N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation

Abstract

<jats:p>Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific <jats:italic>N</jats:italic>- and <jats:italic>O</jats:italic>-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively <jats:italic>O</jats:italic>-glycosylated N-terminal region. Five novel and five known <jats:italic>O</jats:italic>-glycosylation sites were identified, carrying mainly core1-type <jats:italic>O</jats:italic>-glycans. In addition, we detected a heavily <jats:italic>O</jats:italic>-glycosylated portion spanning from Thr<jats:sub>82</jats:sub>-Ser<jats:sub>121</jats:sub> with up to 16 <jats:italic>O</jats:italic>-glycans attached. Likewise, all known six <jats:italic>N</jats:italic>-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released <jats:italic>N</jats:italic>-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications.</jats:p>

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

“…plA1AT has three N-glycosylation sites [4], where natural A2G2S2 structures are not essential for biological activity but enhance in vivo half-life and in vitro protein stability [10,11]. plC1INH is thought to be one of the most heavily glycosylated plasma proteins and harbors ten O-linked and six N-linked glycan structures [12]. The six N-linked glycan moieties with A2G2S2 as predominant structure have been shown to be increase serum half-life and are reported to increase in vivo efficacy [13][14][15].…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Incomplete sialylation can cause mAb in vivo titers which are decreased by 50% compared to their fully sialylated counterpart 14 days after mAb injection (Naso, Tam, Scallon, & Raju, 2010). Unfavorable glycosylation is a major reason for the small number of approved biopharmaceuticals from yeast, plant, and insect platforms (Walsh, 2014 Stavenhagen et al, 2018;van Veen et al, 2012;Zhang, Luo, & Zhang, 2016). Furthermore, glycosylation is also particularly in the focus during the production of "biosimilar" biopharmaceuticals.…”

“…While other human-like PTMs, such as γ-carboxylation, phosphorylation and tyrosine sulfation can also be challenging during the production of therapeutic proteins, the most critical quality attribute is glycosylation, as indicated by the available documents from regulatory agencies across the world, for example the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA; EMEA, 2014a;2014b;FDA, 2012a;2012b;2012c;FDA, 2014). Many cell platforms were therefore engineered toward human-like glycosylation (Stavenhagen et al, 2018). For certain proteins, untypical N-glycosylation may also be beneficial, for example immunoglobulin G (IgG) with Man3-GlcNAc2 trimannosyl chitobiose units for patients with systemic lupus erythematosus (Lood et al, 2012).…”

Site-specific N- and O-glycosylation analysis of atacicept

“…22,23 This approach has been shown to be very useful for a variety of glycoproteins, in particular for O-glycosylation site identification. 18 Depending on the experimental conditions, most of the protein is trimmed, resulting in small peptides around the glycosylation site, which also allows detection of sites within regions that only have a few or no cleavage sites for standard proteases. A wide range of glycopeptides were detected using this approach ( Supplementary Figure 1).…”

“…16 Glycopeptidecentered approaches may be combined with released glycan analysis, in order to obtain more detailed glycan structural information. 17,18 The analysis of intact glycoproteins provides complementary information on overall glycoform distributions and relative abundances of the corresponding proteoforms for comprehensive characterization. 19,20 A comparison of glycan-, glycopeptide-and glycoprotein-based MS approaches has been recently shown for the analysis of IgG Fc-glycosylation.…”