Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes.
A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.
Department of Molecular Genetics, University of Texas Southwestern, Medical Center, Dallas 75235.
P A
PA
Johnston
F X
FX
Yu
G A
GA
Reynolds
H L
HL
Yin
C R
CR
Moomaw
C A
CA
Slaughter
T C
TC
Südhof
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D010586:Phagocytes / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin
D000199:Actins; D002135:Calcium-Binding Proteins; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D014357:Trypsin