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Glycosite mapping and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

ABSTRACT: Mucin-domain glycoproteins are densely O-glycosylated and play key 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 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 dynamic (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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Alexandra Steigmeyer

LAB HEAD: Stacy A. Malaker

PROVIDER: PXD039583 | Pride | 2023-09-27

REPOSITORIES: Pride

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			Action	DRS
	220523_TML_TIM4RD_SmE_ETD.raw	Raw
	220523_TML_TIM4RD_SmE_EThcD.raw	Raw
	220523_TML_TIM4_SmE_ETD.raw	Raw
	220523_TML_TIM4_SmE_EThcD.raw	Raw
	220615_ADS_TIM1_IMPa_ETD.raw	Raw

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