Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization.
Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the alpha 3-chain first globular domain) that was measured at 35.40 +/- 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the beta 3-chain globular domain IV) that measured 45.20 +/- 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 +/- 6.28 nm from the PM. The laminin 5 gamma 2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 +/- 1.65 nm from the keratinocyte PM. Taken together with previous gamma 2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27 degrees from the horizontal lamina lucida (LL)/LD border and propose that the gamma 2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D001485:Basement Membrane / Q000378:metabolism / Q000648:ultrastructure; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D004817:Epidermis / Q000378:metabolism* / Q000648:ultrastructure; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D017979:Gold Colloid / Q000737:chemistry; D006801:Humans; D007106:Immune Sera / Q000737:chemistry / Q000276:immunology; D015603:Keratinocytes / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron / Q000379:methods; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D014018:Tissue Distribution; D000097794:Kalinin
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000939:Epitopes; D017979:Gold Colloid; D007106:Immune Sera; D021122:Protein Subunits; D000097794:Kalinin