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.