Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y

<div><p>Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed <i>N-</i>glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput <i>N</i>-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases.</p></div

IgY <i>N</i>-glycan assignment.

<p>(A) HILIC UPLC profile of undigested <i>N</i>-glycans from serum IgY. Profiles are standardised against a dextran hydrolysate (GU). The HILIC chromatogram was separated into 40 peaks. (B) Unfractionated IgY profile was subjected to exoglycosidase digestions. (C) IgY glycans were separated according number of sialic acids on WAX HPLC and (D) each WAX fraction was then subjected to HILIC UPLC (Final IgY Structural assignments are listed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159859#pone.0159859.s003" target="_blank">S1 Table</a>).</p

Summary of <i>N-</i>glycans identified from IgY purified form avian serum.

<p>Summary of <i>N</i>-glycans released from IgY purified from serum. The HILIC chromatogram was separated into 40 peaks and structural assignment carried made using established methods (Royle <i>et al</i>., 2008) and the software tool GlycoBase (<a href="https://glycobase.nibrt.ie" target="_blank">https://glycobase.nibrt.ie</a>). Nomenclature used is according to Royle <i>et al</i>., 2008 and Harvey <i>et al</i>., 2009 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159859#pone.0159859.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159859#pone.0159859.ref025" target="_blank">25</a>]. Shown here are the most abundant glycans identified–glycans assigned to peaks with % area great than 5%- highlighted in grey is the most abundant glycan(s) within that particular peak. For full glycan assignment see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159859#pone.0159859.s003" target="_blank">S1 Table</a>. *Glycan nomenclature.</p

Comparison of the different properties of IgG and IgY.

<p>Comparison of the different properties of IgG and IgY.</p

IgY Purification.

<p>IgY purified from chicken serum was resolved on 12% SDS-PAGE gels and visualised by staining with InstantBlue (left). The resolved proteins were also transferred to nitrocellulose membranes and the presence of the heavy chains at approximately 65–68 kDa and the light chains at 25 kDa can be seen after probing with an anti-IgY H+L-HRP-tagged antibody (right). L: PageRuler Plus Prestained protein ladder.</p

Structures of Immunoglobulin’s G, E and Y and their <i>N</i>-glycosylation sites.

<p>IgG (left) is composed of 2 identical heavy chains that each comprise a variable domain (V_H) and three constant domains (C_γ1, C_γ2 and C_γ3) with a single carbohydrate site (purple star). In contrast, the additional constant domain in IgE (middle) is much more heavily glycosylated than IgG, with 7 <i>N-</i>glycosylation sites, however, one site (Asn 264) is unoccupied (yellow star) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159859#pone.0159859.ref007" target="_blank">7</a>]. IgY is also comprised of four constant domains per heavy chain (Cv1-Cv4), with two carbohydrate sites (right). The flexible hinge region found in IgG is absent in IgE and IgY and thus may restrict their flexibility in comparison to IgG.</p