title: 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.
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Lopez
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada.
authors.forename: Lauren
authors.initials: L
authors.lastname: Girard
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Buist
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada.
authors.forename: Andrey Giovanni Gomes
authors.initials: AG
authors.lastname: de Oliveira
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada.
authors.forename: Edward
authors.initials: E
authors.lastname: Bodnar
authors.affiliation: INSERM UMR 10-64, Institut de Transplantation Urology Nephrology (ITUN), Université de Nantes, Nantes, France.
authors.forename: Apolline
authors.initials: A
authors.lastname: Salama
authors.affiliation: INSERM UMR 10-64, Institut de Transplantation Urology Nephrology (ITUN), Université de Nantes, Nantes, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Soulillou
authors.affiliation: Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T 2N2, Canada. Helene.Perreault@umanitoba.ca.
authors.forename: Hélène
authors.identifier: http://orcid.org/0000-0003-4665-6120
authors.initials: H
authors.lastname: Perreault
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009438:Neuraminic Acids / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013552:Swine
chemical_list: D007074:Immunoglobulin G; D009438:Neuraminic Acids; C032592:N-glycolylneuraminic acid
keywords: Glycosylation; Immunoglobulin; MALDI-TOF-MS; N-Glycolylneuraminic acid; Pig; Xenoantigen