The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture.
Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.
Colombatti
A
A
Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, Aviano, Italy.
Bonaldo
P
P
Volpin
D
D
Bressan
G M
GM
D000818:Animals; D001011:Aorta / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D013710:Tendons / Q000378:metabolism*
D003470:Culture Media; D008562:Membrane Glycoproteins; C030340:elastin microfibril interface located protein