The Assembly of Laminin-5 Subunits (*)
- From the Department of Dermatology, Stanford University School of Medicine, Stanford, California 94305
- ¶ To whom correspondence should be addressed. Tel.: 415-723-7843; Fax: 415-723-8762.
Abstract
Laminin-5 is a heterotrimer composed of α3, β3, and 
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 α3, β3, and 
2 monomers, a β3
2 heterodimer, and an α3β3
2 heterotrimer. The presence of the β3
2 heterodimer, but not heterodimers containing an α3 chain and any of the other chains, was suggestive of assembly of laminin-5
proceeding from a β3
2 heterodimer to an α3β3
2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant β3 and 
2 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 β3
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.
Footnotes
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↵§ Partially supported by a scholarship from the Uehara Memorial Foundation.
-
↵* This work was supported by NIAMS Grants AR41045-03 and AR19537. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- HJEB
-
Herlitz junctional epidermolysis bullosa
- DMEM
-
Dulbecco's modified Eagle's medium
- PAGE
-
polyacylamide gel electrophoresis
- rβ
-
recombinant β3
- r
-
recombinant
2.
-
- Received May 8, 1995.
- Revision received July 20, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
