Interpretation of the electron microscopical appearance of collagen fibrils from corneal stroma

The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea.