Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine

Abstract

The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.

Characterization of Whole and Fragmented Wild-Type Porcine IgG

“…As the tryptic digestion products of whole wild-type porcine IgG antibodies have been characterized by MALDI-MS [2,3] and ESI-MS and MS/MS (new results presented in this report), data from these different workflows serve as comparative benchmarks between intact and fragmented IgG samples. Overlaps and differences allow to identify peptides and glycopeptides as originating from either the Fc or Fab portions, and database searches [22] can verify if these sequences are already available in the literature or need to be determined de novo.…”

“…There have been reports on the mass spectrometric (MS) analysis of pig immunoglobulins (IgG) in relationship with use in a xenotransplantation context [1][2][3][4]. These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2,3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies.…”

“…There have been reports on the mass spectrometric (MS) analysis of pig immunoglobulins (IgG) in relationship with use in a xenotransplantation context [1][2][3][4]. These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2,3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies. Glycoproteomic workflows resulted in the identification of many peptides that could be matched with the conserved gamma portion of the heavy chains of some of the 11 subtypes of pig IgG [5], including N-glycopeptides EEQFNSTYR and EAQFNSTYR [3].…”

“…These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2,3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies. Glycoproteomic workflows resulted in the identification of many peptides that could be matched with the conserved gamma portion of the heavy chains of some of the 11 subtypes of pig IgG [5], including N-glycopeptides EEQFNSTYR and EAQFNSTYR [3]. No specific information was given on the variable portions of neither Fab nor Fc components, as most of such assignments would have had to be attributed de novo.…”

Quantitative and qualitative changes in anti‐Neu5Gc antibody response following rabbit anti‐thymocyte IgG induction in kidney allograft recipients

“…12,[16][17][18] Non-immunosuppressed patients with severe burns treated by engineered pig skin dressings develop a long-lasting increase in their anti-Neu5Gc antibody levels. 14 Similarly, an intravenous (IV) injection of rabbit anti-human thymocyte IgGs, which display α-Gal and Neu5Gc carbohydrate antigens detectable by mass spectrometry analysis, 19,20 also induce a vigorous immune response against α-Gal and Neu5Gc antigens in non-immunosuppressed patients causing serum sickness in almost all cases. 21,22 In contrast, kidney recipients receiving ATG as an induction treatment in association with a cocktail of modern immunosuppressive agents develop a late anti-Neu5Gc response, and this response is stronger in patients who develop an early serum sickness disease (SSD).…”