Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

Konstantinidi, Andriana; Nason, Rebecca; Čaval, Tomislav; Sun, Lingbo; Sørensen, Daniel Madriz; Furukawa, Shoei; Ye, Zilu; Vincentelli, Renaud; Narimatsu, Yoshiki; Vakhrushev, Sergey Y.; Clausen, Henrik

doi:10.1016/j.jbc.2022.101784

Cited by 23 publications

(36 citation statements)

References 92 publications

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“…Note that the MUC1 TR contains two bis- O -glycosites and one isolated glycosite. We previously showed that all five O -glycosites are fully O -glycosylated when expressed in glycoengineered HEK293 cells and that BT4244 efficiently cleaves the Tn glycoform of this MUC1 reporter with predominant cleavage in between the bis- O -glycan at the VTSA and GSTA motifs 24 , 37 . We tested the wt glycoform of the MUC1 TR reporter (containing a mixture of mono and disialylated core 1 and core 2 structures), and three engineered more homogeneous glycoforms with mSTa, T, and Tn O -glycans.…”

Section: Resultsmentioning

confidence: 95%

“…This suggests that AM0627 and BT4244 should behave very similarly in terms of recognition towards bis- O -glycan and that BT4244 likely cleaves glycopeptides containing bis- O -glycans. In fact, it has been recently shown that BT4244 acts on glycopeptides with bis- O -glycans, in particular G S*T* A and V T*S* A motifs of the Tn-MUC1-TR reporter, while it is inactive in a single PD T* R O -glycosite 24 , 37 (S* or T* denotes a GalNAc-glycosylated Ser and Thr, respectively). With respect to the other mucinases, while ZmpB and ZmpC do not have an aromatic residue close to the sugars at the G subunits, IMPa contains two threonines (Thr775 and Tyr776) that will likely clash with both the GalNAc and Gal at the G1 and G2 subunits, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…1a, b ). Mucinases and O -glycoproteases appear to be promiscuous in terms of substrate peptide sequence specificity 4 , although refinement of substrate peptide sequences is still in progress 24 .…”

Section: Introductionmentioning

confidence: 99%

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Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

et al. 2022

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Mucinases of human gut bacteria cleave peptide bonds in mucins strictly depending on the presence of neighboring O-glycans. The Akkermansia muciniphila AM0627 mucinase cleaves specifically in between contiguous (bis) O-glycans of defined truncated structures, suggesting that this enzyme may recognize clustered O-glycan patches. Here, we report the structure and molecular mechanism of AM0627 in complex with a glycopeptide containing a bis-T (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycan, revealing that AM0627 recognizes both the sugar moieties and the peptide sequence. AM0627 exhibits preference for bis-T over bis-Tn (GalNAcα1-O-Ser/Thr) O-glycopeptide substrates, with the first GalNAc residue being essential for cleavage. AM0627 follows a mechanism relying on a nucleophilic water molecule and a catalytic base Glu residue. Structural comparison among mucinases identifies a conserved Tyr engaged in sugar-π interactions in both AM0627 and the Bacteroides thetaiotaomicron BT4244 mucinase as responsible for the common activity of these two mucinases with bis-T/Tn substrates. Our work illustrates how mucinases through tremendous flexibility adapt to the diversity in distribution and patterns of O-glycans on mucins.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

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Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

et al. 2022

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“…Mucin glycoproteins are attractive targets for this approach since their structures and biological activities are equally dependent on their protein and glycan constituents. We and others have characterized bacterial proteases with peptide-and glycan-dependent cleavage motifs that render them highly selective for densely O-glycosylated mucin domains [14][15][16][17][18] . Therefore, we chose mucinases, specifically the pan mucinase StcE, as an initial candidate for evaluation as a peptide-and glycan-selective mucin degrader.…”

Section: Discussionmentioning

confidence: 99%

“…We and others have characterized proteases from the bacterial kingdom with selectivity for mucin domains [14][15][16][17][18] . These "mucinases" act through recognition of joint peptide-and glycanmotifs, which have been mapped using mass spectrometry of cleavage products.…”

Section: Mucinase Treatment Undermines Mucin-driven Survival Pathways...mentioning

confidence: 99%

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

Pedram

Shon

Tender

et al. 2022

Preprint

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Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of substrates that can be selectively degraded, we designed degraders which are dependent on both peptide sequence and glycosylation status of the target protein. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in murine models of breast cancer progression. This work establishes a blueprint for the development of biologics which degrade specific glycoforms of cell surface proteins.

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Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage

Downs,

Curran,

Zaia

et al. 2023

Anal Bioanal Chem

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Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

Cited by 23 publications

References 92 publications

Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage

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