Laminin-5 is a heterotrimer composed of ␣3, ␤3, 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 ␣3, ␤3, and ␥2 monomers, a ␤3␥2 heterodimer, and an ␣3␤3␥2 heterotrimer. The presence of the ␤3␥2 heterodimer, but not heterodimers containing an ␣3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a ␤3␥2 heterodimer to an ␣3␤3␥2 heterotrimer. We showed, by cotransfection experiments using fulllength recombinant ␤3 and ␥2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of ␣3 chain expression. In the SCC-25 cell fraction, the ␣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 ␣3 being limiting for heterotrimer assembly, with rapid association of the ␣3 chain with ␤3␥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.Laminin-5 (kalinin/nicein) is an epithelium-specific laminin subtype and is a component of the anchoring filament of the lamina lucida in the basement membrane of the skin (1). The anchoring filament is the bridge between hemidesmosomes and the lamina densa region of the basement membrane and is believed to mediate the adhesion of the epithelium to the basement membrane. Laminin-5 is therefore one of the primary adhesion proteins holding together the epidermis and the dermis. Laminin-5 is initially synthesized in a cell-associated form, estimated to be 460 kDa, and is composed of three polypeptides: the 200-kDa (␣3), 145-kDa (␤3), and 155-kDa (␥2) chains (2). The three chains are presumed to form a cruciform structure where the chains are bound by disulfide linkages (2). The two heterotrimeric forms in keratinocyte cell-conditioned culture medium are derived from the cellular form by extracellular processing. A 440-kDa medium form results from the 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).A number of variant laminin subunits, in addition to those of laminin-5, are assembled in the rough endoplasmic reticulum. N-Linked oligosaccharide processing of some of these has been shown to occur within the Golgi apparatus prior to secretion (3, 4). These variant chains are produced in different cell types (5, 6); for exa...

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the α3β3γ2 heterotrimer assembles intracellularly via a β3γ2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein–protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.