Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.
BACKGROUND
Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF.


METHODS
Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay.


RESULTS
We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity.


CONCLUSIONS
Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.
Mohamad
Saharuddin Bin
SB
Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Minamijyosanjimacho-2, Tokushima, 770-8506 Japan.
Nagasawa
Hideko
H
Uto
Yoshihiro
Y
Hori
Hitoshi
H
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D006801:Humans; D008262:Macrophage Activation / Q000187:drug effects; D016215:Macrophage-Activating Factors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014809:Vitamin D-Binding Protein / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism
D016215:Macrophage-Activating Factors; D014809:Vitamin D-Binding Protein; C101908:vitamin D-binding protein-macrophage activating factor; D013481:Superoxides; D009439:Neuraminidase; D001616:beta-Galactosidase