STRUCTURAL-ANALYSIS OF PROTEOGLYCAN MACROPHAGE-COLONY-STIMULATING FACTOR

Proteoglycan macrophage colony-stimulating factor (PG-M-CSF) was recen tly reported as a high molecular type of macrophage colony stimulating factor (M-CSF). We analyzed its structure by determining the expressi on of mutant M-CSF cDNA in Chinese hamster ovary cells. PG-M-CSF conta ined two types of molecules, a homodimeric 150-200-kDa subunit and a h eterodimeric form of a 43-kDa subunit and the 150-200-kDa subunit. The 150-200-kDa subunit carries a chondroitin sulfate chain, and its amin o terminal amino acid sequence was identical to that of the 43-kDa sub unit, 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 par t of the precursor sequence at its carboxyl terminus removed in the 43 -kDa subunit by proteolytic processing. The expression of mutagenized cDNA, in which Arg(220) was replaced by an alanine residue, resulted i n the disappearance of the 43-kDa subunit but not that of the 150-200- kDa subunit, indicating that Arg(220)-Pro-Pro-Arg is essential to proc ess 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 Arg (412). We also showed that the chondroitin sulfate binding site in the 150-200-kDa subunit was Ser(277), since conversion of Ser(277) to the alanine residue resulted in complete loss of the chondroitin sulfate substitution.