γ2 Chain of Laminin-5 Is Recognized By Monoclonal Antibody GB3

Abstract

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.

Laminin-5 in the progression of carcinomas

“…Later its 190 -200 kD ␣3 and 155 kD ␥2 chains are posttranslationally processed by proteolysis to 160 and 105 kD polypeptides, respectively. 49,50 BASEMENT MEMBRANE IN CARCINOMAS The role of the BM in carcinoma biology is not well understood. Based mostly on morphologic data, the loss of BM continuity has been associated with increasing malignancy.…”

“…85 It has been suggested that the cleavage of laminin ␥2 chain by matrix metalloprotease-2 to 80 kDa fragment enhances breast epithelial cell motility. 81 The latter mechanism as a promotor in the invasion of colorectal carcinomas has been excluded by showing that there is no small cleaved polypeptides but only the normal 155,000 and 105,000 kD polypeptides 50 from the laminin ␥2 chain in carcinoma tissues. 61 Also, it has been recently suggested that the MT1-MMP plays a role in regulating the cell migration over laminin-5.…”

Functional Re-expression of Laminin-5 in Laminin-γ2-deficient Human Keratinocytes Modifies Cell Morphology, Motility, and Adhesion

“…Immunofluorescence analysis using the mAb GB3, which binds to a conformational epitope in the laminin ␣3␤3␥2 heterotrimer (5,29), displayed a strong intracellular reactivity with the transfected LSV5 cells (Fig. 3).…”

“…This polypeptide associated with the endogenous laminin ␣3 and ␤3 chains and formed a heterotrimeric ␣3␤3␥2 complex secreted extracellularly that was recognized by three distinct antibodies raised against each individual chain of laminin-5 and by mAb GB3. Because chain assembly into a complex preserving the native conformation of laminin-5 is required for binding with GB3 (5,29), GB3 recognition of the heterotrimer secreted by LSV5 cells in the culture medium and in the extracellular matrix suggests that the restored synthesis of ␥2 chain results in production of properly assembled laminin-5 molecules.…”

Immunomapping of Basement Membrane Zone Macromolecules in Canine Salt-Split Skin.

“…This was a generous gift from Dr. Jonathan Jones (Department of Cellular and Molecular Structures, Northwestern University, Chicago, IL). The monoclonal antibody against type VII collagen raised from mouse, EBA2 [18] and monoclonal antibody to laminin 5, GB3 [7], were also used. Rabbit anti-mouse laminin antibody was purchased from Collaborative Biomedical Products/Becton Dickinson Labware (Bedford, MA).…”