MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.     

The Role of Glycosyltransferases in Colorectal Cancer

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell–cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.     

Antibody Recognition of Cancer Cells via Glycan Surface Engineering

Stimulation of the body's immune system toward tumor cells is now well recognized as a promising strategy in cancer therapy. Just behind cell therapy and monoclonal antibodies, small molecule-based strategies are receiving growing attention as alternatives to direct immune response against tumor cells. However, the development of small-molecule approaches to modulate the balance between stimulatory immune factors and suppressive factors in a targeted way remains a challenge. Here, we report the cell surface functionalization of LS174T cancer cells with an abiotic hapten to recruit antibodies to the cell surface. Metabolic glycoengineering followed by covalent reaction with the hapten results in antibody recognition of the target cells. Microscopy and flow cytometry studies provide compelling evidence that metabolic glycoengineering and small molecule stimulators can be combined to direct antibody recognition.     

人CD4分子及其治疗性抗体的研究进展

在正常情况下,人CD4分子能协助机体免疫系统进行免疫防御、免疫自稳和免疫监视;但在一定条件下,又与某些疾病的发生发展密切相关。目前,已研制出几种抗CD4治疗性抗体,并在一些疾病的临床研究中获得了成效。本文就人CD4分子的结构、功能、与疾病的关系以及抗CD4治疗性抗体的研究进展作一综述。     

Synthesis and Immunomodulatory Activity of Fluorine-Containing Bisphosphonates

Immune checkpoint blockade using anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibodies (mAbs) has revolutionized cancer treatment. However, many types of cancer do not respond and for those that do, only a minority of patients achieve durable remissions. Therefore, oncoimmunologists are working to develop adoptive cell therapies for non-hematopoietic tumors by harnessing immune effector cells such as αβ T cells and γδ T cells. In contrast to conventional αβ T cells that recognize peptides in the context of MHC class I or II molecules, γδ T cells expressing Vγ2Vδ2 T cell receptors (also termed Vγ9Vδ2) are stimulated by isoprenoid metabolites (phosphoantigens) such as isopentenyl diphosphate in a butyrophilin-3A1-dependent manner. Vγ2Vδ2 T cells kill almost all types of tumor cells that have been treated with bisphosphonates. In this study, we synthesized a series of fluorine-containing bisphosphonates based on current drugs and found that they stimulated Vγ2Vδ2 T cell killing of tumor cells. A fluorine-containing prodrug analogue of zoledronate where phosphonate moieties were masked with pivaloyloxymethyl groups markedly enhanced Vγ2Vδ2 T-cell-mediated cytotoxicity, and also promoted the expansion of peripheral blood Vγ2Vδ2 T cells. These results demonstrate that a prodrug of a fluorine-containing zoledronate analogue can sensitize tumor cells for killing as well as expand Vγ2Vδ2 T cells for adoptive cell therapy.     

Minimalist Antibodies and Mimetics: An Update and Recent Applications

The immune system utilizes antibodies to recognize foreign or disease‐relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small‐molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post‐translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics.     

Two and three-dimensional pattern recognition of organized surfaces by specific antibodies

Understanding molecular recognition of supramolecules for solid substrates is essential for designing chemical sensors and molecular devices. The rules of molecular recognition are well established at the level of single molecules. However, during the transition from molecular-scale devices to macroscopic devices, issues concerning control over recognition that are well-established at the molecular level become much more complex. Hopefully, the conceptual and practical considerations reported here will clarify some of these issues. The immune system uses antibodies to identify molecular surfaces through molecular recognition. Antibodies are thus appropriate tools to study the rules of macromolecule-surface interactions, and this was done using crystal surfaces as substrates. Crystals can be formed or introduced into organisms and should be thus treated by the organism as any other intruder, by eliciting antibodies specific to their surfaces. A structure-recognizing antibody is defined here as complementary to a certain ordered supramolecular organization. It can be considered as a mold bearing in its binding site memory of the organization against which it was elicited. On the surface of a crystal composed of relatively small organic molecules, an antibody binding site would encompass an array of 10-20 molecular moieties. The antibody binding site would not detect one molecule, but rather a two- or three-dimensional molecular arrangement on the surface, similar to a macromolecular surface. The complementarity between antibody binding site and surface is supported by stereoselective supramolecular interactions to the repetitive structural motifs that are exposed at the surface. A procedure was developed in order to isolate monoclonal antibodies that specifically recognize a certain crystalline surface. The procedure was applied in particular to crystals of cholesterol monohydrate, of 1,4-dinitrobenzene, and of the tripeptide (S)leucine-(S)leucine-(S)tyrosine (LLY). A series of antibodies were selected and studied, three of which provided reliable specific antibody-antigen structural models. The three docking models show an astounding geometrical and chemical match of the antibody binding sites on the respective crystal surfaces. We also showed that antibodies are intrinsically capable of recognition at the length scale necessary for detection of chirality. Once the structural parameters determining the antibody specificity to the target surfaces are characterized, the antibodies may be conceivably used as reporters of the existence and location of target domains with similar structure in biological milieus. In this context, we developed and characterized monoclonal antibodies specific to crystalline mixed monolayers of cholesterol and ceramide, fundamental building blocks of lipid microdomains in cellular membranes. When used on cells, one antibody indeed labels cell membrane domains composed of cholesterol and ceramide. The fundamental contribution of the approach developed here may be in the antibody ability to report on the structural organization of paracrystalline domains that cannot be determined by other means. Alternatively, structure-recognizing antibodies may be conceivably used to carry information or build connections to specific targets, which may offer interesting developments in medicine or electronics.     

Crystal Structure of a Soluble Form of Human CD73 with Ecto‐5′‐Nucleotidase Activity

CD73 is a dimeric ecto‐5′‐nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto‐5′‐nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer‐dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.     

Biological Evaluation of Multivalent Lewis X–MGL‐1 Interactions

Myeloid C‐type lectin receptors (CLRs) expressed by antigen‐presenting cells are pattern‐recognition receptors involved in the recognition of pathogens as well as of self‐antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin–carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le^X) epitope on its interaction with the CLR macrophage galactose‐type lectin‐1 (MGL‐1). Glycan arrays, including N‐glycan structures with terminal Le^X, were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL‐1‐hFc fusion protein showed up to tenfold increased binding to multiantennary N‐glycans displaying Le^X structures, compared to monovalent Le^X trisaccharide. Multivalent presentation of Le^X on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le^X‐OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate–lectin interactions can be exploited to modulate immune responses.     

Stem Cell and Macrophage Roles in Skeletal Muscle Regenerative Medicine

In severe muscle injury, skeletal muscle tissue structure and functionality can be repaired through the involvement of several cell types, such as muscle stem cells, and innate immune responses. However, the exact mechanisms behind muscle tissue regeneration, homeostasis, and plasticity are still under investigation, and the discovery of pathways and cell types involved in muscle repair can open the way for novel therapeutic approaches, such as cell-based therapies involving stem cells and peripheral blood mononucleate cells. Indeed, peripheral cell infusions are a new therapy for muscle healing, likely because autologous peripheral blood infusion at the site of injury might enhance innate immune responses, especially those driven by macrophages. In this review, we summarize current knowledge on functions of stem cells and macrophages in skeletal muscle repairs and their roles as components of a promising cell-based therapies for muscle repair and regeneration.     

Immunoprofiling: An Encouraging Method for Predictive Factors Examination in Lung Cancer Patients Treated with Immunotherapy

The efficiency of immunotherapy using monoclonal antibodies that inhibit immune checkpoints has been proven in many clinical studies and well documented by numerous registration approaches. To date, PD-L1 expression on tumor and immune cells, tumor mutation burden (TMB), and microsatellite instability (MSI) are the only validated predictive factors used for the qualification of cancer patients for immunotherapy. However, they are not the ideal predictive factors. No response to immunotherapy could be observed in patients with high PD-L1 expression, TMB, or MSI. On the other hand, the effectiveness of this treatment method also may occur in patients without PD-L1 expression or with low TMB and with microsatellite stability. When considering the best predictive factor, we should remember that the effectiveness of immunotherapy relies on an overly complex process depending on many factors. To specifically stimulate lymphocytes, not only should their activity in the tumor microenvironment be unlocked, but above all, they should recognize tumor antigens. The proper functioning of the anticancer immune system requires the proper interaction of many elements of the specific and non-specific responses. For these reasons, a multi-parameter analysis of the immune system at its different activity levels is considered a very future-oriented predictive marker. Such complex immunological analysis is performed using modern molecular biology techniques. Based on the gene expression studies, we can determine the content of individual immune cells within the tumor, its stroma, and beyond. This includes all cell types from active memory cytotoxic T cells, M1 macrophages, to exhausted T cells, regulatory T cells, and M2 macrophages. In this article, we summarize the possibilities of using an immune system analysis to predict immunotherapy efficacy in cancer patients. Moreover, we present the advantages and disadvantages of immunoprofiling as well as a proposed future direction for this new method of immune system analysis in cancer patients who receive immunotherapy.     

Glycosphingolipid and Glycosylphosphatidylinositol Affect Each Other in and on the Cell

Glycosphingolipid (GSL) and glycosylphosphatidylinositol (GPI) are the two major glycolipids expressed by eukaryotic cells, and their metabolisms share the same machineries. Moreover, both GSLs and GPI-anchored proteins (GPI-APs) are localized in the cholesterol-rich regions, namely the lipid rafts, of the cell membrane, where many other signaling molecules are compartmentalized as well. Therefore, the interaction between GSLs and GPI-APs and their interactions with other molecules in the lipid rafts are inevitable. This review is focused on the influences of GSLs and GPI-APs on each other's biosynthesis, trafficking, cell membrane distribution, and biological functions, such as signal transduction.     

Automated Glycan Assembly of Plant Oligosaccharides and Their Application in Cell-Wall Biology

The plant cell wall provides the richest available resource of fermentable carbohydrates and biobased materials. The main component of plant cell walls is cellulose, which is the most abundant biomolecule on earth. Apart from cellulose, which is constructed from relatively simple β-1,4-glucan chains, plant cell walls also contain structurally more complex heteropolysaccharides (hemicellulose and pectin), as well as lignin and cell-wall proteins. A detailed understanding of the molecular structures, functions, and biosyntheses of cell-wall components is required to further promote their industrial use. Plant cell-wall research is, to a large degree, hampered by a lsack of available well-defined oligosaccharide samples that represent the structural features of cell-wall glycans. One technique to access these oligosaccharides is automated glycan assembly; a technique in which monosaccharide building blocks are, similarly to automated peptide and oligonucleotide chemistry, successively added to a linker-functionalized resin in a fully automated manner. Herein, recent research into the automated glycan assembly of different classes of cell-wall glycans used as molecular tools for cell-wall biology is discussed. More than 60 synthetic oligosaccharides were prepared and printed as microarrays for screening monoclonal antibodies that recognize plant cell-wall polysaccharides. The synthesized oligosaccharides have also been used to investigate glycosyltransferases and glycoside hydrolases, which are involved in synthesis and degradation of plant cell walls, as well as for the analysis of cell-wall-remodeling enzymes.     

Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order–disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order–disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.     

Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N‐Glycan Profile of Mouse Embryonic Fibroblast

The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell‐surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N‐glycans of glycoproteins. To test the feasibility of a glycoblotting‐based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase‐deficient mouse embryonic fibroblast GM3(?/?). GM3(?/?) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o‐series gangliosides involving GM1‐b and GD1‐α increased dramatically, whereas a‐/b‐series gangliosides were predominantly detected in wild‐type (WT) cells. We also discovered that glycoprotein N‐glycan profiles of GM3(?/?) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N‐glycan biosynthesis. The present approach allows for high‐throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N‐glycans of glycoproteins.     

Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo‐oligosaccharide from Thermomonas hydrothermalis

Thermomonas hydrothermalis is a Gram‐negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell‐wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram‐negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure‐dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system—an antagonistic effect, as shown in studies of LPS structure–activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo‐oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.     

Synthesis of a Glycopeptide Vaccine Conjugate for Induction of Antibodies Recognizing O‐Mannosyl Glycopeptides

In spite of the clear importance of protein O‐mannosylation in brain glycobiology, tools are lacking for specific detection, enrichment, and identification of proteins containing these modifycations. We envisioned inducing antibodies that specifically recognize O‐mannose glycans on proteins and peptides. With this in mind, we prepared a glycopeptide vaccine construct containing the N‐acetyllactosamine‐extended mannose motif Galβ1‐4GlcNAcβ1‐2ManαThr, found as a common core structure on almost all mammalian O‐mannosyl glycoproteins identified. O‐mannose glycosylated amino acid building blocks and the corresponding glycopeptides were prepared by chemical synthesis and then conjugated to an immune carrier protein. After administration of the synthetic vaccine into rabbits, strong immune responses were obtained. Further evaluation by ELISA neutralization experiments and glycopeptide microarrays showed that the induced antibodies were highly specific to the glycopeptide antigen.     

Fc-Binding Antibody-Recruiting Molecules Targeting Prostate-Specific Membrane Antigen: Defucosylation of Antibody for Efficacy Improvement**

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc-binding antibody-recruiting molecules (Fc-ARMs). Fc-ARMs can theoretically recruit most endogenous antibodies, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc-ARMs that target prostate cancer (Fc-ARM-Ps). Fc-ARM-Ps recruited antibodies to cancer cells expressing prostate-specific membrane antigen but did so with lower efficiency compared with Fc-ARMs targeting the folate receptor. Upon recruitment by Fc-ARM-P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N-glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc-ARM strategy.     

TCR Recognition of Peptide–MHC-I: Rule Makers and Breakers

T cells are a critical part of the adaptive immune system that are able to distinguish between healthy and unhealthy cells. Upon recognition of protein fragments (peptides), activated T cells will contribute to the immune response and help clear infection. The major histocompatibility complex (MHC) molecules, or human leukocyte antigens (HLA) in humans, bind these peptides to present them to T cells that recognise them with their surface T cell receptors (TCR). This recognition event is the first step that leads to T cell activation, and in turn can dictate disease outcomes. The visualisation of TCR interaction with pMHC using structural biology has been crucial in understanding this key event, unravelling the parameters that drive this interaction and their impact on the immune response. The last five years has been the most productive within the field, wherein half of current unique TCR–pMHC-I structures to date were determined within this time. Here, we review the new insights learned from these recent TCR–pMHC-I structures and their impact on T cell activation.