Detection of chondroitin sulfate‐binding proteins on the membrane

Abstract

Chondroitin sulfate is a ubiquitous component of proteoglycans that is present both in the extracellular matrix and at the cell surface of various tissues. Until recently, chondroitin sulfate has attracted less attention than heparan sulfate and dermatan sulfate, owing to the limited number of known chondroitin sulfate-binding proteins. To determine the biological function of chondroitin sulfate, biotinylated probes were prepared and used to search for binding proteins. Chondroitin sulfates A, C, D, and E were biotinylated through either the uronic acid or the residual core peptide. Lysates from mouse Lewis lung carcinoma (3LL) cells were blotted onto a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the membrane was treated with the biotinylated chondroitin sulfates. Among the chondroitin sulfate variants, the E type showed the most intense bands upon visualization of the membrane with avidin-conjugated alkaline phosphatase and the appropriate substrates. The binding of chondroitin sulfate E to proteins in the cell lysate was not affected by the A, C or D variants but was reduced by treatment with dermatan sulfate. Lysates from 3LL cells were also treated with biotinylated chondroitin sulfate E and, after two-dimensional (2-D) electrophoresis and blotting, several chondroitin sulfate E-binding proteins including lamins and heterogeneous nuclear ribonucleoproteins were identified by mass spectrometry.

Surface functionalization of biomaterials with tissue-inductive artificial extracellular matrices

“…Their sulfate groups as well as their carboxyl groups are suggested to interact with mineral structures in bone such as HA [94,95]. CS is able to bind and modulate proteins like tumor necrosis factor or fibronectin [96][97][98] which might result in an enhanced attachment of cells on the implant surface [95] and an improved healing process.…”

Proteomic Analysis of Potential Keratan Sulfate, Chondroitin Sulfate A, and Hyaluronic Acid Molecular Interactions

“…Attachment of biotin to free carboxy groups in the residual core peptide was performed by using the EDC protocol. 55,56 KS (1 mg) was dissolved in 0.5 mL 0.1 M MES ((2-N-morpholino)ethanesulfonic acid; pH 4.5), containing 2 mg/mL biotin-LC-hydrazide and 20 mg/mL EDC. The mixture was rotated end-over-end at room temperature for 24 hours.…”

“…Purified CSA was labeled (EZ-link Biotin-LC-Hydrazide; Thermo Scientific), and biotin was attached to the carboxyl groups of uronic acid residues along the CSA GAG chain, by using the EDC protocol, 55,56 beginning with 2 mg CSA dissolved in 1 mL 0.1 M MES (pH 4.5) containing 2 mg/mL biotin-LC-hydrazide and 20 mg/mL EDC. The yield of the biotinylated CSA (bCSA) was 1.7 mg.…”

Immobilization of chondroitin sulfate to lipid membranes and its interactions with ECM proteins

“…Independent of their origin, GAGs are highly compatible with various biological functions such as stabilizing growth factors, mediating cellular processes [3] and regulating atherogenesis [4]. For example, chondroitin sulfate (CS) has recently gained increased interest because of its role in bone and cartilage tissue and its interactions with growth factors and proteins like type I collagen, the most abundant protein in the ECM [5][6][7]. Growth factors, for example bone morphogenetic protein 2 (BMP-2), are frequently used in research, primarily with the aim of stimulating bone growth and regeneration [8][9][10][11].…”