Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing.
The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV-1 cells expressing a short M-CSF clone secrete only 24 kd recombinant M-CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M-CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M-CSF protein heterogeneity. Most secreted MIA PaCa-2 M-CSF reacts with long-clone-specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa-2 M-CSF size heterogeneity. In addition, cell lysates also contain larger M-CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M-CSF protein heterogeneity results from both pre- and post-translational processing.
Shadle
P J
PJ
Department of Protein Chemistry, Cetus Corporation, Emeryville, California 94608.
Aldwin
L
L
Nitecki
D E
DE
Koths
K
K
D015153:Blotting, Western; D002846:Chromatography, Affinity; D003115:Colony-Stimulating Factors / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008264:Macrophages / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D011863:Radioimmunoassay; D014407:Tumor Cells, Cultured
D003115:Colony-Stimulating Factors; D012333:RNA, Messenger