Flotillin 2 is distinct from epidermal surface antigen (ESA) and is associated with filopodia formation.
ECS-1, a monoclonal antibody (MoAb) raised to cultured human keratinocytes, stains the intercellular glycocalyx with a pemphigus-like pattern and recognizes a 35-kDa epidermal surface antigen (ESA) on Western blotting of keratinocyte extracts. When ECS-1 MoAb was used to screen a keratinocyte expression library, a unique cDNA was identified that predicted a 42-kDa globular protein of unknown function. This putative ESA was conserved between mice and humans and was encoded by a gene on chromosome 17q11-12 in linkage with neurofibromin. Homology between the cDNA sequence has been reported with flotillin 1, a caveolae associated protein, as well as Reggie 1 and 2, neuronal proteins expressed during axonal regeneration present in activated GPI-anchored cell adhesion molecules in non-caveolar-associated micropatches. In order to determine whether the cDNA predicted protein and ECS-1 antigen were identical, we compared ECS-1 with the immunoreactivity of a new antibody raised to the cDNA fusion protein in epidermis and cultured cells. The cDNA fusion protein was expressed in bacteria and in cos cells with his, FLAG, and EGFP reporter tags and by stable transfection as an EGFP fusion protein. The fusion protein and native protein of 42 kDa were detected by the new antibody, but not by the original ECS-1. Thus, the ECS-1 antigen, ESA (35 kDa), is clearly distinct from the protein predicted by the cDNA (renamed flotillin 2). Stable transfection of ESA/flotillin 2 fusion protein in cos cells induced filopodia formation and changed epithelial cells to a neuronal appearance. Thus, the function of flotillin 2 may resemble that of the goldfish optic nerve neuronal regeneration proteins, Reggie 1 and 2.
Hazarika
P
P
Department of Dermatology, University of Texas Medical School and Department of Medical Specialties, Section of Dermatology, MD Anderson Cancer Center, Houston, Texas 77030, USA.
Dham
N
N
Patel
P
P
Cho
M
M
Weidner
D
D
Goldsmith
L
L
Duvic
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004817:Epidermis / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015603:Keratinocytes / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence; D011554:Pseudopodia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D051242:Caveolin 1; D022461:Caveolins; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C106354:flotillins