The assembly of laminin-5 subunits
Autor: | Charlotte F. Nelson, Warren K. Hoeffler, C. Kathy Wang, Eugene A. Bauer, Chihiro Matsui |
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Rok vydání: | 1995 |
Předmět: |
Keratinocytes
Glycosylation Immunoprecipitation Protein Conformation Biochemistry Models Biological law.invention chemistry.chemical_compound Laminin law Tumor Cells Cultured Humans Secretion Molecular Biology Gel electrophoresis biology Tunicamycin Cell Biology Cell Fraction Recombinant Proteins Molecular Weight Kinetics chemistry biology.protein Biophysics Recombinant DNA Epidermolysis Bullosa Junctional Cell Adhesion Molecules Protein Processing Post-Translational |
Zdroj: | The Journal of biological chemistry. 270(40) |
ISSN: | 0021-9258 |
Popis: | 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. |
Databáze: | OpenAIRE |
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