scholarly journals Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

2020 ◽  
Vol 295 (42) ◽  
pp. 14430-14444 ◽  
Author(s):  
Mariano Malamud ◽  
Gustavo J. Cavallero ◽  
Adriana C. Casabuono ◽  
Bernd Lepenies ◽  
María de los Ángeles Serradell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Protein Glycosylation ◽  
Immunomodulatory Activity ◽  
Glycosylation Pattern ◽  
Immunostimulatory Activity ◽  
Lectin Receptors ◽  
Lectin Receptor ◽  
Raw264.7 Macrophages ◽  
Lactobacillus Kefiri

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

scholarly journals Ultrastructural localization of lectin receptors on the surface of the rat retinal pigment epithelium. Decreased sensitivity of the avidin-biotin method due to cell surface charge.

1984 ◽  
Vol 32 (8) ◽  
pp. 862-868 ◽  
Author(s):  
R M Pino
Keyword(s):  
Bone Marrow ◽  
Retinal Pigment Epithelium ◽  
Cell Surface ◽  
Retinal Pigment Epithelial ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ultrastructural Localization ◽  
Cell Surfaces ◽  
Lectin Receptors ◽  
Lectin Receptor

Monosaccharides on the apical processes of the retinal pigment epithelium were examined using lectin-affinity cytochemical methods. Lectin receptor sugars were localized with lectin-horseradish peroxidase (HRP) and lectin-ferritin conjugates as well as with biotinylated lectins, avidin, and biotinylated HRP. In contrast, only wheat germ agglutinin (WGA) receptors were identified with biotinylated WGA followed by avidin-ferritin or free avidin and biotinylated ferritin. Labeling with avidin-ferritin subsequent to biotinylated lectin treatment was dependent upon the source and lot of the reagent. These findings are similar to those reported for the endothelium of bone marrow sinusoids (Pino RM: Am J Anat, 169:259, 1984). Since both the retinal pigment epithelial and bone marrow sinusoidal surfaces are highly anionic (negative), we investigated the possibility that the charge of the lectin reagents and cell surfaces might affect the localization of monosaccharides on cell surfaces. Analytical isoelectric focusing revealed that biotinylated ferritin and some avidin-ferritins are highly anionic, while the other lectin reagents have more cationic (positive) components. Based on this information, a less charged biotinylated ferritin marker was made that made it possible to localize biotinylated lectins bound to the cell surface.

scholarly journals Transcriptome Analysis Reveals Possible Immunomodulatory Activity Mechanism of Chlorella sp. Exopolysaccharides on RAW264.7 Macrophages

Marine Drugs ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 217
Author(s):  
Siwei Wu ◽  
Hongquan Liu ◽  
Siyu Li ◽  
Han Sun ◽  
Xiumiao He ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Enrichment Analysis ◽  
Immunomodulatory Activity ◽  
Pathway Enrichment Analysis ◽  
Immune Activity ◽  
Lectin Receptor ◽  
Chlorella Sp ◽  
Raw264.7 Macrophages ◽  
Immune Related Genes ◽  
The Impact

In this study, the exopolysaccharides of Chlorella sp. (CEP) were isolated to obtain the purified fraction CEP4. Characterization results showed that CEP4 was a sulfated heteropolysaccharide. The main monosaccharide components of CEP4 are glucosamine hydrochloride (40.8%) and glucuronic acid (21.0%). The impact of CEP4 on the immune activity of RAW264.7 macrophage cytokines was detected, and the results showed that CEP4 induced the production of nitric oxide (NO), TNF-α, and IL-6 in a dose-dependent pattern within a range of 6 μg/mL. A total of 4824 differentially expressed genes (DEGs) were obtained from the results of RNA-seq. Gene enrichment analysis showed that immune-related genes such as NFKB1, IL-6, and IL-1β were significantly upregulated, while the genes RIPK1 and TLR4 were significantly downregulated. KEGG pathway enrichment analysis showed that DEGs were significantly enriched in immune-related biological processes, including toll-like receptor (TLR) signaling pathway, cytosolic DNA-sensing pathway, and C-type lectin receptor signaling pathway. Protein–protein interaction (PPI) network analysis showed that HSP90AB1, Rbx1, ISG15, Psmb6, Psmb3, Psmb8, PSMA7, Polr2f, Rpsa, and NEDD8 were the hub genes with an essential role in the immune activity of CEP4. The preliminary results of the present study revealed the potential mechanism of CEP4 in the immune regulation of RAW264.7 macrophages, suggesting that CEP4 is a promising immunoregulatory agent.

scholarly journals Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ingeborg Streng-Ouwehand ◽  
Nataschja I Ho ◽  
Manja Litjens ◽  
Hakan Kalay ◽  
Martine Annemarie Boks ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Glycan Structure ◽  
Antigen Uptake ◽  
Lectin Receptors ◽  
Cross Presentation ◽  
Lectin Receptor ◽  
Intracellular Storage ◽  
Glycan Modification

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies.

Differential Signaling Patterns of Stimulated Bone Marrow-Derived Dendritic Cells under α1-Antitrypsin–Enriched conditions

2021 ◽  
pp. 104281
Author(s):  
Eyal Ozeri ◽  
Peleg Rider ◽  
Shoham Rigbi ◽  
Galit Shahaf ◽  
Iulia I. Nita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Differential Signaling
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