determining region results in a 100-fold affinity increase for gp120, which correlates with a similar increase in neutralization capacity. These results imply that in vivo IgM to IgG isotype switch and affinity maturation may be important for protection and long-term survival in certain HIV-1-infected individuals.

We analyzed VDJ and VJ rearrangements in IgM-secreting B lymphocytes from a cow infected with bovine leukemia virus (BLV). BLV causes expansion of CD5(+) and IgM(+) B lymphocytes regardless of antigen specificity. The data showed that single point mutations contribute to the diversification of IgM antibodies. The most striking observation, however, is that approximately 9% of theVDJ rearrangement in IgM-secreting B cells encode an exceptionally long third complementarity-determining region of the heavy chain (CDR3H; 56 to 61 amino acids) with multiple cysteine residues. Such an exceptionally long CDR3H is the first ever to be documented for an antibody in a species. These VDJ rearrangements encode functional IgM antibodies as some of these show polyspecific reactivity. The presence of even-numbered cysteine residues in the CDR3H may provide hitherto unknown configurational ability to the antigen combining site via intra-CDR3H disulfide bridging. In addition, the VDJ rearrangements encoding exceptionally long CDR3H paired with either novel V(lambda)1 or V(x)1x genes, earlier noted not to be expressed. Overall, these experiments provide evidence that somatic hypermutations and generation of an exceptionally long CDR3H contribute to the diversification of IgM antibodies in cattle.

A mouse with an inactivated joining chain locus was produced by gene targeting in embryonic stem cells by deleting the first exon. Heterozygote (J+/-) and homozygote (J-/-) offspring from these mice showed normal total serum immunoglobulin levels and a normal peripheral B cell compartment when compared to wild-type littermates. The distribution of serum immunoglobulin isotypes in serum was different; IgA levels were elevated while IgM levels were reduced in J-/- mice as compared to wild-type mice. High molecular weight serum IgM was reduced in J+/- and J-/- mice and instead found in oligomeric form of undefined structure. Furthermore, serum IgM from J+/- and J-/- mice showed a reduced ability to activate complement. The number of splenic and bone marrow IgM plaque-forming cells were reduced in unimmunized J+/- as well as in J-/- mice. Furthermore, the number of plaque-forming cells was reduced in B cells from both J+/- and J-/- mice after stimulation with lipopolysaccharide in vitro. The perturbation of IgM production in J-/- mice appears to affect a late stage of differentiation, because cells with intracellular IgM were readily detected both in vivo and in vitro. Finally, after immunization with T-dependent or T-independent antigens the IgM component of the immune response was reduced in J-/- mice while only a marginal reduction of the IgG response was detected.

Using a degenerate oligonucleotide primer specific for immunoglobulin (Ig) constant type 1 (C-1 set) domain genes, products were amplified by the reverse transcriptase-polymerase chain reaction from nurse shark spleen cDNA. The deduced protein sequence of one of these clones reveals a novel Ig class in cartilaginous fish. A complete mRNA could encode a mature protein bearing an amino-terminal variable (V) domain, followed by six C-1 set domains, and ending in a carboxy-terminal tail typical of secreted IgM, IgA, and the new antigen receptor (NAR). The two amino-terminal C domains are orthologous to IgX (or IgR), an Ig heavy (H) chain class in the skate, and the last four domains are homologous to the carboxy-terminal four domains of NAR. We designate this "chimeric" Ig class IgNARC for Ig new antigen receptor from cartilaginous fish. Like NAR, but unlike shark IgM, IgNARC is encoded by very few V and C genes which apparently are not closely linked. The number of bands that hybridize with exon-specific probes varies with genomic DNA from individual sharks, suggestive of different numbers of IgNARC genes in different animals. A protein of approximately 95 kDa, which is likely to be the IgNARC H chain, is immunoprecipitated with both light chain-specific monoclonal antibodies and with antisera generated to a peptide comprising the IgNARC carboxy-terminal tail. We conclude that the arsenal of secreted antigen receptors in cartilaginous fish is greater than previously believed. In addition, our data cast doubt on the dogma that IgM is the primordial Ig isotype.

Previous studies on IgM secretion demonstrated a role for the mu chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of mu and alpha heavy chains prompted us to investigate the role of cysteine-mediated, retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, alpha 2L2 subunits, but not mu2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the alpha chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.

Membrane-bound immunoglobulins of the IgM and IgD class are expressed on the B cell surface in association with a disulfide-linked heterodimer consisting of alpha and beta subunits. While the alpha component of the IgM antigen receptor (IgM-alpha, 34 kDa) is encoded by the B cell-specific gene mb-1, the gene coding for IgD-alpha (35 kDa) has not yet been identified. We show here that the alpha component of the IgD antigen receptor is also encoded by the mb-1 gene. The difference in molecular weight between IgM-alpha and IgD-alpha thus seems to be due to post-translational modifications of the mb-1 gene product. We also demonstrate that the previously described myeloma variant J558L delta m2.6 expresses an alternative form of the IgD antigen receptor, which does not contain an alpha/beta heterodimer.

Beside the immunoglobulin (Ig) heavy and light chains the murine B cell receptor of the IgM class contains a heterodimer of two transmembrane proteins (IgM-alpha and Ig-beta). By N-terminal sequencing of IgM-alpha and Ig-beta we have identified the genes encoding these proteins as mb-1 and B29, respectively. Both genes are B cell specific and have been previously cloned from B minus T cell subtractive cDNA libraries. We have constructed expression vectors of the two genes and demonstrate that expression of the mb-1 and B29 genes can influence the surface expression of IgM in micron-transfected myeloma cells. From the known sequences of the IgM-alpha and Ig-beta proteins and from the results of previous transfection experiments with various vectors expressing the mu chain we have developed a structural model of the B cell antigen receptor of class IgM which we compare with that of the T cell antigen receptor.

The inducible B cell lymphoma, CH12, and its in vitro adapted subclone, CH12-LBK, produce immunoglobulins of identical sequence, specificity and isotype, with equivalent affinities for the hapten trimethyl ammonium. However, the hemolytic efficiencies of the antibody secreted by the two cell lines are quite different. Antibody preparations from lipopolysaccharide-stimulated CH12 cells lyse erythrocytes six- to ten times more effectively than antibody preparations of the same concentration from CH12-LBK cells. Both cell lines secrete polymeric IgM, but while CH12-LBK cells secrete predominantly the canonical pentameric IgM, CH12 cells secrete a mixture of pentamers and hexamers. High-efficiency complement-dependent cytolysis is associated with hexameric IgM, which has a specific activity that is approximately twenty times higher than that of the pentameric form. J chain protein is found in the secreted IgM of both cell lines, but is associated only with the pentameric IgM and not with the hexameric form, nor with any intermediate polymers smaller than a pentamer. A deficit in, or the inaccessibility of, J chain protein appears to facilitate hexamer formation. These experiments confirm previously published data showing that J chain is not necessary either for assembly or secretion of polymeric IgM, and suggest instead that J chain may be important in regulating the lytic efficiency of polymeric IgM by controlling the IgM pentamer/hexamer ratio. The experiments further suggest a mechanism, in addition to isotype switching and somatic mutation, by which the biological efficiency of antibodies from a single clone of B cells can be regulated.