A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Abstract

Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

Deciphering O-glycoprotease substrate preferences with O-Pair Search

“…Figure 6c shows that the sequence motif does not change much, but that some sialylated core-1 O-glycans can be tolerated for cleavage by OgpA at P1'. That said, the number of identifications substantially decreased (vide infra), matching the reports of lower OgpA Finally, we sought to use our approach to generate the cleavage motif of immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa, which has been explored in several recent studies, 35,39,40 and to evaluate its performance in O-glycoproteomic experiments. Like OgpA, IMPa is known to cleave immediately N-terminal to O-glycosylated threonine and serine residues, but it does not have the intolerance for sialylated glycans like OgpA.…”

“…Recent work used synthetic peptides to investigate the importance residues at the P1 position of the substrate peptide for the IMPa's activity, which showed minimal influence from amino acids adjacent to the cleavage site despite the presence of proline-specific recognition domain that may target the protease to an O-glycosylated P-T/S motif. 40 This work relied on beamCID data only, though, limiting its ability to localize O-glycosites for protease motif generation. To add to these studies, we first used non-specific and semi-tryptic searches to generate putative cleavage motifs for IMPa (Supplemental Figure 5).…”

“…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 protease AM0627, AM0908, and AM1514 from Akkermansia mucinophila; [27][28][29][30][31][32][33] zinc-metallo-endopeptidase 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. 30,31,[41][42][43] Perhaps the most immediate utility for O-glycoproteases is their use in glycoproteomic GE ÄKTA Pure FPLC.…”

A previously uncharacterized O-glycopeptidase from Akkermansia muciniphila requires the Tn-antigen for cleavage of the peptide bond

“…It has quite broad specificity for both peptide sequence and glycan structure that is known to cleave immediately N-terminal to site of O-glycosylation (10,19). The product of CAT activity on the Tn antigen peptide displayed similar mobility to that of the IMPa thus also suggesting that CAT cleaved Nterminal to the site of O-glycosylation.…”

Deciphering O-glycoprotease substrate preferences with O-Pair Search

“…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 protease AM0627, AM0908, and AM1514 from Akkermansia mucinophila; [27][28][29][30][31][32][33] zinc-metallo-endopeptidase 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. 30,31,[41][42][43] Perhaps the most immediate utility for O-glycoproteases is their use in glycoproteomic GE ÄKTA Pure FPLC.…”