MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS

Abstract

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Fc-Linked IgG N-Glycosylation in FcγR Knock-Out Mice

“…BALB/c and C57BL/6 mice are well-known to have polymorphic positions in IgG (Keane et al, 2011;Yalcin et al, 2011), which we proposed as candidates for IgG N-glycan regulation (Krištić et al, 2018). This might explain the different IgG N-glycosylation profiles of the two strains reported here, which were also observed in previous LC-MS studies of murine IgG N-glycosylation (de Haan et al, 2017;Zaytseva et al, 2018). Moreover, it is well-known that immune responses in BALB/c mice are more dominated by Th2 cytokines, whereas immune responses in C57BL/6 mice are FIGURE 4 | Comparison of fragment crystallizable (Fc)-linked immunoglobulin G (IgG) N-glycosylation in C57BL/6 and BALB/c mice.…”

“…In contrast to IgG1, IgG2a/c, and IgG2b, IgG3 interact only very weakly with known FcγRs but is the most effective isotype to activate complement Bruhns, 2012;Sörman et al, 2014). Interestingly, there are known differences between mouse strains in the protein sequences of IgG1 and IgG2a/c heavy chains that occur in the proximity of the Fc N-glycosylation site (Zhang et al, 2012;Maresch and Altmann, 2016;de Haan et al, 2017;Zaytseva et al, 2018), and it has been hypothesized that the amino acid sequence of the heavy chain can impact the Fc glycosylation profile (Lund et al, 1996;Krištić et al, 2018).…”

“…Together with the fact that the two glycan moieties attached to each Fc domain can vary due to asymmetric glycosylation, this results in several hundred possible different IgG Fc glycosylation variants (Shields et al, 2002;Shinkawa et al, 2003;Arnold et al, 2007;Kao et al, 2015;Quast et al, 2015). In addition, recent studies in mice and humans suggest that different IgG subclasses show subclass-specific glycosylation patterns (Kao et al, 2015;de Haan et al, 2017;Zaytseva et al, 2018). More importantly, for some of these IgG glycosylation variants, an altered IgG activity was noted due to a modulation of binding to FcγRs Kao et al, 2015).…”

Maternal asthma is associated with persistent changes in allergic offspring antibody glycosylation

“…Subclass‐specific mouse IgG N‐glycosylation analysis was performed as described previously . Briefly, IgG was captured from 15 µL of murine serum using protein G affinity beads in PBS.…”

“…Subclass-specific mouse IgG N-glycosylation analysis was performed as described previously. 25 Briefly, IgG was captured from 15 µL of murine serum using protein G affinity beads in PBS. Proteins interacted with the beads while being shaken for 1.5 hours, after which the beads were washed seven times with 1.5 mL PBS.…”