Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension

David Gibson, Sorcha Finnegan, Gwen Manning, Mark W. Duncan, Stephen Pennington, Terry Moore, Madeleine Rooney

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Background Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. Objectives We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy. Methods SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry. Results Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids. Conclusions The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.
LanguageEnglish
Title of host publicationAnnals of the Rheumatic Diseases
Volume71
DOIs
Publication statusPublished - 2013

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Vitamin D-Binding Protein
Juvenile Arthritis
Glycosylation
Synovial Fluid
Proteome
C-Reactive Protein
Mass Spectrometry
Proteins
Inflammation
Macrophage Activation
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
N-Acetylneuraminic Acid
Post Translational Protein Processing
Hydrophobic and Hydrophilic Interactions
Fluorescent Dyes
Immunoprecipitation
ROC Curve
Liquid Chromatography
Autoimmune Diseases
Arthritis

Cite this

Gibson, D., Finnegan, S., Manning, G., Duncan, M. W., Pennington, S., Moore, T., & Rooney, M. (2013). Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension. In Annals of the Rheumatic Diseases (Vol. 71) https://doi.org/10.1136/annrheumdis-2012-eular.2252
Gibson, David ; Finnegan, Sorcha ; Manning, Gwen ; Duncan, Mark W. ; Pennington, Stephen ; Moore, Terry ; Rooney, Madeleine. / Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension. Annals of the Rheumatic Diseases. Vol. 71 2013.
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abstract = "Background Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. Objectives We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy. Methods SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry. Results Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids. Conclusions The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.",
author = "David Gibson and Sorcha Finnegan and Gwen Manning and Duncan, {Mark W.} and Stephen Pennington and Terry Moore and Madeleine Rooney",
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Gibson, D, Finnegan, S, Manning, G, Duncan, MW, Pennington, S, Moore, T & Rooney, M 2013, Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension. in Annals of the Rheumatic Diseases. vol. 71. https://doi.org/10.1136/annrheumdis-2012-eular.2252

Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension. / Gibson, David; Finnegan, Sorcha; Manning, Gwen; Duncan, Mark W.; Pennington, Stephen ; Moore, Terry; Rooney, Madeleine.

Annals of the Rheumatic Diseases. Vol. 71 2013.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

TY - GEN

T1 - Glycosylation of vitamin D binding protein reduced in juvenile idiopathic arthritis patients at risk of disease extension

AU - Gibson, David

AU - Finnegan, Sorcha

AU - Manning, Gwen

AU - Duncan, Mark W.

AU - Pennington, Stephen

AU - Moore, Terry

AU - Rooney, Madeleine

PY - 2013

Y1 - 2013

N2 - Background Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. Objectives We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy. Methods SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry. Results Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids. Conclusions The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.

AB - Background Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. Objectives We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy. Methods SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry. Results Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids. Conclusions The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.

U2 - 10.1136/annrheumdis-2012-eular.2252

DO - 10.1136/annrheumdis-2012-eular.2252

M3 - Conference contribution

VL - 71

BT - Annals of the Rheumatic Diseases

ER -