The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope

Abstract

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…However, when we used the CE result to calculate the product yield we found 6.1 ± 0.1 mg/L of viscumin heterodimer in the elution fraction corresponding to 7.2 ± 0.1 mg/kg fresh leaf biomass (AM ± SD, n = 2) and a purity of 0.54 g/g TSP (Table ). We concluded that the direct CE‐derived yield and purity seemed more likely than the ~1.2 mg/kg and 0.08 g/g, respectively determined by the indirect SPR assay (Figure S2), because the latter can be affected by differential glycosylation patterns of proteins (Uray, Mizuno, Inazu, Goto, & Hudecz, ). For example, epitope recognition and binding kinetics of mAbs can differ for glycosylated and nonglycosylated products.…”

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV–Cell Interaction and Functional Activation of Endothelial Cells

“…Additional evidence of the enzyme treatment was the reduction in all of the EV markers analyzed (i.e., CD9, CD63, CD90), indicating the presence of sensitive epitopes for PNGase-F. In fact, the antibody-binding alterations due to glycosylation were already reported [ 52 , 53 ]. The high diversity of the glycans affected by PNGase-F treatment precluded any identification of the specific targets involved in the analyzed functions.…”

Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients’ Sera

“…This is the case of the α2,3-SA PSA glycoforms for prostate cancer [ 72 ], core fucosylated-AFP glycoforms (AFP-L3) for hepatocellular carcinoma [ 73 ] or CA125-STn for ovarian cancer [ 74 ]. In addition, this knowledge could be useful for therapies targeting MSLN using specific antibodies [ 28 , 41 ], because their affinity could be dependent on the type of MSLN glycans, as it has been described for other glycoproteins [ 75 , 76 ].…”