The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.
Mucin domains are densely O-glycosylated modular protein domains which might be present in all kinds of cell floor and secreted proteins. Mucin-domain glycoproteins are recognized to be key gamers in a number of human ailments, particularly most cancers, whereby mucin expression and glycosylation patterns are altered.
Mucin biology has been tough to check on the molecular stage, partially, as a result of strategies to govern and structurally characterize mucin domains are missing. Here, we exhibit that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE’s distinctive properties to enhance sequence protection, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry.
We additionally discovered that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from sufferers with ovarian most cancers.
Finally, utilizing StcE, we found that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as organic ligands, whereas the associated receptor Siglec-9 doesn’t. Mucin-selective proteolysis, as exemplified by StcE, is due to this fact a robust instrument for the examine of mucin area construction and operate.
Mutations within the uridine diphosphate glucosyltransferase 76G1 gene end in completely different contents of the foremost steviol glycosides in Stevia rebaudiana.
In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was proven to catalyze a minimum of eight completely different glucosylation steps, together with the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this examine, the buildup of steviolbioside, Reb B, stevioside (ST) and Reb A in additional than 140 samples of stevia leaves collected from completely different areas in China had been analyzed by high-performance liquid chromatography (HPLC), and 5 genotypes, ‘N01-N05’, with considerably completely different ranges of the abovementioned glycosides had been found.
Mutations within the UGT76G1 gene cloned from cDNAs from these 5 genotypes had been recognized, and the features of the recombinant UGT76G1 variants had been ascertained by including steviolbioside and ST substrates. In addition, homology modeling and molecular docking had been used to elucidate the functional variations between variants and UGT76G1.
Comparing the sequences of the 5 variants ‘N01-N05’ with UGT76G1 (AY345974.1) revealed that base substitutions weren’t noticed in ‘N01’. By distinction, ‘N02’ exhibited 9 single nucleotide polymorphisms (SNPs) and 9 related amino acid substitutions or insertions with notable variations within the protein construction; nonetheless, an enzyme assay confirmed comparable functionalities between the variant and UGT76G1.
In ‘N03’, 49 SNPs and 29 related amino acid substitutions or insertions had been recognized and proven to induce important variations within the protein construction, particularly within the binding pocket, ensuing within the lack of performance of this variant within the enzyme assay.
These outcomes had been in settlement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in ‘N04’ and an insertion of a 68 base fragment in ‘N05’ had been discovered, and each produced a untimely protein with none catalytic exercise. Therefore, UGT76G1, which is important to the content material of primary steviol glycosides, must be a key gene marker for the molecular breeding of Stevia rebaudiana.
Our investigations additionally revealed the placement and orientation of energetic teams of the receptors and donors within the UGT76G1 enzyme, which play key roles in determining whether or not the enzyme has any enzymatic exercise.