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Introduction: Antibody Fc regions harbour the binding sites for receptors that mediate effector functions following antigen engagement by the Fab regions. An extended "hinge" region in IgG allows flexibility between Fab and Fc, but in both the most primitive antibody, IgM, and in the evolutionarily more recent IgE, the hinge is replaced by an additional domain pair in the homodimeric six-domain Fc region. This permits additional flexibility within the Fc region, which has been exploited by nature to modulate antibody effector functions. Thus, in pentameric or hexameric IgM, the Fc regions appear to adopt a planar conformation in solution until antigen binding causes a conformational change and exposes the complement binding sites. In contrast, IgE-Fc principally adopts an acutely bent conformation in solution, but the binding of different receptors is controlled by the degree of bending, and there is allosteric communication between receptor binding sites., Methods: We sought to trace the evolution of Fc conformational diversity from IgM to IgE via the intermediate avian IgY by studying the solution conformations of their Fc regions by small-angle X-ray scattering. We compared four extant proteins: human IgM-Fc homodimer, chicken IgY-Fc, platypus IgE-Fc, and human IgE-Fc. These are examples of proteins that first appeared in the jawed fish [425 million years ago (mya)], tetrapod (310 mya), monotreme (166 mya), and hominid (2.5 mya) clades, respectively., Results and Discussion: We analysed the scattering curves in terms of contributions from a pool of variously bent models chosen by a non-negative linear least-squares algorithm and found that the four proteins form a series in which the proportion of acutely bent material increases: IgM-Fc < IgY-Fc < plIgE-Fc < huIgE-Fc. This follows their order of appearance in evolution. For the huIgM-Fc homodimer, although none are acutely bent, and a significant fraction of the protein is sufficiently bent to expose the C1q-binding site, it predominantly adopts a fully extended conformation. In contrast, huIgE-Fc is found principally to be acutely bent, as expected from earlier studies. IgY-Fc, in this first structural analysis of the complete Fc region, exhibits an ensemble of conformations from acutely bent to fully extended, reflecting IgY's position as an evolutionary intermediate between IgM and IgE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Calvert, Nyamboya, Beavil and Sutton.)

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab') 2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab') 2 subunits is much slower than the other four F(ab') 2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of IgMs, composed of 21 human protein subunits correctly assembled into pentamers. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kallolimath, Palt, Föderl-Höbenreich, Sun, Chen, Pruckner, Eidenberger, Strasser, Zatloukal and Steinkellner.)

Immunoglobulin M (IgM) is the first antibody to emerge during embryonic development and the humoral immune response 1 . IgM can exist in several distinct forms, including monomeric, membrane-bound IgM within the B cell receptor (BCR) complex, pentameric and hexameric IgM in serum and secretory IgM on the mucosal surface. FcμR, the only IgM-specific receptor in mammals, recognizes different forms of IgM to regulate diverse immune responses 2-5 . However, the underlying molecular mechanisms remain unknown. Here we delineate the structural basis of the FcμR-IgM interaction by crystallography and cryo-electron microscopy. We show that two FcμR molecules interact with a Fcμ-Cμ4 dimer, suggesting that FcμR can bind to membrane-bound IgM with a 2:1 stoichiometry. Further analyses reveal that FcμR-binding sites are accessible in the context of IgM BCR. By contrast, pentameric IgM can recruit four FcμR molecules to bind on the same side and thereby facilitate the formation of an FcμR oligomer. One of these FcμR molecules occupies the binding site of the secretory component. Nevertheless, four FcμR molecules bind to the other side of secretory component-containing secretory IgM, consistent with the function of FcμR in the retrotransport of secretory IgM. These results reveal intricate mechanisms of IgM perception by FcμR., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient concentrating and purifying of IgM from hybridoma culture supernatants. Hybridoma culture supernatants containing IgMs were diluted 3-fold with 10 mM phosphate buffer (pH 7.0) and passed through the column. During this process, zirconia particles selectively adsorbed these IgMs, and most of the contaminating proteins flowed out into the flow-through. The adsorbed IgMs were easily eluted with a small volume of 400 mM phosphate buffer (pH 8.0), and high-concentration IgM solutions were prepared. Subsequent simple processing using a Capto™ Core 400 cartridge column provided highly purified IgM. The operation is easy, and the activity of IgM is maintained because the purification process is performed using only neutral ranges of phosphate buffers. Here, we showed that anti-globoside and anti-CDw75 IgM purified by this method can be used to stain cervical cancer and Burkitt lymphoma cells that specifically express these respective tumor-associated carbohydrate antigens., Competing Interests: Declaration of competing interest The National Institute of Advanced Industrial Science and Technology holds patents (JP6172687B2, US9796785B2, and EP2993184B1) that relate to the materials reported in this article. The authors declare no other competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The B cell receptor (BCR) initiates immune responses through antigen recognition. We report a 3.3-angstrom cryo-electron microscopy structure of human immunoglobulin M (IgM)-BCR in the resting state. IgM-BCR comprises two heavy chains, two light chains, and the Igα/Igβ heterodimer. The ectodomains of the heavy chains closely stack against those of Igα/Igβ, with one heavy chain locked between Igα and Igβ in the juxtamembrane region. Extracellular interactions may determine isotype specificity of the BCR. The transmembrane helices of IgM-BCR form a four-helix bundle that appears to be conserved among all BCR isotypes. This structure contains 14 glycosylation sites on the IgM-BCR ectodomains and reveals three potential surface binding sites. Our work reveals the organizational principles of the BCR and may facilitate the design of antibody-based therapeutics.

Molecular structures provide a road map for understanding and controlling B cell receptor activation.

The B cell receptor (BCR) complex plays a critical role in B cell development and immune responses. The assembly mechanisms underlying the BCR complex remain unknown. We determined the cryo-electron microscopy (cryo-EM) structures of human IgG-BCR and IgM-BCR, which consist of membrane-bound immunoglobulin molecules (mIg) and Igα/β subunits at a 1:1 stoichiometry. Assembly of both BCR complexes involves their extracellular domains, membrane-proximal connection peptides, and transmembrane (TM) helices. The TM helices of mIgG and mIgM share a conserved set of hydrophobic and polar interactions with Igα/β TM helices. By contrast, the IgG-Cγ3 and IgM-Cμ4 domains interact with extracellular Ig-like domains of Igα/β through head-to-tail and side-by-side modes, respectively. This work reveals the structural basis for BCR assembly and provides insights into BCR triggering.

Objectives: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context., Methods: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available., Results: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies., Conclusions: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

As central effectors of the adaptive immune response, immunoglobulins, or antibodies, provide essential protection from pathogens through their ability to recognize foreign antigens, aid in neutralization, and facilitate elimination from the host. Mammalian immunoglobulins can be classified into five isotypes-IgA, IgD, IgE, IgG, and IgM-each with distinct roles in mediating various aspects of the immune response. Of these isotypes, IgA and IgM are the only ones capable of multimerization, arming them with unique biological functions. Increased valency of polymeric IgA and IgM provides high avidity for binding low-affinity antigens, and their ability to be transported across the mucosal epithelium into secretions by the polymeric immunoglobulin receptor allows them to play critical roles in mucosal immunity. Here we discuss the molecular assembly, structure, and function of these multimeric antibodies.

Antibodies of the immunoglobulin M (IgM) class represent the frontline of humoral immune responses. They are secreted as planar polymers in which flanking µ 2 L 2 "monomeric" subunits are linked by two disulfide bonds, one formed by the penultimate cysteine (C575) in the tailpiece of secretory µ chains (µ s tp) and the second by C414 in the Cµ3. The latter bond is not present in membrane IgM. Here, we show that C575 forms a non-native, intra-subunit disulfide bond as a key step in the biogenesis of secretory IgM. The abundance of this unexpected intermediate correlates with the onset and extent of polymerization. The rearrangement of the C-terminal tails into a native quaternary structure is guaranteed by the engagement of protein disulfide isomerase ERp44, which attacks the non-native C575 bonds. The resulting conformational changes promote polymerization and formation of C414 disulfide linkages. This unusual assembly pathway allows secretory polymers to form without the risk of disturbing the role of membrane IgM as part of the B cell antigen receptor., (© 2021 The Authors.)

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination., Competing Interests: I have read the journal’s plicy and the authors of this manuscript have the following competing interests: BW holds a patent entitled ‘Enhancing serological assays via fusion proteins’ US10101327B2 describing the fusion protein based technology described in the COVID-19 Multiplex Assay. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N -glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans . A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through a highly conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity., Competing Interests: Declaration of interests All authors are employees of Genentech, Inc., a member of the Roche Group, and may hold stock and options., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF ( p < 0.01) or GF ( p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM ( p < 0.01), IgG ( p < 0.05), IL-2 ( p < 0.01), IL-4 ( p < 0.01), IL-6 ( p < 0.01), and IFN-γ ( p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines., Competing Interests: Disclosures: C.D. Thouvenel, M.F. Fontana, J. Netland, A.T. Krishnamurty, Y. Chen, D. Rawlings, and M. Pepper reported a patent to serial # appl. no. 16/319,755: "Identification and production of high affinity IgM antibodies and derivatives thereof," pending. N. King reported personal fees from Icosavax, Inc. and grants from Pfizer outside the submitted work. No other disclosures were reported., (© 2021 Thouvenel et al.)

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent for the production system used to make the protein for this study in the cytoplasm of E. coli using a sulfhydryl oxidase and an isomerase is held by the University of Oulu: Method for producing natively folded proteins in a prokaryotic host (patent no. 9238817; date of patent January 19, 2016; inventor: L.W.R.)., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protective effect of Tagetes erecta flowers essential oils was investigated on oxidative stress, immune response, inflammation, and apoptosis against N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats. Essential oil were extracted from Tagetes erecta flowers and analyzed using gas chromatography-mass spectrometry (GC-MS). For observing a protective effect against MNNG induced gastric cancer, we divided rats into 4 groups (group A to D) having 10 rats in each group. Performed various experiments and measured a different parameters to investigate antioxidant activity, immune response, anti-inflammatory and anti-apoptotic activity. The levels of malondialdehyde were markedly increased in the presence of N-methyl-N'nitro-N-nitroguanidine, whereas, the antioxidant activities of superoxide dismutase, and catalase were lowered in the treated rats in contrast with the control. Intervention with TEEO to gastric cancer-induced rats upregulated the redox status and the activity of the immune system to decrease cancer risk. The proinflammatory cytokines (IL-6 and TNF-α) secretions that were induced by MNNG were markedly inhibited by TEEO. Administration of TEEO also significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positive gastric cancer cells, expression of mRNA of caspase-3, and Bax. Whereas, the expression of Bcl-2 was increased. Additionally, downregulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and IκBα degradation and the nuclear factor-κB (NF-κB) p65 expression in tissues of the stomach of MNNG-induced-rats were markedly elevated due to TEEO. This suggested possession of TEEO with a protective shield against MNNG induced gastric cancer by the exertion of antioxidative stress, anti-apoptotic response, the anti-inflammatory response through Nrf2/HO-1, and NF-κB signaling pathways.

Multivalent interactions frequently occur in biological systems and typically provide higher binding affinity and selectivity in target recognition than when only monovalent interactions are operative. Thus, taking inspiration by nature, bivalent or multivalent nucleic acid aptamers recognizing a specific biological target have been extensively studied in the last decades. Indeed, oligonucleotide-based aptamers are suitable building blocks for the development of highly efficient multivalent systems since they can be easily modified and assembled exploiting proper connecting linkers of different nature. Thus, substantial research efforts have been put in the construction of dimeric/multimeric versions of effective aptamers with various degrees of success in target binding affinity or therapeutic activity enhancement. The present review summarizes recent advances in the design and development of dimeric and multimeric DNA-based aptamers, including those forming G-quadruplex (G4) structures, recognizing different key proteins in relevant pathological processes. Most of the designed constructs have shown improved performance in terms of binding affinity or therapeutic activity as anti-inflammatory, antiviral, anticoagulant, and anticancer agents and their number is certainly bound to grow in the next future.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila . These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.

Chlamydophila Pneumoniae: , a common cause of respiratory tract infections, rarely leads to serious conditions. A 13-year-old boy with serologically confirmed C. pneumoniae infection presented with pneumonia complicated by pericardial and bilateral pleural effusions. He had a large haemorrhagic pericardial effusion from which 1000 ml of fluid was aspirated over 10 days and a right haemorrhagic pleural effusion which required a chest drain and the removal of 700 ml over 5 days. The addition of clarithromycin to ceftriaxone appeared to enhance recovery. As far as we are aware, this is the first report in the English literature of massive haemorrhagic pericardial and pleural effusions in children owing to C. pneumoniae infection.

While studying the human public IgM igome as represented by a library of 224,087 linear mimotopes, three exact matches to peptides in the proteins of SARS-CoV-2 were found: two in the open reading frame 1ab and one in the spike protein. Joining the efforts to fast track SARS-CoV-2 vaccine development, here we describe briefly these potential epitopes in comparison to mimotopes representing peptides of SARS-CoV, HCoV 229E and OC43., (Copyright © 2020 Shivarov, Petrov and Pashov.)

Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cμ 1 of the IgMH of catfish (IgMHCμ 1 mAb) was developed in a rabbit model using sequence information from bighead catfish ( Clarias macrocephalus ). The IgMHCμ 1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish ( Clarias gariepinus ) and their hybrid ( C. macrocephalus × C. gariepinus ), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ 1 mAb successfully recognized IgM + cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ 1 mAb was further used to quantify the percentage of IgM + cells among PBLs through flow cytophotometry. The IgM + cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0-39.9%, 45.6-53.2%, and 58.7-60.0%, respectively. Furthermore, the IgMHCμ 1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish., Competing Interests: The authors declare no conflicts of interest.

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012&#95;GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function., Competing Interests: The authors have declared that no competing interests exist.

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo-electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During evolution C-terminal peptide extensions were added to proteins on the gene level. These convey additional functions such as interaction with partner proteins or oligomerisation. IgM antibodies and molecular chaperones are two prominent examples discussed., (© 2019 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur-containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl-tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl- and phenylalanyl-tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl-tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review., (© 2019 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

FcμR is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fcα/μR and pIgR, but unlike these two other IgM receptors which also bind IgA, FcμR exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/FcμR interaction mainly involves the Cμ4 domains with possible contributions from either Cμ3 or Cμ2. To define the binding site more precisely, we generated three recombinant IgM-Fc proteins with specific mutations in the Cμ3 and Cμ4 domains, as well as a construct lacking the Cμ2 domains, and analyzed their interaction with the extracellular Ig-like domain of FcμR using surface plasmon resonance analysis. There is a binding site for FcμR in each IgM heavy chain. Neither the absence of the Cμ2 domains nor the quadruple mutant D340S/Q341G/D342S/T343S (in Cμ3 adjacent to Cμ2) affected FcμR binding, whereas double mutant K361D/D416R (in Cμ3 at the Cμ4 interface) substantially decreased binding, and a single mutation Q510R (in Cμ4) completely abolished FcμR binding. We conclude that glutamine at position 510 in Cμ4 is critical for IgM binding to FcμR. This will facilitate discrimination between the distinct effects of FcμR interactions with soluble IgM and with the IgM BCR., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Immunoglobulin M (IgM) antibodies are considered as promising biopharmaceutical drugs in the future despite recombinant production is quite challenging as incomplete polymer formation or nucleic acid adherence can decrease the quality of the IgM preparation. Therefore, we defined densitometric and chromatographic methods as suitable tools to analyze the polymer distribution and the remaining nucleic acid content after initial IgM purification. Additionally, the quality of the glycosylation pattern is an important parameter for biological safety and efficacy.

Nanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 μm, and linked to diseases including Alzheimer's and Parkinson's. We used NTA to measure the concentration and hydrodynamic size of gold nanorods (10 nm diameter, 35-250 nm length) and myosin (2 nm diameter, 160 nm length), as models of rod-like particles. Measured hydrodynamic diameters of gold nanorods were consistent with theoretical calculations, as long as particle concentration and solution conditions were controlled. Myosin monomers were invisible by NTA, but a small population of aggregates was detected. We combined NTA results with other light scattering data to gain insight into number and size distribution of protein solutions containing both monomer and aggregates. Finally, we demonstrated the utility of NTA and its limitations by characterizing aggregates of alpha-synuclein. Of note is the use of NTA to detect a change in morphology from compact to elongated by analyzing the ratio of hydrodynamic size to intensity., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin M (IgM) type antibodies play a significant role in complement activation, cellular debris clearance and cell quality control, and have the potential to be used as a therapeutic or targeting/delivery antibody. However, this potential has not been explored thoroughly due to its high molecular weight, polymeric structure and large number of glycosylation sites. Site-specific antibody-drug-conjugates (ADC) are considered the next generation protein biotherapeutic drugs and currently all, in clinical trials and approved, are of the IgG isotype. As existing methods for the development and characterization of IgG-ADCs are not compatible with IgM-ADC, we describe a platform methodology suitable for site specific IgM-ADC using a chemoenzymatic method targeting the glycans on the IgM. Azide functionalized sialic acids were incorporated onto IgM glycans using sialyltransferase for biocompatible conjugation using "click" chemistry. The number of azide groups incorporated onto the IgM glycans were characterized by mass spectrometry of the enzymatically released glycans and glycopeptides. Quantitation of the azide incorporation showed an azide antibody ratio of 8 (glycan data) and 6-10 (glycopeptide data) which translates to a high drug antibody ratio based on IgG-ADC standards. This platform methodology can be readily adapted for any human IgM produced in a mammalian cell expression system., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Waldenstrom macroglobulinemia (WM) is a rare type of non-Hodgkin lymphoma. The diagnosis of WM is established by the presence of lymphoplasmacytic lymphoma in the bone marrow or other organs, a monoclonal IgM paraproteinemia and the recurrent MYD88 L265P somatic mutation. Some patients with WM can be asymptomatic, in which case treatment is not indicated. However, most patients with WM will become symptomatic during the course of the disease, due to anemia, hyperviscosity, neuropathy, or other processes, necessitating therapy. Current treatment options for symptomatic WM patients include alkylating agents, proteasome inhibitors and anti-CD20 monoclonal antibodies. The approval of the oral Bruton tyrosine kinase (BTK) inhibitor ibrutinib alone and in combination with rituximab has expanded the treatment options for WM patients. The present Perspective would focus on exciting treatment strategies under development for WM patients, such as proteasome inhibitors (e.g., ixazomib), BTK inhibitors (e.g., acalabrutinib, zanubrutinib, vecabrutinib), BCL2 inhibitors (e.g., venetoclax), and anti-CXCR4 antibodies (e.g., ulocuplumab), among others. It is certainly an exciting time for WM therapy development with novel and promising treatment options in the horizon.

Immunoassays are widely used for detection and quantification of analytes in biological samples, but are vulnerable to analytical errors caused by interfering sample substances. Of particular interest are endogenous anti-animal antibodies that may bind to the immunoassay antibodies and cause erroneous test results. This phenomenon is a hazard to patient safety in both human and veterinary medicine. Here, we demonstrate that anti-mouse antibodies in dogs bind selectively to different regions of the murine IgG molecule, cross-react with IgG from different species, and consist of all major antibody classes present in canine serum (IgA, IgG and IgM). The antibody characteristics varied among individuals and their prevalence differed between two dog breeds. The selective binding to different IgG regions suggests that the antibodies might not originate from immunization through exposure to mice or other species. These findings show that canine anti-mouse antibodies are highly heterogeneous in nature and therefore require a combination of strategies to be counteracted.

Background & Objectives: Kyasanur forest disease (KFD) is an infectious disease discovered in Karnataka State of India in 1957; since then, the State has been known to be enzootic for KFD. In the last few years, its presence was observed in the adjoining five States of the Western Ghats of India. The present study was conducted to understand the kinetics of viral RNA, immunoglobulin M (IgM) and IgG antibody in KFD-infected humans for developing a diagnostic algorithm for KFD., Methods: A prospective follow up study was performed among KFD patients in Sindhudurg district of Maharashtra State, India. A total of 1046 suspected patients were tested, and 72 KFD patients were enrolled and followed for 17 months (January 2016 to May 2017). Serum samples of KFD patients were screened for viral RNA, and IgM and IgG antibodies., Results: KFD viral positivity was observed from 1 st to 18 th post-onset day (POD). Positivity of anti-KFD virus (KFDV) IgM antibodies was detected from 4 th till 122 nd POD and anti-KFDV IgG antibodies detected from 5 th till 474 th POD. A prediction probability was determined from statistical analysis using the generalized additive model in R-software to support the laboratory findings regarding viral kinetics., Interpretation & Conclusions: This study demonstrated the presence of KFD viral RNA till 18 th POD, IgM antibodies till 122 nd POD and IgG till the last sample collected. Based on our study an algorithm was recommended for accurate laboratory diagnosis of KFDV infection. A sample collected between 1 and 3 POD can be tested using KFDV real-time reverse transcriptase polymerase chain reaction (RT-PCR); between 4 and 24 POD, the combination of real-time RT-PCR and anti-KFDV IgM enzyme-linked immunosorbent assay (ELISA) tests can be used; between POD 25 and 132, anti-KFDV IgM and IgG ELISA are recommended., Competing Interests: None

Antenatal milk anti-influenza antibodies may provide additional protection to newborns until they are able to produce their own antibodies. To evaluate the relative abundance of milk, we studied the antibodies specific to influenza A in feeds and gastric contents and stools from preterm infants fed mother's own breast milk (MBM) and donor breast milk (DBM). Feed (MBM or DBM) and gastric contents (MBM or DBM at 1 h post-ingestion) and stool samples (MBM/DBM at 24 h post-ingestion) were collected, respectively, from 20 preterm (26-36 weeks gestational age) mother-infant pairs at 8-9 days and 21-22 days of postnatal age. Samples were analyzed via ELISA for anti-H1N1 hemagglutinin (anti-H1N1 HA) and anti-H3N2 neuraminidase (anti-H3N2 NA) specificity across immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) isotypes. The relative abundance of influenza A-specific IgA in feeds and gastric contents were higher in MBM than DBM at 8-9 days of postnatal age but did not differ at 21-22 days. Anti-influenza A-specific IgM was higher in MBM than in DBM at both postnatal times in feed and gastric samples. At both postnatal times, anti-influenza A-specific IgG was higher in MBM than DBM but did not differ in gastric contents. Gastric digestion reduced anti-H3N2 NA IgG from MBM at 21-22 days and from DBM at 8-9 days of lactation, whereas other anti-influenza A antibodies were not digested at either postnatal times. Supplementation of anti-influenza A-specific antibodies in DBM may help reduce the risk of influenza virus infection. However, the effective antibody dose required to induce a significant protective effect remains unknown.

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.)

This study demonstrates the preparation of photosensitively orientated and crosslinked proteinous polymeric shell having quantum dot based nanocrystals through Amino acid Decorated and Light Underpinning Conjugation Approach (ANADOLUCA). ANADOLUCA is based on photo-electron transfer method and uses these decorated nanocrystals for specifically and effectively recognition and detection of Immunoglobulin M in the aqueous environment. The conjugation method effectively provides an orientation of affinity pairs on the surface of quantum dots nanocrystals. This photosensitive ruthenium-based amino acid monomer is a synthetic and inexpensive material for the preparation of bioconjugates. The nanocrystals give advantages for using a wide pH and temperature range. The construction and preparation method is applicable to silica materials, superparamagnetic particles, quantum dots, carbon nanotubes, Ag/Au nanoparticles, Au surfaces, and polymeric materials. This prepared proteinous polymeric shell decorated nanocrystals are of great potential in applications in life sciences and can be used in infection case studies or allergy symptoms.

Biological hydrogels (biogels) are selective barriers that restrict passage of harmful substances yet allow the rapid movement of nutrients and select cells. Current methods to modulate the barrier properties of biogels typically involve bulk changes in order to restrict diffusion by either steric hindrance or direct high-affinity interactions with microstructural constituents. Here, we introduce a third mechanism, based on antibody-based third party anchors that bind specific foreign species but form only weak and transient bonds with biogel constituents. The weak affinity to biogel constituents allows antibody anchors to quickly accumulate on the surface of specific foreign species and facilitates immobilization via multiple crosslinks with the biogel matrix. Using the basement membrane Matrigel® and a mixture of laminin/entactin, we demonstrate that antigen-specific, but not control, IgG and IgM efficiently immobilize a variety of individual nanoparticles. The addition of Salmonella typhimurium-binding IgG to biogel markedly reduced the invasion of these highly motile bacteria. These results underscore a generalized strategy through which the barrier properties of biogels can be readily tuned with molecular specificity against a diverse array of particulates. STATEMENT OF SIGNIFICANCE: Biological hydrogels (biogels) are essential in living systems to control the movement of cells and unwanted substances. However, current methods to control transport within biogels rely on altering the microstructure of the biogel matrix at a gross level, either by reducing the pore size to restrict passage through steric hindrance or by chemically modifying the matrix itself. Both methods are either nonspecific or not scalable. Here, we offer a new approach, based on weakly adhesive third-party molecular anchors, that allow for a variety of foreign entities to be trapped within a biogel simultaneously with exceptional potency and molecular specificity, without perturbing the bulk properties of the biogel. This strategy greatly increases our ability to control the properties of biogels at the nanoscale, including those used for wound healing or tissue engineering applications., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Alloimmunization against the high-frequency Vel blood group antigen may result in transfusion reactions or hemolytic disease of fetus and newborn. Patients with anti-Vel alloantibodies require Vel-negative blood but Vel-negative individuals are rare (1:4000). Identification of Vel-negative donors ensures availability of Vel-negative blood; however, accurate Vel blood group typing is difficult due to variable Vel antigen expression and limited availability of anti-Vel typing sera. We report the production of a recombinant anti-Vel that also identifies weak Vel expression., Study Design and Methods: A recombinant anti-Vel monoclonal antibody was produced by cloning the variable regions from an anti-Vel-specific B cell isolated from an alloimmunized patient into a vector harboring the constant regions of immunoglobulin (Ig)G1-kappa or IgM-kappa. Antibody Vel specificity was tested by reactivity to SMIM1-transfected HEK293T cells and by testing various red blood cells (RBCs) of donors with normal, weak, or no Vel expression. High-throughput donor screening applicability was tested using an automated blood group analyzer., Results: A Vel-specific IgM class antibody was produced. The antibody was able to distinguish between Vel-negative and very weak Vel antigen-expressing RBCs by direct agglutination and in high-throughput settings using a fully automated blood group analyzer and performed better than currently used human anti-Vel sera. High-throughput screening of 13,288 blood donations identified three new Vel-negative donors., Conclusion: We generated a directly agglutinating recombinant anti-Vel IgM, M3F5S-IgM, functional in manual, automated agglutination assays and flow cytometry settings. This IgM anti-Vel will improve diagnostics by facilitating the identification of Vel-negative blood donors., (© 2019 AABB.)

Changes in serum glycans discriminate between disease statuses in cancer. A similar connection has not been established in the context of infectious diseases such as tuberculosis (TB). The inflammation arising from infection by Mycobacterium tuberculosis may affect host protein glycosylation, thereby providing information about disease status in TB. A mouse model of infection was used to study glycoprotein N-glycosylation in serum. Following digestion of serum glycoproteins with peptide- N -glycosidase F (PNGase F), released glycans were permethylated and analyzed by multidimensional mass spectrometry (MS). Conditions included naive or Mycobacterium bovis BCG-vaccinated animals, which were either uninfected or infected with M. tuberculosis MS results were validated by lectin blotting. We found that both glycoprotein fucosylation and sialylation were particularly sensitive to M. tuberculosis infection. We observed that M. tuberculosis infection elevates serum IgM levels and induces changes in glycosylation that could inform about the disease. IMPORTANCE We demonstrate that M. tuberculosis infection influenced host protein glycosylation in a mouse model. The mechanism by which infection modifies glycans in serum proteins is not understood. Investigation of the regulation of such modifications by M. tuberculosis opens a new field that could lead to the discovery of novel biomarkers. Validation of such findings in human samples will reveal the clinical relevance of these findings., (Copyright © 2019 Kumagai et al.)

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (Lys A200 Asp-Lys A201 Asp, Arg B108 Asp-Arg B109 Glu, Tyr B175 Leu, and Lys C170 Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The Lys A200 Asp-Lys A201 Asp and Lys C170 Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the Arg B108 Asp-Arg B109 Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The Tyr B175 Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Three different immunoglobulin (Ig) isotypes, namely IgM, IgD, and IgT/IgZ have been described in most teleost, among which IgM and IgT are considered crucial in systematic and mucosal immunity, respectively. However, some teleost have no IgT/IgZ and it is unclear how other Ig isotypes interact to perform immune-protective roles in both systematic and mucosal sites. In this study, the complete cDNA sequences of IgM and IgD heavy chains were cloned and analyzed from yellow catfish (Pelteobagrus fulvidraco). The full-length cDNA of Pf-IgM and Pf-IgD heavy chains contained an open reading frame (ORF) of 1710 and 2991 bp encoding a predicted protein of 570 and 997 amino acids, respectively. Tissue-specific expression analysis indicated that both IgM and IgD were highly expressed in kidney and spleen, and higher expression levels were found at zygote and 13th day post hatching during early development. Multiple sequence alignment and phylogenetic analysis showed IgM and IgD of yellow catfish are closely related to other fish of Siluriformes. Moreover, we also constructed the infection model of yellow catfish with bacteria (Flavobacterium columnare G 4 ) for the first time to study the function of Pf-IgM and Pf-IgD heavy chain genes in immune response. Quantitative real-time PCR (qRT-PCR) showed that significantly up-regulated expression of Pf-IgM was not only detected in liver and spleen, but also in mucosal tissues including skin and intestine, while Pf-IgD was just significantly increased in liver and spleen, which might suggest the main immune-protecting roles of IgM in mucosal tissues of yellow catfish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antigen binding to B-cell antigen receptors (BCRs) followed by signaling initiates the humoral immune response. The signaling is intimately coupled to nanoclustering of BCRs and their sorting to specific membrane domains, a process that is ruled by interactions between the BCR transmembrane domain and lipids. While the structure of the extracellular domains of BCRs has been resolved, little is known about the configuration of the constituting four immunoglobulin domains spanning the membrane. Here, we modeled the structure of the transmembrane (TM) domain of the IgM B-cell receptor using self-assembly coarse-grained molecular dynamics simulations. The obtained quaternary structure was validated against available experimental data and atomistic simulations. The IgM-BCR-TM domain configuration shows a 1:1 stoichiometry between the homodimeric membrane-bound domain of IgM (mIgM) and a Ig-α/Ig-β heterodimer. The mIgM homodimer is based on an asymmetric association of two mIgM domains. We show that a specific site of the Ig-α/Ig-β heterodimer is responsible for the association of IgM-BCRs with lipid rafts. Our results further suggest that this site is blocked in small-sized IgM-BCR clusters. The BCR TM structure provides a molecular basis for the previously suggested dissociation activation model of B-cell receptors. Self-assembly molecular dynamics simulations at the coarse-grained scale here proved as a versatile tool in the study of receptor complexes.

Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied. A striking decrease in the level of occupancy at the Asn-565 glycosite was observed in dimeric IgM compared to that in monomeric IgM, and no glycan occupancy of Asn-565 was observed in tetrameric IgM. Glycopeptide analysis with liquid chromatography-electrospray ionization tandem mass spectrometry revealed mainly complex-type glycans with substantial heterogeneity, with neutral; monosialyl-, disialyl- and trisialylated; and fucosyl-and non-fucosyl-oligosaccharides conjugated to grass carp serum IgM. Glycan variation at a single site was greatest at the Asn-262 glycosite. Unlike IgMs in other species, only traces of complex-type and no high-mannose glycans were found at the Asn-565 glycosite. Matrix-assisted laser desorption ionization analysis of released glycans confirmed the overwhelming majority of carbohydrates were of the complex-type. These results indicate that grass carp serum IgM exhibits unique N-glycan features and highly processed oligosaccharides attached to individual glycosites.