Features of N-Glycosylation of Immunoglobulins from Knockout Pig Models

Buist, Marjorie; Komatsu, Emy; López, Paúl; Girard, Lauren; Bodnar, Edward; Salama, Apolline; Sachs, David H.; Galli, Cesare; Perota, Andrea; Conchon, Sophie; Judor, Jean Paul; Concordet, Jean‐Paul; Lazzari, Giovanna; Soulillou, Jean Paul; Perreault, Hélène

doi:10.4172/2155-9872.1000333

Cited by 5 publications

(21 citation statements)

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“…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.…”

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…The two groups of antibody fragments of primary interest are the antigen-binding fragments (Fab) and class-defining crystallizable fragments (Fc). The hinge region of immunoglobulins (IgGs) is readily accessible to proteolytic attack by enzymes [9,10], and cleavage at that point produces F(ab') 2 or Fab fragments and the Fc fragment. Papain is a nonspecific thiol-endopeptidase and has a sulfhydryl group in its active site, which must be reduced for activity.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Whole and Fragmented Wild-Type Porcine IgG

Nelson¹,

Bacala²,

Gigolyk³

et al. 2019

Recent Advances in Analytical Chemistry

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Glycoproteomic analyses of tryptic (glyco)peptides from wild-type (WT) porcine IgG were performed. In a first protocol, intact antibody was digested with trypsin, followed by glycopeptide enrichment and liquid chromatographytandem MS (HPLC-MS/MS). This procedure allowed to detect N-glycopeptides observed previously (Lopez, P. G. et al., Glycoconj. J. 2016, 33 (1), 79), plus other non-reported N-glycopeptides. The method provided useful information but did not allow to discern between Fab (antigen-binding region) and Fc (constant region, fragment crystallizable) peptides/glycopeptides. In a second scheme, glycoproteomic analysis was attempted for Fab and Fc fragments obtained by papain and Fabulous™ hydrolysis. Usually employed for milligram amounts of antibodies, the papain and Fabulous™ protocols were adapted to 200 μg of WT IgG. Fab and Fc fragments were separated by size-exclusion (SEC) HPLC. Fractions collected were reanalyzed by gel electrophoresis (SDS-PAGE). Bands were excised, and fragments digested in-gel, followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and HPLC/MS-MS. In the protocol no glycopeptide enrichment was involved, that is, whole tryptic digests were analyzed. Fc N-glycopeptides were identified, and greater numbers of non-glycosylated peptides were tabulated. Very few peptides overlapped between Fc and Fab, as most peptides were clearly from Fc or Fab. HPLC-MS/MS detected more sialylated glycoforms than MALDI-TOF-MS. Sections of Fab and Fc were assigned de novo, through a database search or manually.

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Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of Whole and Fragmented Wild-Type Porcine IgG

Nelson¹,

Bacala²,

Gigolyk³

et al. 2019

Recent Advances in Analytical Chemistry

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Rousse¹,

Salama²,

Ben-Arye

et al. 2019

Eur J Clin Investigation

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Antibodies of non‐human mammals are glycosylated with carbohydrate antigens, such as galactose‐α‐1‐3‐galactose (α‐Gal) and N‐glycolylneuraminic acid (Neu5Gc). These non‐human carbohydrate antigens are highly immunogenic in humans due to loss‐of‐function mutations of the key genes involved in their synthesis. Such immunogenic carbohydrates are expressed on therapeutic polyclonal rabbit anti‐human T‐cell IgGs (anti‐thymocyte globulin; ATG), the most popular induction treatment in allograft recipients. To decipher the quantitative and qualitative response against these antigens in immunosuppressed patients, particularly against Neu5Gc, which may induce endothelial inflammation in both the graft and the host. We report a prospective study of the antibody response against α‐Gal and Neu5Gc‐containing glycans following rabbit ATG induction compared to controls. We show a drop in the overall levels of anti‐Neu5Gc antibodies at 6 and 12 months post‐graft compared to the pre‐existing levels due to the major early immunosuppression. However, in contrast, in a cross‐sectional study there was a highly significant increase in anti‐Neu5Gc IgGs levels at 6 months post‐graft in the ATG‐treated compared to non‐treated patients(P = 0.007), with a clear hierarchy favouring anti‐Neu5Gc over anti‐Gal response. A sialoglycan microarray analysis revealed that the increased anti‐Neu5Gc IgG response was still highly diverse against multiple different Neu5Gc‐containing glycans. Furthermore, some of the ATG‐treated patients developed a shift in their anti‐Neu5Gc IgG repertoire compared with the baseline, recognizing different patterns of Neu5Gc‐glycans. In contrast to Gal, Neu5Gc epitopes remain antigenic in severely immunosuppressed patients, who also develop an anti‐Neu5Gc repertoire shift. The clinical implications of these observations are discussed.

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Liquid chromatography–tandem mass spectrometry glycoproteomic study of porcine IgG and detection of subtypes

Battellino

Bacala

Gigolyk

et al. 2021

Rapid Comm Mass Spectrometry

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Rationale While high‐throughput proteomic methods have been widely applied to monoclonal antibodies and human immunoglobulin gamma (IgG) samples, less information is available on porcine IgG. As pigs are considered one of the most suitable species for xenotransplantation, it is important to characterize IgG amino acid sequences and glycosylation profiles, which is the focus of this study. Methods Three different purified porcine IgG samples, including wild‐type and knockout species, were digested with trypsin and enriched for glycopeptides. Digestion mixtures were spiked with a mixture of six standard peptides. Analysis was performed using electrospray ionization liquid chromatography–tandem mass spectrometry (MS/MS) in standard MS/MS data‐dependent acquisition mode on a hybrid triple quadrupole time‐of‐flight mass spectrometer. Results To facilitate the classification of subtypes detected experimentally, UniprotKB database entries were organized using comparative alignment scores. Sequences were grouped based on 11 different subtypes as translated from GenBank entries. Proteomic searches were accomplished automatically using specialized software, whereas glycoprotein searches were performed manually by monitoring the extracted chromatograms of diagnostic MS/MS glycan fragments and studying their corresponding mass spectra; 40–50 non‐glycosylated peptides and 4–5 glycosylated peptides were detected in each sample, with several glycoforms per sequence. Conclusions Proteomic analysis of porcine IgG is complicated by factors such as the presence of several subtypes, redundant heavy chain (HC) sequences in protein databases, and the lack of consistent cross‐referencing between databases. Aligning and comparing HC sequences were necessary to eliminate redundancy. This study highlights the complexity of pig IgG and shows the importance of MS in proteomics and glycoproteomics.

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Features of N-Glycosylation of Immunoglobulins from Knockout Pig Models

Cited by 5 publications

References 0 publications

Characterization of Whole and Fragmented Wild-Type Porcine IgG

Characterization of Whole and Fragmented Wild-Type Porcine IgG

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

Liquid chromatography–tandem mass spectrometry glycoproteomic study of porcine IgG and detection of subtypes

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