Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.
Xylose is the optimal substrate for β4GalT7, an essential enzyme in GAG biosynthesis, but analogs act as effective inhibitors.
We previously reported that the xyloside 2-(6-hydroxynaphthyl) -D-xylopyranoside (XylNapOH), in contrast to 2-naphthyl -D-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH-and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH-and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/ dermatan sulfate nor the heparan sulfate derived from CCD1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH-and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate.
Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase β4GalT7. Some β-d-xylosides, such as 2-naphthyl β-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for β4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with β-glycosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle differences in orientation of the xylose moiety.
Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remain poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from the human placenta, tumor tissues and cancer cells and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well.
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