The assembly of laminin-5 subunits

Abstract

Laminin-5 is a heterotrimer composed of α3,β3, and γ2 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 α3, β3, and γ2 monomers, a β3γ2 heterodimer, and an α3β3γ2 heterotrimer. The presence of the β3γ2 heterodimer, but not heterodimers containing an α3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a β3γ2 heterodimer to an α3β3γ2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant β3 and γ chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of α3 chain expression. In the SCC-25 cell fraction, the α3 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 α3 being limiting for heterotrimer assembly, with rapid association of the α3 chain with β3γ2 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.