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Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients., (© The Author(s) 2021. Published by Oxford University Press.)

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%–86% of DS-epi1 activity at 10 μM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients. [ABSTRACT FROM AUTHOR]

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N -sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N -linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi. [ABSTRACT FROM AUTHOR]

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva , which has a variety of biological properties. Ulvan lyase degrades ulvan through a β-elimination mechanism which cleaves the β-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97 kDa, and it had the highest catalytic activity at 45 °C and pH 8.0 in Tris–HCl buffer. The K m and V max values were 2.24 mg · mL−1 and 2.048 μmol · min−1 · mL−1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20 ~ 30 °C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides. [ABSTRACT FROM AUTHOR]

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan. [ABSTRACT FROM AUTHOR]

N -glycolylated carbohydrates are amino sugars with an N -glycolyl amide group. These glycans have not been well studied due to their surprising rarity in nature in comparison with N -acetylated carbohydrates. Recently, however, there has been increasing interest in N -glycolylated sugars because the non-human sialic acid N - glycolylneuraminic acid (Neu5Gc), apparently the only source of all N -glycolylated sugars in deuterostomes, appears to be involved in xenosialitis (inflammation associated with consumption of Neu5Gc-rich red meats). Xenosialitis has been implicated in cancers as well as other diseases including atherosclerosis. Furthermore, metabolites of Neu5Gc have been shown to be incorporated into glycosaminoglycans (GAGs), resulting in N -glycolylated GAGs. These N -glycolylated GAGs have important potential applications, such as dating the loss of the Neu5Gc-generating CMAH gene in humans and being explored as a xenosialitis biomarker and/or estimate of the body burden of diet-derived Neu5Gc, to understand the risks associated with the consumption of red meats. This review explores N -glycolylated carbohydrates, how they are metabolized to N -glycolylglucosamine and N -glycolylgalactosamine, and how these metabolites can be incorporated into N -glycolylated GAGs in human tissues. We also discuss other sources of N -glycolylated sugars, such as recombinant production from microorganisms using metabolic engineering as well as chemical synthesis. [ABSTRACT FROM AUTHOR]

Introduction The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. Methods GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. Results and Conclusions Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS. [ABSTRACT FROM AUTHOR]

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N -linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N -linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum , will allow delineation of the N-glycosylation pathway in this organism. [ABSTRACT FROM AUTHOR]

Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the β-1,4 glycosidic linkage of chondroitin sulfate (CS) by β-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g. approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4- O -sulfated N -acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6- O -sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the k cat/ K m of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4- O -sulfated and 6- O -sulfated GalNAc residues. The distance between Arg500 and the 4- O -sulfate group was 0.8 Å shorter than that between Arg500 and the 6- O -sulfated group. Moreover, it is likely that the 6- O -sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4- O -sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I. [ABSTRACT FROM AUTHOR]

Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell (NSC) presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knockout (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both wild-type and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1 / 2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for NSCs to thrive, which is critical for the design of future stem cell therapies. [ABSTRACT FROM AUTHOR]

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)