Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species.
Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.
Suzu
S
S
Biochemical Research Laboratory, Morinaga Milk Industry Co, Ltd., Kanagawa, Japan.
Ohtsuki
T
T
Yanai
N
N
Takatsu
Z
Z
Kawashima
T
T
Takaku
F
F
Nagata
N
N
Motoyoshi
K
K
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight
D006025:Glycosaminoglycans; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA; D005341:Fibrinolysin