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Biochem. J. (2001) 357 (437–445) (Printed in Great Britain)

Phosphorylation of human plasma a2-Heremans–Schmid glycoprotein (human fetuin) in vivo

Åsa C. HAGLUND*1, Bo EK† and Pia EK*

*Department of Medical Biochemistry and Microbiology, Biomedical Centre, University of Uppsala, Box 582, SE-751 23 Uppsala, Sweden, and †Department of Plant Biology, The Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden

A fraction of a₂-Heremans–Schmid (a₂-HS) glycoprotein (human fetuin) isolated from plasma was phosphorylated at serine-120 and serine-312 as shown by MS and peptide fragment sequencing after tryptic digestion. Serine-312-containing peptides were phosphorylated to 77% as determined from relative peak heights in the mass spectrum, which together with the phosphorylation of serine-120 implies a molar degree of phosphorylation of at least 1. Approximately 20% of the circulating fetuin plasma pool was phosphorylated to approx. 1mol of phosphate/mol of protein. The remainder did not contain phosphate, resulting in an average phosphorylation degree for the protein in plasma of approx. 0.2 mol/mol. The isolated a₂-HS glycoprotein was a heterodimer in which the entire C-terminal part of the connecting peptide including threonine-321 was present, but traces of C-terminally trimmed connecting peptide fragments were also found. The short B-chain was O-glycosylated to approx. 40%, whereas the N-glycosylation of asparagine-138 and asparagine-158 seemed to be 100%. This finding, for the first time, that circulating human plasma fetuin is partly phosphorylated, implies that the effects of phosphorylated a₂-HS glycoprotein on insulin signal transduction seen in different cell systems could be relevant to its physiological function in vivo.

Key words: glycosylation, MS, phosphate analysis, phosphopeptide analysis.

Abbreviations used: 7-AHA, 7-aminoheptanoic acid, AHSG, a₂-Heremans–Schmid glycoprotein; Ga(III)-IDA, gallium(III)-iminodiacetic acid.

¹To whom correspondence should be addressed (e-mail asa.haglund@imbim.uu.se).

Received 13 November 2000/26 February 2001; accepted 12 April 2001

The Biochemical Society, London © 2001

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