Notes

Low-grade side-line infection influences mental faculties pathology within the AppNL-G-Fmouse type of Alzheimer's disease.
Hyaluronan (HA), a major component of the extracellular matrix in vertebrate tissues, provides structural and functional integrity to cells and organs. Biological functions of HA are dependent on the molecular size of HA and the interaction with a wide range of HA-binding proteins, i.e., hyaladherins. In this book chapter, we introduce hyaladherins and focus on HYBID (Hyaluronan-binding protein involved in hyaluronan depolymerization, alias KIAA1199 and CEMIP), which is one of the hyaladherins and plays a central role in HA degradation in human dermal and arthritic synovial fibroblasts. The protocols describe the preparation of the stable transfectants expressing HYBID, the assays of HYBID-mediated HA depolymerization, and the binding assay of HYBID to HA. These methods will be helpful to further study the HYBID-mediated biological activities and its relevance on HA degradation and turnover under various physiological and pathological conditions such as wound healing, ageing, arthritis, and cancer.C-type lectins bind to carbohydrate structures in a Ca2+-dependent manner. Some transmembrane forms of lectins act as innate immune receptors and induce signal transduction pathways in macrophages and dendritic cells (DCs). Expressing these receptors in cells bearing a reporter gene is a useful tool to investigate ligand binding and recognition. However, it cannot be used to quantify the precise affinity of the interaction, and the involvement of other proteins remains a possibility. Direct binding between a receptor and its ligand can be investigated using an immunoglobulin receptor (Ig)-fused soluble protein. This binding can be assessed using enzyme-linked immunosorbent assays and flow cytometry, and the fusion protein may also be used in a glycan array. In this chapter, we explain the generation of Ig fusion proteins and subsequent binding assays using these proteins.Cell-cell interactions mediated by selectins and their ligand glycans play pivotal roles in a variety of biological processes represented by leukocyte recruitment to inflammatory sites, lymphocyte homing, and extravasation of cancer cells. The interactions are enhanced at least partly through the upregulation of the selectin-ligand glycan expression, which is observed, for instance, during the activation of leukocytes or epithelial-mesenchymal transition of cancer cells. Selectin-binding assays such as cell adhesion assay or rolling assay have long been used to directly evaluate the activity of these cells in the selectin-mediated processes. In this chapter, we introduce a highly quantitative assay by flow cytometry using recombinant selectin-Ig(Fc) chimera proteins, showing our procedure and tips for E-selectin-binding assay of colon cancer cells undergoing epithelial-mesenchymal transition.Native and recombinant collectins are purified by using mannan-agarose and an anti-collectin antibody column. The use of sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies against human mannan-binding lectin (MBL) enables elucidation of the collectin concentration in the blood, serum, and plasma. The collectin sugar specificity is demonstrated by determining the concentration of saccharide required to inhibit sugar binding by 50% in a saccharide-binding assay. Biological analyses including the complement-dependent hemolysis test and several other methods are used to evaluate collectin.Siglecs are transmembrane receptor-like vertebrate lectins that recognize glycans containing sialic acid. Most Siglecs also interact with intracellular signal transduction molecules, and modulate immune responses. Recombinant soluble Siglecs fused with the fragment crystallizable (Fc) region of immunoglobulin G (Siglec-Fc) are a versatile tool for the investigation of Siglec functions. We describe protocols for the production of recombinant Siglec-Fc, the analysis of expression of Siglec ligands by flow cytometry, and the identification of the Siglec ligand candidates based on proximity labeling.Siglecs are known to be bound and regulated by membrane molecules that display specific sialic acid-containing ligands and are present on the same cell (cis-ligands). Because of the low-affinity binding of Siglecs to the glycan ligands, conventional methods such as immunoprecipitation are not suitable for identification of Siglec cis-ligands. Here we describe efficient and specific labeling of cis-ligands of CD22 (also known as Siglec-2) on B lymphocytes by proximity labeling using tyramide. This method may also be applicable to labeling of cis-ligands of other Siglecs.Although cell-based protein expression systems enable us a certain amount of protein suitable for subsequent biological experiments to be obtained, aggregates of the protein of interest are sometimes encountered during the purification procedure. Pentraxin 3 (PTX3), a member of the pentraxin family that is classified as a carbohydrate-binding protein based on its structure, comprises one of the humoral arms of the pattern recognition receptors that play an important role in the innate immune response. PF543 PTX3 comprises two domains; an N-terminal domain and a C-terminal domain. The C-terminal domain containing pentraxin signature has similar biological functions as other pentraxins such as C-reactive protein (CRP) and serum amyloid-P component (SAP). On the other side, the N-terminal domain is specific to PTX3. A supply of the PTX3 protein in full length or partial fragments is thus essential for the elucidation of its biological functions. Here we describe the expression and purification of recombinant PTX3. An arginine-containing buffer is essential for the elution of bacterially expressed PTX3 N-terminal domain to minimize aggregation. This method allows high-yield purification of full-length or domain-fragment recombinant PTX3 proteins for biological study.S-nitrosylation, which involves the coupling of an NO group to the reactive thiol of Cys residue(s) in a polypeptide, is an important posttranslational modification detected in a variety of proteins. Here, we present the S-nitrosylation of recombinant galectin-2 (Gal-2) using S-nitrosocysteine and the measurement of the molecular ratio of S-nitrosylation of Cys residues in the Gal-2 protein.
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