Spatial distributions of sulfur-rich proteins in cornifying epithelia

We have used energy-filtered electron microscopy and electron energy loss spectroscopy (EELS) to characterize the distributions of sulfur-rich proteins in granular layer cells of squamous stratifying epithelia and their redistribution in the cornified layer, with particular attention to assembly of the cornified cell envelope (CE). Our measurements provide quantitative information that complements highly specific but qualitative data from immunocytochemistry. Spatial distributions of sulfur, phosphorus, and nitrogen were mapped in unstained thin sections of mouse epidermis and forestomach, using a postcolumn energy filter. Nitrogen images were indicative of total protein while phosphorus images provided a control to validate the algorithms used to calculate the elemental maps. Sulfur was found at high levels in round L-granules in the granulocyte cytoplasm and in the cornified CE, correlating with the presence of the protein, loricrin ( approximately 7% Cys/Met residues). EELS confirmed these observations quantitatively: either L-granules consist exclusively of loricrin or any additional components must have an equally high net sulfur content. These data also confirm the large fraction (approximately 75%) of loricrin in the CE, as inferred from modeling of its amino acid composition. We also observed extracellular deposits between cornified squames in fetal mouse epidermis that we call peripheral granules. Their sulfur content is at least as high as that of L-granules but they do not label with anti-loricrin antibodies, suggesting the presence of another sulfur-rich protein.