Detection and Characterization of Phosphorylation, Glycosylation, and Fatty Acid Bound to Fetuin A in Human Blood.

Markéta Kovářová,Andreas Peter,Konstantinos Kantartzis,Hans-Ulrich Häring,Erwin Schleicher,Norbert Stefan,Andreas Birkenfeld,Triantafyllos Didangelos,Hubert Kalbacher

doi:10.3390/jcm10030411

Abstract

The hepatokine fetuin A (Fet A) has been associated with diverse pathological states such as insulin resistance, type 2 diabetes, macrovascular disease, and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern was determined by gas chromatography. Using the antibody, we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 h. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser312 phosphorylation was not associated with glycosylation. The phosphorylation pattern did not change during an oral glucose tolerance test (0–120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly-unsaturated fatty acids. Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A.

Highlights

In human adults, fetuin A (Fet A, synonymous with α2-Heremanns-Schmid- glycoprotein) is expressed and secreted into the circulation predominantly by the liver [1,2]
Since Fet A interacts with fatty acids to bind to toll-like receptor (TLR) 4 to trigger an inflammatory cellular response [28], we studied the pattern of fatty acids bound to Fet A
Based on previous results showing that only the phosphorylated but not unphosphorylated Fet A inhibits insulin signal transduction, we hypothesized that the serum or plasma pFet A may be the biological form of Fet A exerting its metabolic function

Summary

Introduction

Fetuin A (Fet A, synonymous with α2-Heremanns-Schmid- glycoprotein) is expressed and secreted into the circulation predominantly by the liver [1,2]. Present in high concentrations in fetal plasma, Fet A levels are rather low in healthy adults ranging from 250–600 μg/mL; reported levels depend to some extent on the assay [3]. Fet A is a ~64 kDa glycoprotein, which consists of an A-chain (282 aa), a B-chain (182 aa) and a connecting peptide (40 aa) harboring the major phosphorylation site [3,4]. Fet A contains two N-linked (Asp 138 and Asp 158) and two O-linked glycosylation sites (Thr 238 and Thr 252) further emphasizing the large heterogeneity of human fetuin A [6]. Numerous reports implicate Fet A in cell adhesion and tumor growth. A previous report has extensively reviewed the role of Fet A in tumor progression and metastasis [9]

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Results

Conclusion