Structural analysis of proteoglycan macrophage colony-stimulating factor.
Proteoglycan macrophage colony-stimulating factor (PG-M-CSF) was recently reported as a high molecular type of macrophage colony-stimulating factor (M-CSF). We analyzed its structure by determining the expression of mutant M-CSF cDNA in Chinese hamster ovary cells. PG-M-CSF contained two types of molecules, a homodimeric 150-200-kDa subunit and a heterodimeric form of a 43-kDa subunit and the 150-200-kDa subunit. The 150-200-kDa subunit carries a chondroitin sulfate chain, and its amino-terminal amino acid sequence was identical to that of the 43-kDa subunit, which is known to form the conventional M-CSF molecule (85-kDa M-CSF). The results obtained with the carboxyl-terminal deleted mutants showed that the PG-M-CSF-specific 150-200-kDa subunit had a large part of the precursor sequence at its carboxyl terminus removed in the 43-kDa subunit by proteolytic processing. The expression of mutagenized cDNA, in which Arg220 was replaced by an alanine residue, resulted in the disappearance of the 43-kDa subunit but not that of the 150-200-kDa subunit, indicating that Arg220-Pro-Pro-Arg is essential to process PG-M-CSF to 85-kDa M-CSF. Truncated mutation analysis showed that the carboxyl terminus of the 150-200-kDa subunit lay downstream of Arg412. We also showed that the chondroitin sulfate binding site in the 150-200-kDa subunit was Ser277, since conversion of Ser277 to the alanine residue resulted in complete loss of the chondroitin sulfate substitution.
Kimura
F
F
Third Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
Suzu
S
S
Nakamura
Y
Y
Wakimoto
N
N
Kanatani
Y
Y
Yanai
N
N
Nagata
N
N
Motoyoshi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011509:Proteoglycans / Q000737:chemistry*
D018076:DNA, Complementary; D011509:Proteoglycans; D016173:Macrophage Colony-Stimulating Factor