Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

Funding Information: This work was supported by Fundação para a Ciência e a Tecnologia, I. P. through iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (to J.C. and J.B.V.) and by US NIH Grants GM130915, GM103390, and GM107012 (to K.W.M.). Funding Information: Mass spectrometry data were provided/obtained by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. We thank the technical assistance of Conceição Almeida, UniMS and Cristina Leitão, ITQB NOVA with the MS and HPAEC-PAD analyses, respectively. We thank Dr. Pedro Lamosa, CERMAX, ITQB NOVA, for the NMR analysis. iBET is acknowledged for access to ITF-DSF equipment. Publisher Copyright: © 2023, The Author(s).Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.publishersversionpublishe

A Biomimetic Synthetic Strategy can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns

Keratan sulfate (KS) is a proteoglycan that is widely expressed in the extracellular matrix of various tissue types where it performs multiple biological functions. KS is the least understood proteoglycan, which in part is due to a lack of panels of well-defined KS oligosaccharides that are needed for structure-binding studies, as analytical standards, to examine substrate specificities of keratinases and for drug development. Here, we report a biomimetic approach that makes it possible to install, in a regioselective manner, sulfates and fucosides on oligo-N-acetyllactosamine (LacNAc) chains to provide any structural element of KS by using specific enzyme modules. It is based on the observation that 1,3-fucosides, 2,6-sialosides and C-6 sulfation of galactose (Gal6S) are mutually exclusive and cannot occur on the same LacNAc moiety. As a result, the pattern of sulfation on galactosides can be controlled by installing1,3-fucosides or 2,6-sialosides to temporarily block certain LacNAc moieties from sulfation by keratan sulfate galactose 6-sulfotransferase (CHST1). The pattern of 1,3-fucosylation and 2,6-sialylation can be controlled by exploiting the mutual exclusivity of these modifications, which in turn controls the sites of sulfation by CHST1. Late-stage treatment with a fucosidase or sialidase to remove blocking fucosides or sialosides provides selectively sulfated KS oligosaccharides. These treatments also unmasked specific galactosides for further controlled modification by CHST1. To showcase the potential of the enzymatic strategy, we have prepared a range of poly-LacNAc derivatives having different patterns of fucosylation and sulfation and several N-glycans decorated by specific arrangements of sulfates

A Biomimetic Synthetic Strategy Can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns

Keratan sulfate (KS) is a proteoglycan that is widely expressed in the extracellular matrix of various tissue types, where it performs multiple biological functions. KS is the least understood proteoglycan, which in part is due to a lack of panels of well-defined KS oligosaccharides that are needed for structure-binding studies, as analytical standards, to examine substrate specificities of keratinases, and for drug development. Here, we report a biomimetic approach that makes it possible to install, in a regioselective manner, sulfates and fucosides on oligo- N-acetyllactosamine (LacNAc) chains to provide any structural element of KS by using specific enzyme modules. It is based on the observation that α1,3-fucosides, α2,6-sialosides and C-6 sulfation of galactose (Gal6S) are mutually exclusive and cannot occur on the same LacNAc moiety. As a result, the pattern of sulfation on galactosides can be controlled by installing α1,3-fucosides or α2,6-sialosides to temporarily block certain LacNAc moieties from sulfation by keratan sulfate galactose 6-sulfotransferase (CHST1). The patterns of α1,3-fucosylation and α2,6-sialylation can be controlled by exploiting the mutual exclusivity of these modifications, which in turn controls the sites of sulfation by CHST1. Late-stage treatment with a fucosidase or sialidase to remove blocking fucosides or sialosides provides selectively sulfated KS oligosaccharides. These treatments also unmasked specific galactosides for further modification by CHST1. To showcase the potential of the enzymatic strategy, we have prepared a range of poly-LacNAc derivatives having different patterns of fucosylation and sulfation and several N-glycans decorated by specific arrangements of sulfates

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties

Improved yield of α-L-arabinofuranosidase by newly isolated Aspergillus niger ADH-11 and synergistic effect of crude enzyme on saccharification of maize stover

Abstract Background In the view of depleting resources and ever-increasing price of crude oil, there is an urge for the development of alternative sources to solve the issue of fuel in the coming years. Lignocellulosic biomass is considered to be the most potential alternative resources for fossil fuel. Bioconversion of cellulosic and hemicellulosic components into fermentable sugars is the key step in producing fuel ethanol from lignocellulose. The enzymatic hydrolysis of lignocellulosic biomass needs a highly balanced composition of cellulases and hemicellulases. Commercial enzymes are usually poor in accessory hemicellulolytic enzymes like α-L-arabinofuranosidase. The addition of such accessory enzymes in combination with cellulase or hemicellulase plays a vital role in improving the total yield of fuel ethanol by enhancing the saccharification yield. Results The newly isolated fungal strain Aspergillus niger ADH-11 produced a maximum of 22.14 U/g of α-L-arabinofuranosidase under solid-state fermentation using wheat bran as the substrate and modified Mandels-Weber medium at 30°C after 180 h of incubation. The optimization of various fermentation parameters was performed by response surface methodology employing Plackett-Burman design followed by Box-Behnken design. The yield of α-L-arabinofuranosidase was enhanced by 2.34-fold after executing statistical optimization of various fermentative parameters. Crude α-L-arabinofuranosidase was found to be highly stable for 3 h at its optimum temperature (55°C) and pH (4.0). The assessment of the crude enzyme extract in saccharification of alkali-treated maize stover revealed that the supplementation of crude α-L-arabinofuranosidase to commercial cellulase and crude xylanase mixture increased the saccharification yield up to 730 mg/g of maize stover. Conclusions The newly isolated A. niger ADH-11 was found to be a potential producer of α-L-arabinofuranosidase. The crude enzyme was active at low pH and high temperature which makes it suitable for various industrial applications such as enzymatic saccharification of lignocellulosic biomass. The supplementation of α-L-arabinofuranosidase enzyme to commercial cellulases and hemicellulases improves the bioconversion of lignocellulosic biomass to a greater extent. </jats:sec