Laminin biosynthesis in the extracellular matrix-producing cell line PFHR9 studied with monoclonal and polyclonal antibodies.
The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.
Lissitzky
J C
JC
Laboratoire de Cancérologie Expérimentale, UA CNRS 1175, Faculté de Médecine Nord, Marseille, France.
Charpin
C
C
Bignon
C
C
Bouzon
M
M
Kopp
F
F
Delori
P
P
Martin
P M
PM
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002460:Cell Line; D011232:Chemical Precipitation; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism*; D007797:Laminin / Q000096:biosynthesis* / Q000276:immunology; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D011487:Protein Conformation; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007797:Laminin; D008715:Methionine