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The assembly of laminin-5 subunits
C Matsui1, C K Wang, C F Nelson
1Department of Dermatology, Stanford University School of Medicine, California 94305, USA.
The Journal of Biological Chemistry
|October 6, 1995
Summary
Laminin-5 assembly initiates with beta 3 gamma 2 heterodimers, followed by alpha 3 chain addition. Alpha 3 synthesis limits heterotrimer formation, with glycosylation not essential for secretion.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Laminin-5, a heterotrimer of alpha 3, beta 3, and gamma 2 chains, is implicated in junctional epidermolysis bullosa.
- It is produced by keratinocytes and the SCC-25 cell line.
Purpose of the Study:
- To elucidate the assembly pathway of Laminin-5.
- To identify rate-limiting steps and the role of glycosylation in Laminin-5 formation.
Main Methods:
- Two-dimensional SDS-PAGE under reducing and non-reducing conditions.
- Cotransfection experiments in a human cell line lacking endogenous Laminin-5.
- Pulse-chase experiments and tunicamycin treatment.
Main Results:
- Laminin-5 assembly involves alpha 3, beta 3, and gamma 2 monomers, beta 3 gamma 2 heterodimers, and the alpha 3 beta 3 gamma 2 heterotrimer.
- Stable beta 3 gamma 2 heterodimers form independently of the alpha 3 chain.
- Alpha 3 chain synthesis appears rate-limiting for heterotrimer assembly.
- All Laminin-5 chains are glycosylated, but N-linked glycosylation is not required for assembly or secretion.
Conclusions:
- Laminin-5 assembly proceeds via a beta 3 gamma 2 heterodimer intermediate.
- The alpha 3 chain is rapidly incorporated into pre-formed heterodimers.
- Glycosylation is not essential for Laminin-5 secretion.