Abstract
A wide spectrum of (autoimmune) diseases such as rheumatoid arthritis (RA), Crohn’s disease, systemic lupus erythematosus, but also other pathologies such as alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and hallmarked by a reduced level of serum Immunoglobulin G (IgG) galactosylation. IgG undergalactosylation is a promising biomarker to assess the severity of disease, monitor therapy efficacy and adjust therapy accordingly. The main hurdle for clinical implementation is the necessity for purifying the antibodies in order to specifically determine IgG Fc glycan galactosylation. We addressed this issue by using endo-β-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) and optimized the reaction conditions in which the enzyme specifically releases the IgG Fc N-glycans in serum. The sample preparation takes two hours and only entails the addition of endoS to serum and subsequent labeling with the fluorophore 8-aminopyrene-1,3,6-trisulphonic acid (APTS). Samples are then readily analyzed on high-throughput DNA sequencers, which are able to analyze up to 96 samples in one hour. We demonstrate in two separate patient cohorts that the degree of IgG undergalactosylation from our assay correlates very well with undergalactosylation scores calculated from N-glycan profiles derived from antibodies that have been purified from serum and deglycosylated with peptide N-glycosidase from Flavobacterium meningosepticum (PNGaseF). The presented new assay thus allows to directly and specifically measure the degree of IgG galactosylation in serum without the requirement of isolating the antibodies, and should help in advancing this biomarker towards clinical implementation.
- Abbreviations
- APTS
- 8-Aminopyrene-1,3,6-trisulfonic acid
- CE
- Capillary electrophoresis
- CE-LIF
- Capillary electrophoresis – laser-induced fluorescence
- CI
- confidence interval
- CRP
- C-reactive protein
- CV
- column volume
- EndoS
- endo-β-N-acetyl-glucosaminidase S from Streptococcus pyogenes = Endoglycosidase S
- ESR
- erythrocyte sedimentation rate
- GlcNAc
- N-acetylglucosamine
- GNT
- GlycoNASHTest
- HBV
- Hepatitis B virus
- IgG
- immunoglobulin G
- K18F
- caspase cleaved cytokeratin-18 fragments
- MS
- mass spectrometry
- NAFLD
- non-alcoholic fatty liver disease
- NAS
- NAFLD activity score
- NASH
- non-alcoholic steatohepatitis
- PNGaseF
- Peptide N-glycosidase from Elizabethkingia meningoseptica (previously Flavobacterium meningosepticum)
- RA
- rheumatoid arthritis
- UGS
- UnderGalactosylation Score
- UPLC
- ultra-performance liquid chromatography
Footnotes
Email addresses Dieter Vanderschaeghe: dieterv123{at}gmail.com, Leander Meuris: Leander.Meuris{at}ugent.vib.be, Hendrik Grootaert: Hendrik.Grootaert{at}ugent.vib.be, Tom Raes: tomraes.tr{at}gmail.com, Annelies Van Hecke: Annelies.Vanhecke{at}ugent.vib.be, Xavier Verhelst: Xavier.Verhelst{at}UGent.be, Frederique Van de Velde: Frederique.Vandevelde{at}UGent.be, Bruno Lapauw: Bruno.Lapauw{at}UZGent.be, Hans Van Vlierberghe: Hans.VanVlierberghe{at}UGent.be, Nico Callewaert: Nico.Callewaert{at}ugent.vib.be
Contact information Correspondence should be addressed to: Nico Callewaert, Unit for Medical Biotechnology, VIB-UGhent University Center for Medical Biotechnology, Technologiepark 927, B-9052 Gent-Zwijnaarde, Tel. +32 9 331 36 30, Fax. +32 9 331 36 09, e-mail: Nico.Callewaert{at}ugent.vib.be