Synthesis and assembly of the synaptic cleft protein S-laminin by cultured cells.
S-laminin, a homologue of the B1 chain of laminin, is concentrated in a subset of basal laminae (BLs), including the BL at the skeletal neuromuscular junction and bears an adhesive site for motoneuron-like cells. Here, we have begun to characterize the native form of the protein. We show that several muscle- and glia-like cell lines synthesize and secrete S-laminin as well as the A, B1, and B2 subunits of the conventional laminin trimer. Experiments using subunit-specific antibodies showed that S-laminin is complexed with the A and B2 subunits of laminin but not with B1, suggesting that S-laminin replaces B1 to form a novel laminin-like trimer. Comparison of material precipitated by different antibodies provided evidence for two immunochemically distinct forms of S-laminin, both of which associate with B2 and A-like subunits. Analysis of tunicamycin-treated cells indicated that N-linked glycosylation is required neither for the selective association of S-laminin with B2 and A subunits nor for the distinction between two forms of S-laminin. Finally, a full-length S-laminin cDNA was constructed and transfected into muscle and non-muscle cells. S-laminin was detected intracellularly in both cell types, in extracellular matrix of muscle cells, and in two immunochemically distinct forms. Thus, the cDNA contains sufficient information to permit assembly, secretion, and post-translational modification of S-laminin.
Green
T L
TL
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Hunter
D D
DD
Chan
W
W
Merlie
J P
JP
Sanes
J R
JR
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005109:Extracellular Matrix / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D007797:Laminin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D046911:Macromolecular Substances; D008961:Models, Structural; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D012260:Ribonucleases
D007797:Laminin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C058783:laminin beta2; D012260:Ribonucleases