Identification of the sterol- and actin-binding domains of plasma vitamin D binding protein (Gc-globulin).
The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.
Haddad
J G
JG
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6149.
Hu
Y Z
YZ
Kowalski
M A
MA
Laramore
C
C
Ray
K
K
Robzyk
P
P
Cooke
N E
NE
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002846:Chromatography, Affinity; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D014176:Protein Biosynthesis; D011817:Rabbits; D013261:Sterols / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000235:genetics
D000199:Actins; D010446:Peptide Fragments; D013261:Sterols; D014809:Vitamin D-Binding Protein; D004247:DNA