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.

中文翻译：

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一种广泛特异性的 O-糖蛋白酶，可改进对含有完整复合 O-聚糖的糖蛋白和糖肽的分析

分析与糖蛋白的丝氨酸/苏氨酸相关的粘蛋白型 O-聚糖在技术上具有挑战性，部分原因是缺乏有效的酶学工具来进行分析。最近，已经描述了几种 O-聚糖特异性内切蛋白酶，它们可以切割与附加聚糖相邻的蛋白质。尽管在理解这些酶的生物化学方面取得了重大进展，但已知的 O-糖蛋白酶具有特异性约束，例如对带有唾液酸化 O-聚糖的糖蛋白的切割效率低、对某些类型的糖蛋白或蛋白质序列偏倚具有高选择性，这限制了它们的分析应用。在这项研究中，我们检查了来自铜绿假单胞菌的免疫调节金属蛋白酶 (IMPa) 的能力. 使用一系列合成肽及其糖型研究肽底物序列选择性及其对 IMPa 活性的影响。我们表明 IMPa 没有特定的 P1 残基偏好，并且可以耐受 P1 位置的大多数氨基酸，天冬氨酸除外。该酶不会在两个相邻的 O-糖位点之间切割，表明 P1 位不允许 O-糖基化的丝氨酸/苏氨酸。O-糖位点两侧少至两个氨基酸的糖肽被 IMPa 特异性切割。最后，IMPa 有效地切割了带有不同复杂性的唾液酸化和唾液酸化 O-聚糖的肽和蛋白质。我们介绍了 IMPa 在一步 O-糖蛋白组学工作流程中的使用，用于对单个纯化的糖蛋白粒细胞集落刺激因子 (G-CSF) 和受体型酪氨酸蛋白磷酸酶 C (CD45) 进行糖谱分析，而无需糖肽富集。在这些示例中，IMPa 能够识别 O-糖位点和每个位点的复杂 O-聚糖结构范围。