The assembly of laminin-5 subunits.
Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.
Matsui
C
C
Department of Dermatology, Stanford University School of Medicine, California 94305, USA.
Wang
C K
CK
Nelson
C F
CF
Bauer
E A
EA
Hoeffler
W K
WK
D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016109:Epidermolysis Bullosa, Junctional / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008970:Molecular Weight; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; D014415:Tunicamycin; D000097794:Kalinin