The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor derived from mammalian cells.
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 peptide fragments indicated the combinations of Cys7-Cys90, Cys48-Cys139, 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-translationally modified in mammalian cells are identical to those of Escherichia coli-derived [3-153]M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.
Yamanishi
K
K
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima.
Yasuda
S
S
Masui
Y
Y
Nishida
T
T
Shindo
Y
Y
Takano
M
M
Ohmoto
Y
Y
Takahashi
M
M
Adachi
M
M
D000478:Alkylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D004220:Disulfides; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor