Binding and Inhibition Assays for Siglecs

Abstract

Protein-carbohydrate interactions are believed to be important in many biological processes involving cellular communication. Although many of the experimental approaches for studying protein-carbohydrate interactions are similar to those commonly employed in protein-protein interactions, several important aspects have to be taken into account in binding assays with lectin-like receptors such as Siglecs, sialic acid-recognizing immunoglobulin-like lectins. This chapter describes experimental approaches using solid-phase assays with sialylated targets and complexed Siglec Fc-chimeras for binding studies or competitive inhibition assays. Such assays are useful to investigate various aspects of Siglec interactions with their carbohydrate-binding partners, such as the specificity and the molecular basis of sialic acid recognition.

Lectin inhibition assays for the analysis of bioactive milk sialoglycoconjugates

“…Inhibition assays for siglecs have been described (Bock & Kelm, 2006). Fetuin, containing both a2,3-and a2,6-linked Sia, was immobilized to a high binding microtitre plate as target glycoconjugate to determine siglec binding, whereas the compounds to be tested are used as competitive inhibitors.…”

“…For this, all samples were diluted two-fold in the final assay mixture, e.g., the stock solution of 10 mg mL À1 would lead to a final sample concentration of 5 mg mL À1 . Bound siglecs were quantified by using fluorescein diphosphate (FDP) as substrate (Bock & Kelm, 2006). Unspecific binding was determined using immobilized asialofetuin as target and subtracted.…”

“…However, such information is available from assays based on binding by lectins, which are a heterogenous group of proteins with the ability to recognize and bind sugars. For example, assays using immobilized carbohydrates or lectins have been very useful to characterize the glycan specificity of lectins and to analyze molecular interactions as well as screening for inhibitory molecules (Bock & Kelm, 2006). Sia binding proteins have been found in various sources (Varki, 1997) including plants that do not contain Sia.…”

Impact of Strong and Weak Lipid-Protein Interactions on the Structure of a Lipid Bilayer on a Gold Electrode Surface

“…The proteins were analysed by ELISA for detecting the Fc part, a binding assay with immobilized fetuin and SDS-PAGE to verify the Sia binding activity. [94] Heterologous Expression and Purification of Myristoylated Recoverin: Expression of recombinant recoverin was essentially done as described previously. [95] Briefly, Escherichia coli strain BL21 was cotransformed with plasmids pET11d and pBB131 containing the genes for recoverin and yeast N-myristoyl-transferase (kind gift of Dr. Jeffrey Gordon), respectively.…”

Bovine Milk Whey for Preparation of Natural N-glycans: Structural and Quantitative Analysis

“…This is in agreement with our finding that the α2-6-sialylated N-glycans derived from bovine milk protein concentrate are more abundant than α2-3-sialylated N-glycans by about 36.2 molar percent, and they are composed of Neu5Ac/ Neu5Gcα2-6Gal1-4GlcNAc and Neu5Acα2-6GalNAc1-4GlcNAc. Previous studies also showed that N-glycans derived from bovine IgM, present in bovine whey [25], mainly terminated with Neu5Ac/Neu5Gcα2-6Gal [26]. These α2-6-linked sialylated glycans could be useful for determination of human-adapted influenza viruses and for study of the properties of several human cancers, including colon, breast and cervix cancers, associated with Siaα2-6Gal1-4GlcNAc level on their cell surface [19,27].…”