Proteolytic processing by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin-A, the major globulin of fetal calf serum.
Bovine fetuin-A is a member of a glycoprotein family with a wide spectrum of functions. Until now the bovine protein has been thought to be a single-chain protein. Recently we have shown that native bovine plasma fetuin-A partially exists as a disulfide-bridged two-chain protein with a heavy N-terminal and a lighter C-terminal chain similar to the structure of human fetuin-A homologue (alpha2HS glycoprotein), and also is partially phosphorylated at residues Ser120, Ser302, Ser305 and Ser306 (Wind et al., Anal. Biochem. 317 (2003) 26-33). Both fetuin-A modifications, the phosphorylation at the four sites as well as the proteolysis which causes longer or shorter light chains (termed lc-1 and lc-2, respectively), are probably brought about by targeted enzymatic activities which still need to be defined. In this study we show that authentic bovine fetuin-A disulfide-bridged two-chain forms, which include the original C-terminus, were liberated from the single-chain precursor by metalloproteinases MMP-3 (stromelysin-1) and MMP-7 (matrilysin), but not by elastase, cathepsin E and cathepsin G. Peptide sequencing suggested cleavage sites chiefly at the Pro277-Ser278 or Arg294-His295 peptide bonds. Fetuin-A radioactive phosphorylation in vitro by protein kinase CK2 caused (32)P incorporation into the fetuin-A light chain lc-1 but not lc-2 or the fetuin-A heavy chain, as revealed by MMP assisted proteolysis. Analysis by nanoESI-MS pinpointed phosphorylation at the native phospho-residues Ser302, Ser305 and Ser306 by increased relative abundance following in vitro phosphorylation. Moreover, CK2 phosphorylation of synthetic C-terminal fetuin-A peptides, used as effective controls to the native protein, strongly implies that CK2 is involved in the in vivo phosphorylation of fetuin-A. The phosphorylation of N-terminally truncated peptide homologs seemed highly dependent on the sequence context N-terminal of the phosphorylation sites, thus providing a likely explanation for the non-phosphorylation of the light chain lc-2 in native fetuin-A.
Kübler
Dieter
D
Mechanisms of Biomolecular Interactions (A060), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. d.kuebler@dkfz.de
Gosenca
Darko
D
Wind
Mathias
M
Heid
Hans
H
Friedberg
Ilan
I
Jahnen-Dechent
Willi
W
Lehmann
Wolf D
WD
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005312:Fetal Blood / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020539:Sequence Analysis, Protein; D012712:Serum Globulins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D060749:alpha-2-HS-Glycoprotein
C556354:AHSG protein, human; D001798:Blood Proteins; D012712:Serum Globulins; D060749:alpha-2-HS-Glycoprotein; D047390:Casein Kinase II; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020783:Matrix Metalloproteinase 7