J. Biochem, 1993, Vol. 114, No. 2 255-262
© 1993 Japanese Biochemical Society
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The Structure of Recombinant Human Carboxy-Terminal-Truncated Macrophage Colony-Stimulating Factor Derived from Mammalian Cells
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd. 463-10 Kagasuno, Kawauchi-cho, Tokushima, Tokushima 771–01
The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor expressed in CHO cells was investigated. The bioactive protein ([-32–153]M-CSF), expressed from a nucleotide sequence that encoded a signal peptide of 32 amino acids and N-terminal amino acids numbers 1–153, was heterogeneous in terms of molecular mass, as analyzed by SDS-PAGE, because of the presence of N-linked sugar moieties. The primary structure of the polypeptide was determined by sequence analysis and amino acid analysis of the fragments obtained from lysylendopeptidase digests of reduced and alkylated M-CSF, and from pepsin digests of the intact molecule. A sugar chain was located only at Asn-122 of the two putative sites of N-glycosylation that were present per subunit. The homodimeric structure appeared to have seven disulfide bonds, formed by inter- or intra-molecular linkages, since there were no free thiol groups in the molecule. The assignment of disulfide bonds by sequence analysis using peptic fragments indicated the combinations of Cys7-Cys90, Cys48-Cysl39, and Cys102-Cys146. Gel-filtration analysis of Ser31 [ -32–153] M-CSF, in which the remaining Cys31 was replaced by Ser and which was expressed in COS cells, suggested that the mutein existed as a monomer. Our study shows that the disulfide-bond pairings of [-32–153] M-CSF that is expressed and post-translation-ally modified in mammalian cells are identical to those of Escherichia co/i-derived [3–153]-M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.