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

Taleb, Víctor; Liao, Qinghua; Narimatsu, Yoshiki; García‐García, Ana; Compañón, Ismael; Borges, Rafael J.; González-Ramírez, Andrés Manuel; Corzana, Francisco; Clausen, Henrik; Rovira, Carme; Hurtado‐Guerrero, R.

doi:10.1038/s41467-022-32021-9

Cited by 27 publications

(38 citation statements)

References 61 publications

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“…[17][18][19][20][21][22] An exciting development in the glycoproteomics field has been the emergence of O-glycoproteases, which are endoproteases requiring a combination of glycan and amino acid sequence characteristics to cleave the peptide backbone of O-glycoproteins. 23 Specific examples include: secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli; [24][25][26] O-endoprotease OgpA (commercially available as OpeRATOR) and M60-like proteases AM0627, AM0908, and AM1514 from Akkermansia mucinophila; [27][28][29][30][31][32][33] zinc-metalloendopeptidase CpaA from several Acinetobacter strains; 34 BT4244 from Bacteroides thetaiotaomicron; 31,32,35 zinc metalloproteinase C (ZmpC) from Streptococcus pneumoniae; 31,36 SmEnhancin from Serratia marcescens; 37,38 and immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. 35,39,40 These enzymes have been adapted as a means to selectively deplete specific classes of O-glycoproteins (e.g., mucin-domain glycoproteins) from live cell populations, in addition to being used in catalytically inactive forms for imaging and enrichment purposes.…”

Section: Introductionmentioning

confidence: 99%

Deciphering O-glycoprotease substrate preferences with O-Pair Search

Riley

Bertozzi

2022

Mol. Omics

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Section: Introductionmentioning

confidence: 99%

Deciphering O-glycoprotease substrate preferences with O-Pair Search

Riley

Bertozzi

2022

Mol. Omics

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“…The structure of SmE, on the other hand, has not yet been elucidated. As such, we aligned structures of all characterized enzymes with a PF13402 catalytic domain, [49][50][51][52] including the SmE structure recently predicted by AlphaFold (Figure S11). 53,54 We then docked a TIM-4-based bisglycosylated peptide into the predicted SmE structure to provide insight into potential substrate recognition.…”

Section: Molecular Modeling Helps Rationalize Different Substrate Sel...mentioning

confidence: 99%

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Chongsaritsinsuk

Steigmeyer

Mahoney

et al. 2023

Preprint

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Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

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“…Mucinases, a type of O-glycoprotease that depends on neighboring O-glycans for activity, play a fundamental role in degrading mucins as an important step for infectivity 11,12 or as a nutrient source. 3 Therefore, mucin-type O-glycans can function by acting as a shield and nutrient source for pathogenic microorganisms and commensals/symbionts, respectively, 13 and have additional roles in protecting proteins from proteolytic cleavage 1,14,15 and mediating binding of protein receptors toward their protein ligands. Due to these functions, mucintype O-glycans may conceivably interact with almost all physiological processes.…”

Section: ■ Introductionmentioning

confidence: 99%

Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation

et al. 2023

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Conspectus N-Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided ...

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

Cited by 27 publications

References 61 publications

Deciphering O-glycoprotease substrate preferences with O-Pair Search

Deciphering O-glycoprotease substrate preferences with O-Pair Search

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation

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