The glycosylation and characterization of the candidate Gc macrophage activating factor.
The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.
Ravnsborg
Tina
T
Institute of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Olsen
Dorthe T
DT
Thysen
Anna Hammerich
AH
Christiansen
Maja
M
Houen
Gunnar
G
Højrup
Peter
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D006020:Glycopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D016215:Macrophage-Activating Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry; D014312:Trisaccharides / Q000737:chemistry; D014809:Vitamin D-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006020:Glycopeptides; D016215:Macrophage-Activating Factors; D020033:Protein Isoforms; D011498:Protein Precursors; D014312:Trisaccharides; D014809:Vitamin D-Binding Protein; D013912:Threonine