Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

Léa Chuzel,Mehul B Ganatra,Christopher H Taron,Erdmann Rapp,Madison L Boisvert,Udo Reichl,René Hennig,Samantha L Fossa,Samanta Cajic

doi:10.1186/s12934-021-01652-w

Abstract

BackgroundSulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific 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 fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans.ResultsOur screen identified a sugar-specific sulfatase that specifically 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 glycan 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.ConclusionThe present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

Highlights

Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation
Functional screening for sulfatases A fosmid library containing large DNA inserts (~ 40 kb) from human gut microbiota was created in E. coli
We showed that F1-ORF13 is a calcium-dependent sulfatase that exclusively removes sulfate from carbon-6 of GlcNAc

Summary

Introduction

Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. There are over 200 glycosyltransferases encoded in the human genome and expression of many varies by cell type, tissue type or in response to environmental factors This results in substantial glycan structural heterogeneity. Over 140 different N-glycan structures have been identified on glycoproteins present in human serum [12]. Adding to this complexity, glycans can be modified with sulfate, phosphate, methyl or acetyl groups on certain sugars. Glycans can be modified with sulfate, phosphate, methyl or acetyl groups on certain sugars These groups are collectively termed ‘post-glycosylation modifications’ (PGMs) and each imparts a variety of biological functions (See [13] for a review)

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