Abstract
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.
Highlights
Processing of the 200-kDa ␣3 chain to 165 kDa, while the 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155-kDa ␥2 chain to 105 kDa [2]
Defects in laminin-5 result in defective anchoring filaments, causing blistering of the skin, and are known to cause Herlitz junctional epidermolysis bullosa (HJEB),1 an autosomal recessive disorder characterized by generalized blister formation at the level of the lamina lucida within the epidermal basement membrane [8, 9]
We determined the steps in laminin-5 chain assembly using endogenous laminin-5 and checked our conclusions using exogenous chains expressed from full-length cDNAs
Summary
Processing of the 200-kDa ␣3 chain to 165 kDa, while the 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155-kDa ␥2 chain to 105 kDa [2]. All three antibodies precipitated an additional band of 250 kDa under reducing conditions
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