Characterization of cell-adhesive proteins secreted by cell lines growing in protein- and lipid-free synthetic medium: mouse L.P3 cells secrete a procollagen molecule with potent cell-attachment activity.
We have previously shown that mouse L.P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we screened and characterized cell-adhesive proteins in the conditioned media (CM's) of a series of cell lines growing in a protein- and lipid-free synthetic medium (P3 cell lines). Although cell-attachment activity of the CM's ranged from undetectable to highly significant, fractionation with affinity columns revealed the presence of significant cell-attachment activity in all CM's. Using cell-attachment assay and immunoassay on blotted filters, various cell-adhesive proteins were detected in the CM's, and most of them were identified as fibronectin, laminin, vitronectin, and collagen. GNCP-like proteins were detected in the CM's of HeLa.P3, JTC-16.P3, L.P3, and JTC-12.P3. There was no relationship between the origin of the cell lines and the cell-adhesive proteins secreted. GNCP purified from L.P3-CM was separated into 120-, 140-, 150-, and 160-kDa proteins on SDS-PAGE, which were judged to be a type of mouse type I procollagen from the following results: (1) they were digested by collagenase, (2) pepsin treatment converted the 150- and 160-kDa proteins into 120- and 140-kDa proteins, (3) they were recognized by anti-type I collagen antiserum, and (4) amino-terminal sequence of the pepsin-digested 140-kDa protein had significant homology with type I collagen. GNCP showed half-maximum activity of cell attachment at 0.03 micrograms/ml, indicating that GNCP was a cell-adhesive protein with an extremely high specific activity compared to other known cell-adhesive proteins.
Morita
H
H
Kihara Institute for Biological Research, Yokohama City University, Japan.
Yasumitsu
H
H
Watanabe
Y
Y
Miyazaki
K
K
Umeda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D002458:Cell Fractionation; D002460:Cell Line; D002846:Chromatography, Affinity; D017364:Collagenases / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005353:Fibronectins / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D006493:Heparin; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000494:pharmacology; D011347:Procollagen / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D011506:Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology
D015815:Cell Adhesion Molecules; D017077:Culture Media, Conditioned; D005353:Fibronectins; D011347:Procollagen; D011506:Proteins; D006493:Heparin; D010434:Pepsin A; D017364:Collagenases