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

Abstract

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.

Large-Scale and Site-Specific Mapping of the Murine Brain O-Glycoproteome with IMPa

“…We also noted that RAX was based on SAX-ERLIC separation mode, and thus, the charge or hydrophilicity of the glycopeptides might affect the retention of some O-glycopeptides, especially for the hydrophilicity since the glycopeptides were trapped in 95% ACN with 1% TFA where the column behaves in the HILIC mode. Comparison of enzymatic cleavages by OgpA and IMPa, and even with newer O-glycoproteases that have been recently discovered such as SmE, 34 on a large scale could also reveal the substrate preference for these O-glycoproteases. Yet, the limiting factor that may arise is the large protein amount required in our method.…”

“…Whether these features arise from exclusivity in terms of differences in methods of analysis, N - versus O -glycosylation pathways or some other biosynthetic uniqueness to these pathways dependent on the proteins or other factors is intriguing but remains to be explored. Of special interest, a comparison of enzymatic cleavages by OgpA and IMPa, and newer O -glycoproteases that have been recently discovered such as SmE, on a large scale would also reveal the substrate preference for these O -glycoproteases. The limiting factor that may arise is the large protein amount required in our method.…”

“…Hence, information on O -glycan sialylation and other features is lost. Several mucin O -glycoproteases have also been reported for O -glycoproteomics; − however, these mainly showed activities toward highly glycosylated mucin domain. , …”

False-Positive Glycopeptide Identification via In-FAIMS Fragmentation

“…This is caused by (a) the absence of positively charged amino acid (Arg, Lys) on the C-terminus, (b) large glycan structures, (c) sequences mostly composed of amino acids with low proton affinity (e.g., Pro, Thr, Ser), and/or (d) long stretches of amino acids that lack basic residues. To determine how these properties might affect FAIMS separation, recombinant podocalyxin was digested by using two different mucinases: SmE and BT4244, both of which cleave N-terminally to glycosylated residues. While SmE can accommodate a variety of glycan structures in both the P1 and P1′ positions, BT4244 has a strong preference for desialylated core 1 structures. , Therefore, the sample digested with BT4244 was co-treated with sialidase.…”

“…To determine how these properties might affect FAIMS separation, recombinant podocalyxin was digested by using two different mucinases: SmE and BT4244, both of which cleave N-terminally to glycosylated residues. While SmE can accommodate a variety of glycan structures in both the P1 and P1′ positions, BT4244 has a strong preference for desialylated core 1 structures. , Therefore, the sample digested with BT4244 was co-treated with sialidase. As SmE digestion produced a wider variety of peptides and resultant identifications, the BT4244 results are primarily located in the Supporting Information.…”