Abundant Glycoprotein Research Articles

34 Alterations in cystic fibrosis peripheral blood mononuclear cells are induced by an increase in intracellular calcium concentration

Objectives: Mucins are abundant glycoproteins in human lungs. It is wellestablished that airway mucin O-glycosylation is aberrant in cystic fibrosis (CF) and involved directly or indirectly in the pathogenesis. As the first, we here investigate and establish the link between protein N-glycosylation and CF. Methods: N-glycosylation of crudely isolated sputum non-mucin proteins of five CF and five non-CF individuals with and without pulmonary infection was mapped using liquid chromatography and tandem mass spectrometry based glycomics and glycoproteomics. The resulting glycoprofiles were qualitatively and quantitatively compared between the patient groups. Results: Despite covering different patient characteristics including CFTR genotypes, age, gender and microbial flora, the sputum N-glycomes showed little interperson and longitudinal variation within the patient groups. Inter-group comparisons revealed that lung infection, primarily caused by P. aeruginosa, extensively altered the CF sputum N-glycosylation to paucimannoside rich profiles with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of the complex N-glycans. The CF genotype in itself yielded fewer sputum N-glycome alterations by slightly increasing the abundance of paucimannose N-glycans in CF relative to pathogen-infected non-CF individuals. Conclusion: We have established that the absence of a functional CFTR and more importantly the bacterial infection of the respiratory tract of CF patients affect their sputum N-glycosylation phenotype. This study provides an important platform to further understand the complex cellular and molecular environment of the respiratory tract in CF. 34 Alterations in cystic fibrosis peripheral blood mononuclear cells are induced by an increase in intracellular calcium concentration M. Averna1, M. Pedrazzi1, F. Cresta2, L. Minicucci2, E. Melloni1. 1University of Genoa, Department of Experimental Medicine − Biochemistry Section, Genova, Italy; 2IRCCS Istituto Giannina Gaslini, Cystic Fibrosis Center, Genova, Italy

Proteominer facilitates high‐throughput proteomic analysis of low abundance proteins in the cartilage secretome (LB39)

Proteomics is increasingly used for biomarker identification. We have developed high‐throughput mass spectrometric (MS) methods to identify proteins in the secretome of articular cartilage explants, which we are utilizing as an in vitro model of joint inflammation. However, the identification of lower abundance proteins of potentially higher biological significance in this model is hampered by the presence of highly abundant glycoproteins in the secretome. Development of protein depletion techniques that allow the identification of low abundance proteins is important for novel biomarker discovery, especially in the context of identifying pathophysiologically relevant biomarkers of joint inflammation and cartilage degradation. Proteominer protein enrichment kits decrease the dynamic range of complex biological samples, allowing improved analytical coverage of the proteome. The objective of this study was to apply high‐throughput MS techniques following proteominer enrichment to expand the range of proteins that can be identified in the secretome of cartilage exposed to proinflammatory cytokines such as IL‐1β. Explant cultures were established in DMEM + 2% Pen/Strep, using metacarpophalangeal joints of horses. Explants were either untreated controls or treated with 10 ng/ml IL‐1β. After incubating for 6 days at 37⁰C in a humidified environment (95% air, 5% CO2) proteins in culture supernatants were concentrated ~24 fold using Centriplus YM‐3 and Centricon‐3 (3000 MWCO) centrifugal filter units. Concentrated samples were processed with a Small Capacity Proteominer™ Protein Enrichment Kit (Bio‐Rad). After elution, samples were reduced, alkylated, acetone precipitated and trypsin digested overnight, before digested peptides were applied to C18 spin columns, followed by nanoLC‐MS/MS analysis on an amaZon speed ETD. Extracellular matrix proteins identified in untreated control secretome included cartilage oligomeric matrix protein (COMP), fibromodulin and fibronectin. Macrophage migration inhibitory factor (MIF), serum amyloid A protein (SAA) and Heat shock 70 kDa protein 1‐like (HSP70) were identified in explants exposed to IL‐1β. Identification of new low abundance proteins in this model will facilitate a greater understanding of inflammatory processes occurring within cartilage.Grant Funding Source: Supported by Biotechnology and Biological Sciences Research Council (BBSRC) (Contract grant number: BB/G018030/1) and the European Commission Framework 7 program (EU FP7; HEALTH.2012.2.4.5–2, project number 305815).

Anti-tumor activity of human IgG1 anti-gp75 TA99 mAb against B16F10 melanoma in human FcgammaRI transgenic mice

Patients suffering from advanced melanoma have a very poor prognosis. Despite recent advances in the understanding of oncogenic mechanisms and therapeutic interventions, the median survival of patients with metastatic disease is less than 12 months. Immunotherapy of melanoma has been intensely investigated and holds great promises.Tyrosinase-related protein-1 or gp75 (TYRP-1/gp75) antigen is a melanosomal polypeptide. It is the most abundant glycoprotein synthesized by pigmented melanocytes and melanomas. It is specific for melanocytes and both primary and metastatic melanomas. In mice, administration of the mouse mAb anti-gp75 TA99 prevents outgrowth of B16F10 melanoma metastases. The activity of TA99 is dependent on the presence and activity of the IgG specific, Fc receptors. TA99 cross-reacts with human gp75, and is currently being used for diagnosis of patients.Here, we sequenced mIgG2a TA99 and found that the locus harboring the endogenous light chain of the fusion partner in the TA99 hybridoma cells is not inactivated, resulting in the production of a mixed pool of mAbs that mitigates binding to gp75. Since human IgG1 (hIgG1) is the most frequently used mAb format in clinical studies, we produced a recombinant hIgG1 TA99 molecule. Whereas it is known that hIgG1 can functionally interact with mouse Fc receptors, we found that hIgG1 TA99 did not exhibit in vivo activity against B16F10 melanoma in wild type C57BL/6 mice. However, results obtained in this study demonstrated anti-tumor activity of hIgG1 TA99 in FcγRIIB knockout mice and in human FcγRI transgenic mice. These results emphasize the need for testing hIgG mAb in mice with functional human FcγRs.

Structural and immunosuppression study for bioactivities of Tamm-Horsfall glycoprotein and uromodulin

IntroductionTamm-Horsfall glycoprotein (THP) and uromodulin are the most abundant glycoproteins in the urine of non-pregnant women and pregnant women, respectively. Today, the structural profile and biological activities of these glycoproteins are controversial. ObjectivesTo investigate the glycoforms and immunosuppressive activities of THP and Uromodulin MethodsUromodulin were isolated from diatomaceous earth filtration, N- and O-glycans were then analysed with mass spectrometer MALDI-TOF/TOF. MTS viability, cytokine and immunophenotyping assays were carried out to investigate the immunomodulatory activities of both THP and uromodulin Results & DiscussionMALDI-TOF/TOF analysis showed that both THP and uromodulin expressed high mannose and complex-type N-glycan carrying sialic acid residues, Sda, LacNAc and LacdiNAc sequence as the capping antennae. Uromodulin tends to express multiple degree of sialylation, in which up to four sialic acid residues were expressed on its tetra-antennary Nglycans. Core 1 and core 2 type O-glycans were observed in THP and uromodulin, with THP expressing more O-glycans. MTS viability assay showed that 125μg/ml of uromodulin and 250μg/ml of THP significantly reduced the PHA-activated PBMC cell viability, to 85.64 ± 5.9 > % and 84.59 ±2.94%, respectively. Uromodulin was 2-fold more active in suppressing PBMC cell viability. Immunophenotyping analysis showed that CD4+ T helper cells, CD8+ cytotoxic T cells, CD56+ NK cells and CD14+ monocytes were suppressed by THP. It induced pro-inflammatory cytokines such as IL-1β, TNF and Th1 cytokine IFN-γ. On the other hand, uromodulin had slightly greater effect in suppressing CD4+ T helper cells and CD8+ cytotoxic T cells. It only induced IL-1β and suppressed both Th1 cytokine IFN-γ and Th2 cytokine IL-10. Hence, uromodulin is more immunosuppressive than THP because it suppressed CD4+ and CD8+ T cells, stimulated IL-1β only and withheld the TNF secretion. ConclusionsGlycosylation changes as observed in THP are suggested to contribute to their differential immunosuppressive property. Developing

Immunomodulatory properties of Tamm-Horsfall glycoprotein (THP) and uromodulin.

Tamm-Horsfall glycoprotein (THP) and uromodulin are the most abundant glycoproteins in non-pregnant women's/men's and pregnant women's urine, respectively. However, the bioactivities of these glycoproteins are still unclear. To evaluate the immunomodulatory properties of THP and uromodulin on human peripheral blood mononuclear cells (PBMC) METHODS: THP and uromodulin isolated with diatomaceous earth filtration were subjected to several bioassays, such as MTS viability assay, immunophenotyping and cytokine analysis. MTS viability assay and immunophenotyping analysis showed that uromodulin has greater inhibitory activities in suppressing PBMC viability and the percentage of CD4⁺ T helper cells and CD8⁺ cytotoxic T cells, compared to that of THP. In cytokine analysis, THP tended to induce pro-inflammatory cytokines such as IL-1β, TNF and Th1 cytokine IFN-γ; while uromodulin only induced IL-1β and suppressed both Th1 cytokine IFN-γ and Th2 cytokine IL-10. These results demonstrate that uromodulin has greater immunosuppressive activities and lower inductive property in relation to activation of immune cells, which provides a more tolerant environment for the developing fetus.

Influenza neuraminidase as a vaccine antigen.

Neuraminidase (NA) is the second most abundant influenza surface glycoprotein and contributes to virus replication in several ways, most notably by removing sialic acids from the host and viral glycoproteins, releasing newly formed virus particles from infected cells. Antibodies that block this enzyme activity restrict virus replication in vitro. This chapter describes foundational epidemiologic and human influenza challenge studies that provide evidence of an association between NA inhibiting antibodies and resistance to disease. Mouse challenge studies show that while NA immunity is infection-permissive, NA-specific antibodies attenuate infection and prevent severe disease. NA immunity is most effective against homologous viruses but there is substantial protection against viruses with a heterologous NA (different lineage within a NA subtype). Monoclonal antibodies specific for conserved antigenic domains of subtype N1 protect against seasonal and pandemic H1N1 as well as H5N1 virus challenge. Clinical studies demonstrate that licensed seasonal vaccines contain immunogenic amounts of NA, but the contribution of this immunity to vaccine efficacy is currently not known. New types of influenza vaccines could be designed to elicit NA immunity. Because NA induces heterologous immunity, it could be an important constituent of universal influenza vaccines that aim to protect against unexpected emerging viruses.

Detection of activated platelets in a mouse model of carotid artery thrombosis with 18F-labeled single-chain antibodies

Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel (18)F PET radiotracer based on this antibody. ScFv(anti-LIBS) and control antibody mut-scFv were reacted with N-succinimidyl-4-[(18)F]fluorobenzoate (S[(18)F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl(3) induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. After incubation with increasing concentrations of (18)F-scFv(anti-LIBS) clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled (18)F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6). Our results show that the novel antibody radiotracer (18)F-scFv(anti-LIBS) is useful for the sensitive detection of activated platelets and thrombosis. We describe the first (18)F variant of a scFv(anti-LIBS) against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.

Autophagy and the Effects of Its Inhibition on Varicella-Zoster Virus Glycoprotein Biosynthesis and Infectivity

Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (P < 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (P < 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing.

Preparation of cysteine-click maltose modified silica as a hydrophilic interaction liquid chromatography material for the enrichment of glycopeptides

Because of the low abundance of glycoprotein and glycopeptide in complex biological samples, it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research. Herein, a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO2). The developed hydrophilic silica was packed into a solid phase extraction (SPE) column, and applied to the highly selective enrichment and identification of N-linked glycopeptides. The Cys-Mal@SiO2 demonstrated better identification capability over Cys@SiO2, Mal@SiO2 and commercial hydrophilic interaction liquid chromatography (HILIC) in glycopeptide enrichment due to the synergistic effect of the two kinds of hydrophilic molecules. In the selective enrichment of tryptic digest from human immunoglobulin G, glycopeptides with higher signal-to-noises were detected by Cys-Mal@SiO2. In addition, 1551 unique glycopeptides with 906 N-glycosylation sites from 466 different N-linked glycoproteins were identified from the proteins extracted from mouse liver after the enrichment with Cys-Mal@SiO2. In contrast, the numbers of identified glycopeptides, glycoproteins and N-glycosylation sites identified by Cys@SiO2 were 211, 67, 127 respectively less than by Cys-Mal@SiO2, and the corresponding numbers were 289, 76, 193 by Mal@SiO2. These results showed that the developed Cys-Mal@SiO2 is a promising affinity material for N-glycoproteomics research of real complex biological samples.

A multilectin affinity approach for comparative glycoprotein profiling of rheumatoid arthritis and spondyloarthropathy

BackgroundArthritis refers to inflammation of joints and includes common disorders such as rheumatoid arthritis (RA) and spondyloarthropathies (SpAs). These diseases differ mainly in terms of their clinical manifestations and the underlying pathogenesis. Glycoproteins in synovial fluid might reflect the disease activity status in the joints affected by arthritis; yet they have not been systematically studied previously. Although markers have been described for assisting in the diagnosis of RA, there are currently no known biomarkers for SpA.Materials and methodsWe sought to determine the relative abundance of glycoproteins in RA and SpA by lectin affinity chromatography coupled to iTRAQ labeling and LC-MS/MS analysis. We also used ELISA to validate the overexpression of VCAM-1, one of the candidate proteins identified in this study, in synovial fluid from RA patients.Results and discussionWe identified proteins that were previously reported to be overexpressed in RA including metalloproteinase inhibitor 1 (TIMP1), myeloperoxidase (MPO) and several S100 proteins. In addition, we discovered several novel candidates that were overexpressed in SpA including Apolipoproteins C-II and C-III and the SUN domain-containing protein 3 (SUN3). Novel molecules found overexpressed in RA included extracellular matrix protein 1 (ECM1) and lumican (LUM). We validated one of the candidate biomarkers, vascular cell adhesion molecule 1 (VCAM1), in 20 RA and SpA samples using ELISA and confirmed its overexpression in RA (p-value <0.01). Our quantitative glycoproteomic approach to study arthritic disorders should open up new avenues for additional proteomics-based discovery studies in rheumatological disorders.

Targeted glycoprotein enrichment and identification in stromal cell secretomes using azido sugar metabolic labeling

Effectively identifying the proteins present in the cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum. Two stromal cell lines were incubated with tetraacetylated sugar-azide analogs for 48 h in serum-free and low-serum conditions. Sugar-azide labeled glycoproteins were covalently linked to alkyne-beads, followed by on-bead trypsin digestion and MS/MS. The resulting glycoproteins were compared between media conditions, cell lines, and azide-sugar labels. Alkyne-bead capture of sugar-azide modified glycoproteins in stromal cell culture media significantly improved the detection of lower abundance secreted glycoproteins compared to standard serum-free secretome preparations. Over 100 secreted glycoproteins were detected in each stromal cell line and significantly enriched relative to a standard secretome preparation. Sugar-azide metabolic labeling is an effective way to enrich for secreted glycoproteins present in cell line secretomes, even in culture media supplemented with serum. The method has utility for identifying secreted stromal proteins associated with cancer progression and the epithelial-to-mesenchymal transition.

Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids -(X-Y-Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways-amide bond and glycosidic bond cleavage-are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.

Identification of New Apolipoprotein-CIII Glycoforms with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry of Human Sera

Apolipoprotein-CIII (apoCIII) is an abundant blood glycoprotein associated with lipoprotein particles. Three different glycoforms have been described, all containing a mucin-type core-1 O-glycosylation with either zero, one or two sialic acids. Changes in the relative abundance of these glycoforms have been observed in a variety of different pathologies. In this study, ultrahigh resolution 15T MALDI Fourier transform ion cyclotron resonance (FTICR) MS was used to analyze apoCIII isoforms in serum protein profiles. For this purpose, serum proteins were purified using both a fully automated RPC18-based magnetic bead method and an RPC4 cartridge-based solid phase extraction method. Six new apoCIII isoforms were identified with low-ppm mass measurement errors and ultrahigh precision. These were characterized by more complex glycan moieties that are fucosylated instead of sialylated. To confirm the glycan moiety and localize the glycosylation site, top-down ESI-FTICR-MS/MS and bottom-up LC-ion trap MS/MS were used. A large variation in the presence and abundance of the fucosylated isoforms was found in a set of 96 serum samples. These findings of fucosylated apolipoprotein-CIII isoforms warrant further research to elucidate the implications these glycoforms may have for the plethora of studies where alterations in apoCIII have been linked to the development of many different pathologies.

Free glycans derived from glycoproteins present in human sera

During the course of studies on the analysis of O-glycans in biological samples, we found that significant amount of free glycans are present in normal human serum samples. The most abundant free glycan was disialo-biantennary glycan typically observed in transferrin which is one of the abundant glycoproteins found in sera. Minor glycans were also considered to be mainly due to transferrin, but some glycans were derived from mucin-type O-glycans, although the amount was quite minute. However, high mannose-type glycans could not be detected at all. Although there have been many reports on the presence of intracellular "free" N-glycans (mainly derived from high mannose-type glycans) generated either from lipid-linked oligosaccharides or from misfolded glycoproteins through endoplasmic-reticulum associated protein degradation pathway, there is little information on the presence of free glycans in extracellular matrix and biological fluids such as serum. This report is the first one which demonstrates the presence of free glycans due to glycoproteins in sera.

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor-binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N-glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) based glycoanalysis (N-glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N-glycosylation pattern of Madin-Darby canine kidney (MDCK) cell-derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum-free growth has only a minor impact on the HA N-glycosylation pattern. Only relative abundances of N-glycan structures are affected. In contrast, host cell adaptation to serum-free suspension growth resulted in significant changes in the HA N-glycosylation pattern regarding the presence of specific N-glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β-propiolactone inactivation resulted-at best-only in minor changes in the relative N-glycan structure abundances of the HA N-glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N-glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum-free suspension growth significantly influenced HA N-glycosylation regarding both, the type of attached glycan structures as well as their abundances.

Evidence for recycling of invariant surface transmembrane domain proteins in African trypanosomes.

Intracellular trafficking is a vital component of both virulence mechanisms and drug interactions in Trypanosoma brucei, the causative agent of human African trypanosomiasis and n'agana of cattle. Both maintaining the surface proteome composition within a life stage and remodeling the composition when progressing between life stages are important features of immune evasion and development for trypanosomes. Our recent work implicates the abundant transmembrane invariant surface glycoproteins (ISGs) in the uptake of first-line therapeutic suramin, suggesting a potential therapeutic route into the cell. RME-8 is a mediator of recycling pathways in higher eukaryotes and is one of a small cohort of intracellular transport gene products upregulated in mammal-infective trypanosomes, suggesting a role in controlling the copy number of surface proteins in trypanosomes. Here we investigate RME-8 function and its contribution to intracellular trafficking and stability of ISGs. RME-8 is a highly conserved protein and is broadly distributed across multiple endocytic compartments. By knockdown we find that RME-8 is essential and mediates delivery of endocytic probes to late endosomal compartments. Further, we find ISG accumulation within endosomes, but that RME-8 knockdown also increases ISG turnover; combined with previous data, this suggests that it is most probable that ISGs are recycled, and that RME-8 is required to support recycling.

Splenic proliferative lymphoid nodules distinct from germinal centers are sites of autoantigen stimulation in immune thrombocytopenia

AbstractTo understand more specific abnormalities of humoral autoimmunity, we studied 31 spleens from immune thrombocytopenia (ITP) patients and 36 control spleens. Detailed analysis identified at least 2 different splenic structures accommodating proliferating B cells, classic germinal centers (GCs), and proliferative lymphoid nodules (PLNs). PLNs were characterized by proliferating Ki67+ B cells close to follicular dendritic cells (FDCs) and lacked polarization into dark and light zones. As opposed to cells in GCs, proliferating B cells in PLN lacked expression of Bcl6. In both PLNs and GCs of ITP spleens, the density of T cells was significantly reduced. Both T follicular helper cells (TFH) and regulatory T cells were reduced within PLNs of ITP spleens suggesting a defect of tolerance related to a loss of T-cell control. Within PLNs of ITP, but not controls, abundant platelet glycoprotein (GP) IIb/IIIa autoantigens was found in IgM containing immune complexes tightly bound to FDCs and closely approximated to proliferating B cells. GPIV was found less often, but not in the same PLNs as GPIIb/IIIa. Autoantigens were not found in the GCs of ITP or controls indicating that PLNs are the sites of autoantigen stimulation in ITP potentially related to a lack of control by T cells and/or the present autoantigen.

Impact of matrix metalloproteinases on inhibition of mineralization by fetuin

Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro. Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay. From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 μm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization. MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1-3(GlcNAcβ1-6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.

Glycodelin-A Stimulates Interleukin-6 Secretion by Human Monocytes and Macrophages through L-selectin and the Extracellular Signal-regulated Kinase Pathway

Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.