Deciphering O-glycoprotease substrate preferences with O-Pair Search

Abstract

O-glycoproteases are an emerging class of enzymes that selectively digest glycoproteins at positions decorated with specific O-linked glycans. O-glycoprotease substrates range from any O-glycoprotein (albeit with specific O-glycan modifications) to only glycoproteins harboring specific O-glycosylated sequence motifs, such as those found in mucin domains. Their utility for multiple glycoproteomic applications is driving the search to both discover new O-glycoproteases and to understand how structural features of characterized O-glycoproteases influence their substrate specificities. One challenge of defining O-glycoprotease specificity restraints is the need to characterize O-glycopeptides with site-specific analysis of O-glycosites. Here, we demonstrate how O-Pair Search, a recently developed O-glycopeptide-centric identification platform that enables rapid searches and confident O-glycosite localization, can be used to determine substrate specificities of various O-glycoproteases de novo from LC-MS/MS data of O-glycopeptides. Using secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli and O-endoprotease OgpA from Akkermansia mucinophila, we explore numerous settings that effect O-glycopeptide identification and show how non-specific and semi-tryptic searches of O-glycopeptide data can produce candidate cleavage motifs that can be used to define new protease cleavage settings that lower search times and improve O-glycopeptide identifications. We use this platform to generate a consensus motif for the recently characterized immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa and show that IMPa is a favorable O-glycoprotease for characterizing densely O-glycosylated mucin-domain glycoproteins.

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

“…To compare the activity of SmE in context with widely used, commercially available O-glycoproteases, we decided to benchmark against both OgpA and ImpA. 21,24,38,39 OgpA was originally identified in Akkermansia muciniphila, a commensal bacterium known to regulate mucin barriers through controlled degradation. 40 As mentioned above, OgpA is reported to cleave N-terminally to O-glycosylated Ser or Thr residues, with highest affinity towards asialylated core 1 species.…”

“…For example, reduced efficiency was seen when P1 was occupied by Arg or Ile, and cleavage was not observed when occupied by Asp. 24,39 To directly compare the activities of OgpA, ImpA, and SmE, mucin-domain glycoproteins TIM-1, -3, -4, GP1bα, and C1-Inh were digested in the presence and absence of sialidase, followed by gel electrophoresis (Figure S5), MS analysis, and manual glycopeptide validation (see SI Tables 1-5 for all annotated glycopeptides).…”