Chuzel et al. Microb Cell Fact (2021) 20:162 https://doi.org/10.1186/s12934-021-01652-w Microbial Cell Factories RESEARCH Open Access Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans Léa Chuzel1,2 , Samantha L. Fossa2, Madison L. Boisvert2, Samanta Cajic1 , René Hennig3 , Mehul B. Ganatra2, Udo Reichl1,4 , Erdmann Rapp1,3 and Christopher H. Taron2* Abstract Background: Sulfate modifcation of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modifcation and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifcations is hampered in part by a lack of enzymes that enable site-specifc detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fuorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. Results: Our screen identifed a sugar-specifc sulfatase that specifcally removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated gly- can but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. Conclusion: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifcations of glycans. The discovered enzymes represent new specifcities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses. Keywords: Sulfatase, Functional metagenomics, Glycoanalytics, Glycan analysis, Human microbiome, Glycan sulfation, N-acetylglucosamine-6-sulfate Background glycans (O-glycans)) residues. It is estimated that more Most eukaryotic proteins are covalently modifed with than half of all mammalian proteins bear some form carbohydrates (“glycans”) at certain asparagine (N-linked of glycosylation [1]. Additionally, the surface of mam- glycans (N-glycans)) or serine/threonine (O-linked malian cells may contain upwards of 10 million N- and O-linked glycans attached to extracellular proteins [1]. Glycans play many important roles in secretory protein *Correspondence: [email protected] structure and function including assisting in protein fold- 2 New England Biolabs, Ipswich, MA 01938, USA ing, trafcking, tissue distribution, and persistence in the Full list of author information is available at the end of the article mammalian bloodstream [2]. As such, glycosylation is © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chuzel et al. Microb Cell Fact (2021) 20:162 Page 2 of 17 considered an intrinsic part of a secretory protein’s over- analysis. Structural characterization of sulfated glycans all structure. Glycan structure itself has wide-ranging has been accomplished using mass spectrometry (e.g., importance in both basic biology and pharmaceutical sci- LC–MS/MS or MALDI-TOF) of released glycans or ence [2–5]. Aberrant glycosylation has been implicated glycopeptides [14, 22–25]. Tese methods can be com- in numerous diseases [6, 7] and changes in N-glycans of promised by poor ionization of sulfated species and the serum glycoproteins has yielded promising disease bio- natural low abundance of sulfated N- and O-glycans. markers [7–9]. Additionally, N-glycan composition can Methods to enrich samples for sulfated glycans could afect the function and efcacy of many glycoprotein aid their analysis. Glycan-specifc binding proteins have drugs like certain monoclonal antibodies, growth fac- recently been used to isolate glycans possessing certain tors and fusion proteins [10, 11], and is a critical quality structural features from complex samples [26–30]. How- attribute that is monitored during drug manufacturing. ever, no sugar sulfate-specifc binding proteins have yet Protein glycosylation is inherently complex due to the been applied to enrich sulfated glycans. Additional ana- manner in which glycans are assembled and processed lytical methods involving multiplexed capillary gel elec- by cells. Glycan synthesis does not occur through copy- trophoresis with laser-induced fuorescence detection ing or interpreting a biological template (like DNA, RNA, (xCGE-LIF) [31–33] and high/ultra-performance liquid or protein synthesis). Instead glycans are synthesized chromatography coupled to fuorescence detector (H/ via complex biosynthetic pathways involving numer- UPLC-FLR) methods [34–37] are also routinely used to ous glycosyltransferases that assemble diferent mono- separate glycans and match their migration- and reten- saccharides into specifc structures. Tere are over 200 tion-times, respectively, to those of known glycans in ref- glycosyltransferases encoded in the human genome and erence databases. Tese methods of glycan analysis often expression of many varies by cell type, tissue type or in use highly specifc exoglycosidases to enzymatically con- response to environmental factors. Tis results in sub- frm the presence or absence of certain sugars [38–40]. stantial glycan structural heterogeneity. For example, However, enzymes that remove site-specifc sugar sulfa- over 140 diferent N-glycan structures have been identi- tion or sulfated sugars are currently missing from the gly- fed on glycoproteins present in human serum [12]. Add- coanalysis toolbox. ing to this complexity, glycans can also be modifed with In this study, we sought to improve the range of ana- sulfate, phosphate, methyl or acetyl groups on certain lytical enzymes available for analysis of sulfated glycans. sugars. Tese groups are collectively termed ‘post-glyco- We successfully applied high-throughput functional sylation modifcations’ (PGMs) and each imparts a vari- metagenomic screening to identify enzymes that manip- ety of biological functions (See [13] for a review). ulate GlcNAc-6-SO4, a sulfated sugar found in some One major type of PGM involves enzymatic modifca- O-glycans and certain mammalian N-glycan outer arms. tion of glycans with sulfate. Sulfation has been observed We devised a novel coupled screening assay using a fuo- on all classes of glycans, including N- and O-glycans. Sul- rescent GlcNAc-6-SO4 analog and an exogenous hexosa- fation occurs in the Golgi where a sulfate group is trans- minidase. In this assay the coupling enzyme can act upon ferred from 3′-phosphoadenosine-5′-phosphosulfate the substrate and release the fuorophore only if the sul- (PAPS) to specifc positions on sugars by sulfotrans- fate group was frst hydrolyzed by an enzyme expressed ferases. Sulfation of N-acetylglucosamine (GlcNAc- from a metagenomic clone. Our screen identifed a sul- 6-SO4), N-acetylgalactosamine (GalNAc-6-SO4 and fatase and hexosaminidase that each act exclusively upon GalNAc-4-SO4), galactose (Gal-3-SO4 and Gal-6-SO4) GlcNAc-6-SO4. We further employed xCGE-LIF-based and mannose (Man-6-SO4) has been observed in N- and glycoanalysis to evaluate these enzymes activity on N-gly- O-glycans [14–20] (Fig. 1). Sulfation of N- and O-glycans can substrates and showed their suitability for structural has been implicated in many biological processes. Sul- characterization and enrichment of N-glycans bearing fated N-glycans are prevalent on both infuenza virus A terminal GlcNAc-6-SO4. neuraminidase and hemagglutinin surface proteins and likely play a role in virus replication and virulence [14, Results Functional screening for sulfatases 21]. Additionally, a GlcNAc-6-SO4 α2-6-sialyl LacNAc N-glycan epitope on B lymphocytes is a preferred ligand A fosmid library containing large DNA inserts (~ 40 kb) for the CD22 receptor, indicating a role in immunoregu- from human gut microbiota was created in E. coli. lation [13]. Recently, sulfated N-glycans present on serum Te library diversity was assessed by analyzing cloned immunoglobulin G were proposed as potential biomark- fragments from 24 randomly picked transformants. ers for rheumatoid arthritis [22]. Sequencing