Naphthoxylosides – Probing the β4GalT7 active site
Sammanfattning: Proteoglycans (PGs) is a class of highly anionic macromolecules that consist of one or more linear polysaccharide chains (glycosaminoglycans, GAGs) covalently attached to a core protein. The biological functions of PGs are mainly due to the interactions of GAG chains with various protein ligands and regulatory factors, such as cytokines and growth factors. PGs and GAGs are critical for a diverse set of biological processes, such as cell growth, cell attachment, cell-cell interactions, and cell differentiation. PG and GAG also play important roles in various stages of cancer. The biosynthesis of GAG chains is initiated by xylosylation of a serine residue in the core protein. The xylosylated protein is then stepwise galactosylated by two galactosyl transferases, and glucuronated to form a linker tetrasaccharide, which later on is elongated to form a GAG chain. However, the biosynthesis can also be initiated by exogenously added xylosides with hydrophobic aglycones, such as naphthoxylosides, which can act as acceptors in the first galactosylation step (i.e. for xylosylprotein β-1,4-galactosyltransferase, polypeptide 7 (β4GalT7)). In order to determine the structural requirements of β4GalT7, and the importance for GAG synthesis, we have synthesized a number of naphthoxyloside analogs. We have further set up an assay for monitoring the galactosylation of naphthoxyloside analogs by β4GalT7. We conclude that the acceptor binding site of β4GalT7 is a shallow pocket, which encloses the sugar moiety, and all of the xylose hydroxyl groups are suggested to take part in hydrogen bonds necessary for efficient galactosylation. Hence, we propose that xylose is the optimal acceptor sugar for β4GalT7. However, we found that some of the naphthoxyloside analogs with modifications in the sugar residue inhibited galactosylation by β4GalT7. Furthermore, the aglycone part of acceptor substrates of β4GalT7 is found to extend out to the surface of the enzyme, which makes the enzyme more tolerant to modifications of the aglycone than of the sugar residue. Some of the naphthoxyloside analogs have also been investigated for their ability to prime GAG chains in cells, and we found that it was possible to transfer the results from the β4GalT7 experiments to cell studies. Thus, the β4GalT7 assay will be an important tool in future studies of GAG biosynthesis.
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