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. 2022 Oct 7;17(10):e0268592.
doi: 10.1371/journal.pone.0268592. eCollection 2022.

Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with CNS inflammation in children

Frederik Ricken  1   2 , Ahu Damla Can  1   2 , Steffen Gräber  2 , Martin Häusler  1 , Willi Jahnen-Dechent  2
Affiliations

Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with CNS inflammation in children

Frederik Ricken et al. PLoS One. .

Abstract

Fetuin-A is a liver derived plasma protein showing highest serum concentrations in utero, preterm infants, and neonates. Fetuin-A is also present in cerebrospinal fluid (CSF). The origin of CSF fetuin-A, blood-derived via the blood-CSF barrier or synthesized intrathecally, is presently unclear. Fetuin-A prevents ectopic calcification by stabilizing calcium and phosphate as colloidal calciprotein particles mediating their transport and clearance. Thus, fetuin-A plays a suppressive role in inflammation. Fetuin-A is a negative acute-phase protein under investigation as a biomarker for multiple sclerosis (MS). Here we studied the association of pediatric inflammatory CNS diseases with fetuin-A glycosylation and phosphorylation. Paired blood and CSF samples from 66 children were included in the study. Concentration measurements were performed using a commercial human fetuin-A/AHSG ELISA. Of 60 pairs, 23 pairs were analyzed by SDS-PAGE following glycosidase digestion with PNGase-F and Sialidase-AU. Phosphorylation was analyzed in 43 pairs by Phos-TagTM acrylamide electrophoresis following alkaline phosphatase digestion. Mean serum and CSF fetuin-A levels were 0.30 ± 0.06 mg/ml and 0.644 ± 0.55 μg/ml, respectively. This study showed that serum fetuin-A levels decreased in inflammation corroborating its role as a negative acute-phase protein. Blood-CSF barrier disruption was associated with elevated fetuin-A in CSF. A strong positive correlation was found between the CSF fetuin-A/serum fetuin-A quotient and the CSF albumin/serum albumin quotient, suggesting predominantly transport across the blood-CSF barrier rather than intrathecal fetuin-A synthesis. Sialidase digestion showed increased asialofetuin-A levels in serum and CSF samples from children with neuroinflammatory diseases. Desialylation enhanced hepatic fetuin-A clearance via the asialoglycoprotein receptor thus rapidly reducing serum levels during inflammation. Phosphorylation of fetuin-A was more abundant in serum samples than in CSF, suggesting that phosphorylation may regulate fetuin-A influx into the CNS. These results may help establish Fetuin-A as a potential biomarker for neuroinflammatory diseases.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Concentrations of fetuin-A in serum and CSF.
Fig 1 shows the results of the concentration measurements of fetuin-A in CSF and serum. The correlation of fetuin-A in serum (y-axis, mg/ml) with an elevated C-reactive protein concentration (x-axis, (A)) and the presence of neuroinflammatory disease (x-axis, (B)) is shown. The relation between fetuin-A in CSF (y-axis, μg/ml) and a disruption of the blood-CSF barrier (x-axis) is displayed in (C). (D) and (E) show the connection of the serum fetuin-A/total serum protein ratio (y-axis) with an intrathecal IgG synthesis (x-axis, (D)) and the disruption of the blood-CSF barrier (x-axis, (E)). In (F), the link between the CSF fetuin-A/total CSF protein ratio (y-axis) and age (x-axis, years) is shown as a scatter plot. (G) and (H) show the correlation between the CSF fetuin-A/serum fetuin-A quotient QFet (y-axis, x103) and the CSF albumin/serum albumin quotient QAlb (x-axis, x103, (G)) and the presence of a blood-CSF barrier disruption (x-axis, (H)). (G) represents a scatter blot double logarithmically. Values of the control group (black) and inflammatory group (white) are plotted separately. (H) shows a boxplot.
Fig 2
Fig 2. Glycosylation patterns of fetuin-A in CSF and serum.
Fig 2 illustrates the results of the glycosylation studies of fetuin-A. (A) and (B) show the glycosylation patterns of fetuin-A with (A) and without (B) double bands. On each membrane, lane 1 shows undigested fetuin-A, lane 2 shows fetuin-A after PNGase-F digestion and lane 3 shows fetuin-A after sialidase-Au digestion. (C) compares the occurrence of double bands in the control group and inflammatory group as a bar chart. (D) displays the kinetic of PNGase-F digestion in serum and CSF. Lane 1 shows undigested fetuin-A, lane 2 shows the pattern after 1 minute of digestion, lane 3 displays the pattern after 10 minutes of digestion and lane 4 shows fetuin-A after 3 hours of PNGase-F digestion.
Fig 3
Fig 3. Phosphorylation patterns of fetuin-A in CSF and serum.
Fig 3 displays the results of the statistical analysis of the phosphorylation studies. (A) and (B) show relative CSF phosphofetuin-A concentrations (y-axis, %) in comparison to the CSF albumin/serum albumin quotient (A, x-axis, x103) and in the presence of neuroinflammatory diseases (B). (C) and (D) show absolute phosphofetuin-A levels in CSF (y-axis, μg/ml) in comparison to the CSF albumin/serum albumin quotient ((C), x-axis, x103) and a blood-CSF barrier disruption ((D), x-axis). (E) and (F) display the results of the linear regression with the relative CSF phosphofetuin-A/relative serum phosphofetuin-A quotient. This quotient is shown on the y-axis. (E) shows a scatter plot with the CSF albumin/serum albumin quotient (x-axis, x103). In (E) the results are shown divided into control group (black) and inflammatory group (white). (F) shows the correlation of the CSF phosphofetuin-A/serum phosphofetuin-A quotient with the presence of a neuroinflammatory disorder (x-axis).
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Grants and funding

This study was supported by the German Research Foundation (DFG, TRR 219, Project ID 322900939 and Project ID 403041552, both awarded to WJ-D) and by the START program of the Faculty of Medicine of the RWTH Aachen University (grant number 129/14, awarded to MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.