Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

Woods, Robert J.; Wu, Shengcheng; Meng, Lu; Gerner‐Smidt, Christian; Sanders, Matthew J.; Ben‐Ayre, Shani Leviatan; Padler‐Karavani, Vered; Yang, Loretta

doi:10.1096/fasebj.2019.33.1_supplement.801.2

Cited by 2 publications

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“…We reasoned that the catalytically inactive point mutant of StcE (StcE E447D ) could function as a universal mucin enrichment tool for mucin domain discovery, similar to how inactive O-glycosidases and engineered sialidases can enrich broadly for O-glycosylated and sialylated glycoproteins, respectively 44 , 45 . Here we show that StcE E447D -conjugated beads selectively enrich mucin-domain glycoproteins from complex cancer cell lysates and from crude ovarian cancer patient ascites fluid.…”

Section: Introductionmentioning

confidence: 99%

Revealing the human mucinome

et al. 2022

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Mucin domains are densely O-glycosylated modular protein domains found in various extracellular and transmembrane proteins. Mucin-domain glycoproteins play important roles in many human diseases, such as cancer and cystic fibrosis, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial O-glycoprotease, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucin-domain glycoproteins. In this work, we leverage inactive StcE to selectively enrich and identify mucin-domain glycoproteins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy is further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitates detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer from in vitro and ex vivo sources.

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Section: Introductionmentioning

confidence: 99%

Revealing the human mucinome

et al. 2022

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Chemoenzymatic synthesis of human natural killer-1-containing glycans and application as serum antibodies probes

Bunyatov,

Wolfert,

Liu

et al. 2023

Nat. Synth

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Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is an autoimmune disease in which IgM autoantibodies target glycoconjugates of peripheral nerves resulting in progressive demyelination. To examine fine specificities of serum IgM autoantibodies and develop a more robust platform for diagnosis and disease monitoring, we describe here a chemoenzymatic approach that readily provided a panel of HNK-1 containing oligosaccharides presented on type 2 oligo-N-acetyl lactosamine (LacNAc) chains typical of glycosphingolipids. The compounds were prepared by a chemoenzymatic strategy in which an oligo-LacNAc structure was assembled enzymatically and then subjected to protecting group manipulation to chemically install a 3-O-sulfate glucuronic acid moiety. The synthetic strategy is highly divergent and made it possible to prepare from key precursors, additional compounds lacking sulfate of HNK-1 and derivatives in which the HNK-1 epitope is replaced by sulfate or sialic acid. The oligosaccharides were printed as a microarray to examine binding specificities of several monoclonal antibodies and serum antibodies of anti-MAG neuropathy patients.Surprisingly, three distinct patient subgroups were identified with variable dependance on the length of the LacNAc chain and sulfation of the glucuronyl moiety. In most cases, a lactoneohexaose backbone was required for binding indicating the antibodies target corresponding glycosphingolipids.Significance statement: A chemoenzymatic strategy is introduced in which a glycan backbone is assembled by glycosyltransferases to give a core oligosaccharide that is subjected to protecting group manipulations and chemical glycosylations to install terminal epitopes. It addresses limitations of enzymatic synthesis when specific glycosyltransferases or glycanmodifying enzymes for terminal epitope synthesis are not readily available. It provided an unprecedented panel of HNK-1 containing oligosaccharides, which was used to develop a glycan microarray that uncovered distinct binding preferences of serum antibodies of anti-MAG patients. The clinical spectrum of IgM monoclonal gammopathy varies substantially and an understanding of binding properties of IgM auto-antibodies will provide opportunities to monitor disease progression and develop personalized treatment options..

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Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

Cited by 2 publications

References 0 publications

Revealing the human mucinome

Revealing the human mucinome

Chemoenzymatic synthesis of human natural killer-1-containing glycans and application as serum antibodies probes

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