Recapitulating the immune system of hemophilia A patients with inhibitors using immunodeficient mice.

BACKGROUND AND AIM: Treating hemophilia A patients who develop inhibitors remains a clinical challenge. A mouse model of hemophilia A can be used to test the efficacy of strategies for inhibitor suppression, but the differences in the immune systems of mice and humans limit its utility. To address this shortcoming, we established a humanized NOD/SCID-IL2rγnull hemophilia A (hu-NSG-HA) mouse model with a severely deficient mouse immune system presenting a patient's adapted immune cells. METHODS AND RESULTS: Through intrasplenic injection with patient inhibitor-positive peripheral blood mononuclear cells (PBMCs), utilizing an adeno-associated viral delivery system expressing human BLyS, and regular FVIII challenge, human C19+ B cells were expanded in vivo to secrete anti-FVIII antibodies. Both the inhibitor and the human anti-FVIII IgG, including the predominant subclasses (IgG1 and IgG4) present in the majority of inhibitor patients, were detected in the mouse model. We further segregated and expanded the different clones of human anti-FVIII-secreting cells through subsequent transplantation of splenocytes derived from hu-NSG-HA mice into another NSG-HA mouse. By transplanting a patient's PBMCs into the NSG-HA mouse model, we demonstrated the success of reintroducing a strong anti-FVIII immune response for a short period in mice with the immune systems of inhibitor-positive patients. CONCLUSION: Our results demonstrate a potential tool for directly obtaining functional human-derived antigen-specific antibodies and antibody-secreting cells, which may have therapeutic value for testing patient-specific immune responses to treatment options to assist in clinical decisions.

Mechanisms and functions of SUMOylation in health and disease: a review focusing on immune cells.

SUMOylation, which is a type of post-translational modification that involves covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to target substrates, regulates various important molecular and cellular processes, including transcription, the cell cycle, cell signaling, and DNA synthesis and repair. Newly synthesized SUMO is immature and cleaved by the SUMO-specific protease family, resulting in exposure of the C-terminal Gly-Gly motif to become the mature form. In the presence of ATP, mature SUMO is conjugated with the activating enzyme E1 through the cysteine residue of E1, followed by transfer to the cysteine residue of E2-conjugating enzyme Ubc9 in humans that recognizes and modifies the lysine residue of a substrate protein. E3 SUMO ligases promote SUMOylation. SUMOylation is a reversible modification and mediated by SUMO-specific proteases. Cumulative studies have indicated that SUMOylation affects the functions of protein substrates in various manners, including cellular localization and protein stability. Gene knockout studies in mice have revealed that several SUMO cycling machinery proteins are crucial for the development and differentiation of various cell lineages, including immune cells. Aberrant SUMOylation has been implicated in several types of diseases, including cancers, cardiovascular diseases, and autoimmune diseases. This review summarizes the biochemistry of SUMO modification and the general biological functions of proteins involved in SUMOylation. In particular, this review focuses on the molecular mechanisms by which SUMOylation regulates the development, maturation, and functions of immune cells, including T, B, dendritic, and myeloid cells. This review also discusses the underlying relevance of disruption of SUMO cycling and site-specific interruption of SUMOylation on target proteins in immune cells in diseases, including cancers and infectious diseases.

Single Site N-Glycosylation of B Cell Maturation Antigen (BCMA) Inhibits γ-Secretase-Mediated Shedding and Improves Surface Retention and Cell Survival.

B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor (TNFR) family, on the cell surface plays a key role in maintaining the survival of plasma cells and malignant as well as inflammatory accessory cells. Therefore, targeting BCMA or disrupting its interaction with ligands has been a potential approach to cancer therapy. BCMA contains a single N-glycosylation site, but the function of N-glycan on BCMA is not understood. Here, we found that the N-glycosylation of BCMA promoted its cell-surface retention while removing the N-glycan increased BCMA secretion through γ-secretase-mediated shedding. Addition of γ-secretase inhibitor prevented nonglycosylated BCMA from shedding and protected cells from dexamethasone and TRAIL-induced apoptosis.

SENP2 restrains the generation of pathogenic Th17 cells in mouse models of colitis.

The molecular mechanisms contributing to the regulation of Th17-mediated inflammation remain underexplored. We here report a SUMO-specific protease (SENP)2-mediated pathway induced in pathogenic Th17 cells that restricts the pathogenesis of inflammatory colitis. SENP2 regulates the maturation of small ubiquitin-like modifiers (SUMO) and recycles SUMO from the substrate proteins. We find higher levels of SENP2 in pathogenic Th17 cells. By deleting Senp2 in T-cell lineages in mice, we demonstrate that the lack of Senp2 exacerbates the severity of experimental colitis, which is linked to elevated levels of GM-CSF+IL-17A+ pathogenic Th17 cells and more severe dysbiosis of the intestinal microbiome. Adoptive transfer experiments demonstrate the cell-autonomous effect of Senp2 in restraining Th17 differentiation and colitis. The enzymatic activity of SENP2 is important for deSUMOylation of Smad4, which reduces Smad4 nuclear entry and Rorc expression. Our findings reveal a SENP2-mediated regulatory axis in the pathogenicity of Th17 cells.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Molecular Basis and Role of Siglec-7 Ligand Expression on Chronic Lymphocytic Leukemia B Cells.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is an immune checkpoint-like glycan recognition protein on natural killer (NK) cells. Cancer cells often upregulate Siglec ligands to subvert immunosurveillance, but the molecular basis of Siglec ligands has been elusive. In this study, we investigated Siglec-7 ligands on chronic lymphocytic leukemia (CLL) B cells. CLL B cells express higher levels of Siglec-7 ligands compared with healthy donor B cells, and enzymatic removal of sialic acids or sialomucins makes them more sensitive to NK cell cytotoxicity. Gene knockout experiments have revealed that the sialyltransferase ST6GalNAc-IV is responsible for the biosynthesis of disialyl-T (Neu5Acα2-3Galß1-3[Neu5Acα2-6]GalNAcα1-), which is the glycotope recognized by Siglec-7, and that CD162 and CD45 are the major carriers of this glycotope on CLL B cells. Analysis of public transcriptomic datasets indicated that the low expression of GCNT1 (encoding core 2 GlcNAc transferase, an enzyme that competes against ST6GalNAc-IV) and high expression of ST6GALNAC4 (encoding ST6GalNAc-IV) in CLL B cells, together enhancing the expression of the disialyl-T glycotope, are associated with poor patient prognosis. Taken together, our results determined the molecular basis of Siglec-7 ligand overexpression that protects CLL B cells from NK cell cytotoxicity and identified disialyl-T as a potential prognostic marker of CLL.

Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2-M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. IMPLICATIONS: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry.

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.

NK cell receptor and ligand composition influences the clearance of SARS-CoV-2.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Marginal Zone B Cells Assist With Neutrophil Accumulation to Fight Against Systemic Staphylococcus aureus Infection.

In addition to regulating immune responses by producing antibodies that confer humoral immunity, B cells can also affect these responses by producing cytokines. How B cells participate in the clearance of pathogenic infections via functions other than the production of pathogen-specific antibodies is still largely unknown. Marginal zone (MZ) B cells can quickly respond to bacterial invasion by providing the initial round of antibodies. After a bloodborne bacterial infection, neutrophils promptly migrate to the MZ. However, the mechanisms regulating neutrophil accumulation in the MZ during the initial phase of infection also remain obscure. Here, we found that MZ B cell-deficient mice are more susceptible to systemic Staphylococcus aureus (S. aureus) infection compared with wildtype mice. The expression levels of interleukin (IL)-6 and CXCL1/CXCL2 in MZ B cells increased significantly in mice at 3-4 h after infection with S. aureus, then decreased at 24 h post-infection. After systemic S. aureus infection, splenic neutrophils express increased CXCR2 levels. Our results from confocal microscopy imaging of thick-section staining demonstrate that neutrophils in wildtype mice form cell clusters and are in close contact with MZ B cells at 3 h post-infection. This neutrophil cluster formation shortly after infection was diminished in both MZ B cell-deficient mice and IL-6-deficient mice. Blocking the action of CXCL1/CXCL2 by injecting anti-CXCL1 and anti-CXCL2 antibodies 1 h before S. aureus infection significantly suppressed the recruitment of neutrophils to the MZ at 3 h post-infection. Compared with peptidoglycan stimulation alone, peptidoglycan stimulation with neutrophil co-culture further enhanced MZ B-cell activation and differentiation. Using a Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET) analysis, we observed evidence of a direct interaction between neutrophils and MZ B cells after peptidoglycan stimulation. Furthermore, neutrophil depletion in mice resulted in a reduced production of S. aureus-specific immunoglobulin (Ig)M at 24 h post-infection. Together, our results demonstrate that MZ B cells regulate the rapid neutrophil swarming into the spleen during the early phase of systemic S. aureus infection. Interaction with neutrophils assists MZ B cells with their differentiation into IgM-secreting cells and contributes to the clearance of systemic bacterial infections.

Resident vs nonresident multipotent mesenchymal stromal cell interactions with B lymphocytes result in disparate outcomes.

Multipotent human mesenchymal stromal cells (MSCs) from multiple organs including the bone marrow (BM) and placenta harbor clinically relevant immunomodulation best demonstrated toward T lymphocytes. Surprisingly, there is limited knowledge on interactions with B lymphocytes, which originate from the BM where there is a resident MSC. With increasing data demonstrating MSC tissue-specific propensities impacting therapeutic outcome, we therefore investigated the interactions of BM-MSCs-its resident and "niche" MSC-and placental MSCs (P-MSCs), another source of MSCs with well-characterized immunomodulatory properties, on the global functional outcomes of pan-peripheral B cell populations. We found that P-MSCs but not BM-MSCs significantly inhibit proliferation and further differentiation of stimulated human peripheral B populations in vitro. Moreover, although BM-MSCs preserve multiple IL-10-producing regulatory B cell (Breg) subsets, P-MSCs significantly increase all subsets. To corroborate these in vitro findings in vivo, we used a mouse model of B-cell activation and found that adoptive transfer of P-MSCs but not BM-MSCs significantly decreased activated B220+ B cells. Moreover, adoptive transfer of P-MSCs but not BM-MSCs significantly decreased the overall B220+ B-cell proliferation and further differentiation, similar to the in vitro findings. P-MSCs also increased two populations of IL-10-producing murine Bregs more strongly than BM-MSCs. Transcriptome analyses demonstrated multifactorial differences between BM- and P-MSCs in the profile of relevant factors involved in B lymphocyte proliferation and differentiation. Our results highlight the divergent outcomes of tissue-specific MSCs interactions with peripheral B cells, and demonstrate the importance of understanding tissue-specific differences to achieve more efficacious outcome with MSC therapy.

O-GlcNAcylation and its role in the immune system.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O-GlcNAc transferase (OGT) and which can be reversibly removed by O-GlcNAcase (OGA). O-GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O-GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O-GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O-GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O-GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O-GlcNAcylation, and discusses the molecular mechanisms by which O-GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.

Aberrant distribution and function of plasmacytoid dendritic cells in patients with ankylosing spondylitis are associated with unfolded protein response.

Although human leucocyte antigen (HLA)-B27 is strongly associated with ankylosing spondylitis (AS), the association of unfolded protein response (UPR) induced by HLA-B27 misfolding in AS remains controversial. Since dendritic cells (DCs) are crucial in induction of AS in HLA-B27-transgenic rats, and plasmacytoid DCs (pDCs) belong to one type of DCs, we here aim to study the relevance of pDCs and UPR in AS. Peripheral pDCs were isolated from 27 HLA-B27(+) AS patients and 37 controls. The bone marrow (BM) and synovium of inflamed hips from AS patients and controls were obtained. We found a significantly higher frequency of pDCs in the peripheral blood, BM, or inflamed synovium of hips, which is associated with the enhanced expression of pDC trafficking molecules, CCR6 and CCL20 in the synovium of AS patients. Functional analysis further revealed that several inflammatory cytokines, including TNFα, IL-6, and IL-23, secreted by pDCs were significantly increased in AS patients as compared with those in controls. Remarkably, protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in UPR was up-regulated in pDCs of AS patients. Notably, PERK inhibitor treatment significantly inhibited the enhanced cytokine production by pDCs of AS patients. Further, the extent of PERK activation was significantly associated with the increased disease severity of AS patients. Our data uncover the aberrant distribution and function of pDCs in AS patients. The up-regulated PERK pathway in UPR of pDCs not only contributes to enhanced cytokine production of pDCs, but also is associated with increased disease activity of AS patients.

HLA-B27-mediated activation of TNAP phosphatase promotes pathogenic syndesmophyte formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of axial inflammation. Over time, some patients develop spinal ankylosis and permanent disability; however, current treatment strategies cannot arrest syndesmophyte formation completely. Here, we used mesenchymal stem cells (MSCs) from AS patients (AS MSCs) within the enthesis involved in spinal ankylosis to delineate that the HLA-B27-mediated spliced X-box-binding protein 1 (sXBP1)/retinoic acid receptor-ß (RARB)/tissue-nonspecific alkaline phosphatase (TNAP) axis accelerated the mineralization of AS MSCs, which was independent of Runt-related transcription factor 2 (Runx2). An animal model mimicking AS pathological bony appositions was established by implantation of AS MSCs into the lumbar spine of NOD-SCID mice. We found that TNAP inhibitors, including levamisole and pamidronate, inhibited AS MSC mineralization in vitro and blocked bony appositions in vivo. Furthermore, we demonstrated that the serum bone-specific TNAP (BAP) level was a potential prognostic biomarker to predict AS patients with a high risk for radiographic progression. Our study highlights the importance of the HLA-B27-mediated activation of the sXBP1/RARB/TNAP axis in AS syndesmophyte pathogenesis and provides a new strategy for the diagnosis and prevention of radiographic progression of AS.

Blimp-1 Contributes to the Development and Function of Regulatory B Cells.

Regulatory B cells (Bregs) are a B cell subset that plays a suppressive role in immune responses. The CD19+CD1dhiCD5+ Bregs that can execute regulatory functions via secreting IL-10 are defined as B10 cells. Bregs suppress autoimmune and inflammatory diseases, whereas they exacerbate infectious diseases caused by bacteria, viruses, or parasites. Notably, the molecular mechanisms regulating the development and functions of Bregs are still largely unknown. Furthermore, the biological impact of Bregs in fungal infection has not yet been demonstrated. Here, we compared the gene expression profiles of IL-10-producing and -non-producing mouse splenic B cells stimulated with lipopolysaccharide (LPS) or anti-CD40 antibody. Blimp-1, a transcription factor known to be critical for plasma cell differentiation, was found to be enriched in the IL-10-producing B cells. The frequency of Blimp-1+ B10 cells was increased in LPS-treated mice and in isolated B10 cells that were stimulated with LPS. Surprisingly, B cell-specific Blimp-1 knockout (Cko) mice, generated by CD19 promoter driven Cre recombinase-dependent deletion of Prdm1 (gene encoding Blimp-1), showed higher frequencies of B10 cells both in the steady state and following injection with LPS, as compared with control littermates. However, B10 cells lacking Blimp-1 failed to efficiently suppress the proliferation of naïve CD4+ T cells primed with anti-CD3 and anti-CD28 antibodies. B10 cells can be stimulated for further differentiation into plasmablasts, and a subset of plasmablasts express IL-10. We found that B10 cells from Cko mice failed to generate both IL-10-non-producing and IL-10-producing plasmablasts. Mechanistically, we found that Blimp-1 can directly suppress Il-10, whereas, in the presence of activated STAT3, Blimp-1 works together with activated STAT3 to upregulate Il-10. Moreover, we also found that B10 cells improve the clearance of Candida albicans infection but worsen the infection mortality. Notably, a lack of Blimp-1 in B10 cells did not change these effects of adoptively transferred B10 cells on fungal infections. Together, our data show that Blimp-1 regulates the generation, differentiation, and IL-10 production of Bregs.

Regulatory mechanisms of B cell responses and the implication in B cell-related diseases.

Terminally differentiated B cell, the plasma cell, is the sole cell type capable of producing antibodies in our body. Over the past 30 years, the identification of many key molecules controlling B cell activation and differentiation has elucidated the molecular pathways for generating antibody-producing plasma cells. Several types of regulation modulating the functions of the important key molecules in B cell activation and differentiation add other layers of complexity in shaping B cell responses following antigen exposure in the absence or presence of T cell help. Further understanding of the mechanisms contributing to the proper activation and differentiation of B cells into antibody-secreting plasma cells may enable us to develop new strategies for managing antibody humoral responses during health and disease. Herein, we reviewed the effect of different types of regulation, including transcriptional regulation, post-transcriptional regulation and epigenetic regulation, on B cell activation, and on mounting memory B cell and antibody responses. We also discussed the link between the dysregulation of the abovementioned regulatory mechanisms and B cell-related disorders.

The KDM4A/KDM4C/NF-κB and WDR5 epigenetic cascade regulates the activation of B cells.

T follicular helper (Tfh) cell-derived signals promote activation and proliferation of antigen-primed B cells. It remains unclear whether epigenetic regulation is involved in the B cell responses to Tfh cell-derived signals. Here, we demonstrate that Tfh cell-mimicking signals induce the expression of histone demethylases KDM4A and KDM4C, and the concomitant global down-regulation of their substrates, H3K9me3/me2, in B cells. Depletion of KDM4A and KDM4C potentiates B cell activation and proliferation in response to Tfh cell-derived signals. ChIP-seq and de novo motif analysis reveals NF-κB p65 as a binding partner of KDM4A and KDM4C. Their co-targeting to Wdr5, a MLL complex member promoting H3K4 methylation, up-regulates cell cycle inhibitors Cdkn2c and Cdkn3. Thus, Tfh cell-derived signals trigger KDM4A/KDM4C - WDR5 - Cdkn2c/Cdkn3 cascade in vitro, an epigenetic mechanism regulating proper proliferation of activated B cells. This pathway is dysregulated in B cells from systemic lupus erythematosus patients and may represent a pathological link.

Examination of the role of galectins in plasma cell differentiation.

Plasma cells are terminally differentiated B cells that develop via the stimulation of mature B cells with various agents such as antigens and mitogens. Recently, we found that plasma cell differentiation can be modulated by galectin-1 and galectin-8; these galectins appear to play additive and redundant roles in promoting the production of antibody. Here, we describe the protocols for how to investigate the roles of galectins in plasma cell differentiation. These methods include the preparation of recombinant galectins from Escherichia coli for exogenously treating primary B cells, generation of galectin_Fc(m) fusion proteins for determining their binding to B cells, introduction of ectopic galectins in primary B cells using retroviral vectors, and inhibition of the binding of galectins to B cells by synthetic disaccharides.

RegPhos 2.0: an updated resource to explore protein kinase-substrate phosphorylation networks in mammals.

Protein phosphorylation catalyzed by kinases plays crucial roles in regulating a variety of intracellular processes. Owing to an increasing number of in vivo phosphorylation sites that have been identified by mass spectrometry (MS)-based proteomics, the RegPhos, available online at http://csb.cse.yzu.edu.tw/RegPhos2/, was developed to explore protein phosphorylation networks in human. In this update, we not only enhance the data content in human but also investigate kinase-substrate phosphorylation networks in mouse and rat. The experimentally validated phosphorylation sites as well as their catalytic kinases were extracted from public resources, and MS/MS phosphopeptides were manually curated from research articles. RegPhos 2.0 aims to provide a more comprehensive view of intracellular signaling networks by integrating the information of metabolic pathways and protein-protein interactions. A case study shows that analyzing the phosphoproteome profile of time-dependent cell activation obtained from Liquid chromatography-mass spectrometry (LC-MS/MS) analysis, the RegPhos deciphered not only the consistent scheme in B cell receptor (BCR) signaling pathway but also novel regulatory molecules that may involve in it. With an attempt to help users efficiently identify the candidate biomarkers in cancers, 30 microarray experiments, including 39 cancerous versus normal cells, were analyzed for detecting cancer-specific expressed genes coding for kinases and their substrates. Furthermore, this update features an improved web interface to facilitate convenient access to the exploration of phosphorylation networks for a group of genes/proteins. Database URL: http://csb.cse.yzu.edu.tw/RegPhos2/

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.