Abstract
Glycosaminoglycans (GAGs), such as hyaluronan, chondroitin sulfate, and heparin, constitute mammalian extracellular matrices. The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bond, while heparin contains 1,4-glycoside bond. Some bacteria target GAGs as means of establishing colonization and/or infection, and bacterial degradation mechanisms of GAGs have been well characterized. However, little is known about the bacterial import of GAGs. Here, we show a GAG import system, comprised of a solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter, in the pathogenic Streptobacillus moniliformis. A genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs as well as the ABC transporter system was found in the S. moniliformis genome. This bacterium degraded hyaluronan and chondroitin sulfate with an expression of the genetic cluster, while heparin repressed the bacterial growth. The purified recombinant Smon0123 exhibited an affinity with disaccharides generated from hyaluronan and chondroitin sulfate. X-ray crystallography indicated binding mode of Smon0123 to GAG disaccharides. The purified recombinant ABC transporter as a tetramer (Smon0121-Smon0122/Smon0120-Smon0120) reconstructed in liposomes enhanced its ATPase activity in the presence of Smon0123 and GAG disaccharides. This is the first report that has molecularly depicted a bacterial import system of both sulfated and non-sulfated GAGs.
Highlights
Extracellular matrices are ubiquitously present in all mammalian tissues and organs and serve as physical scaffolds for cellular constituents, cell differentiation, homeostasis, and tissue formation[1]
Hyaluronate and heparan sulfate lyases encoded by the cluster are classified in polysaccharide lyase (PL) families 8 and 12, respectively, as per the Carbohydrate Active enZyme (CAZy) database[23], while all unsaturated glucuronyl hydrolase (UGL) are solely members of the glycoside hydrolase (GH) family 88 according to the database
The S. moniliformis ATP-binding cassette (ABC) transporter was demonstrated to be active on various unsaturated GAG disaccharides, unsaturated sulfate group-free chondroitin and hyaluronan disaccharides and uniquely unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc (C∆6S), which is an unsuitable substrate for phosphotransferase system (PTS)
Summary
Extracellular matrices are ubiquitously present in all mammalian tissues and organs and serve as physical scaffolds for cellular constituents, cell differentiation, homeostasis, and tissue formation[1]. Uronate and amino sugar, in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bonds, while residues in heparin and heparan sulfate are linked by 1,4-glycoside bonds. With the exception of hyaluronan, these GAGs frequently contain sulfate groups in uronate and/or amino sugar residues. In pathogenic Gram-positive bacteria, such as staphylococci and streptococci, GAGs (e.g., hyaluronan and chondroitin sulfate) are depolymerized to unsaturated disaccharides by extracellular or cell-surface polysaccharide lyase (HysA) through a β-elimination reaction[6,7,8] (Fig. 1). Our previous reports indicated that resultant unsaturated GAG disaccharides with C=C double bonds at non-reducing termini are degraded to constituent monosaccharides (i.e., unsaturated uronate and amino sugar) by unsaturated glucuronyl hydrolase (UGL) through hydration of C=C double bonds in the cytoplasm[9, 10]. Because ABC transporters incorporate substrates into the cytoplasm without any modifications[20], we hypothesized that GAGs sulfated at C-6 position should be viable substrates for ABC transporters, which is confirmed by this study
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