A comparative biochemical and ultrastructural study of proteoglycan–collagen interactions in corneal stroma. Functional and metabolic implications

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Abstract
Corneas of mouse, rat, guinea pig, rabbit, sheep, cat, dog, pig and cow were quantitatively analysed for water, hydroxyproline, nucleic acid, total sulphated polyanion, chondroitin sulphate/dermatan sulphate and keratan sulphate, several samples or pools of tissues from each species being used. Ferret cornea was similarly analysed for water and hydroxyproline on one pool of eight corneas. Pooled frog (38) and ferret (eight) corneas and a single sample of human cornea were qualitatively examined for keratan sulphate and chondroitin sulphate/dermatan sulphate by electrophoresis on cellulose acetate membranes. Nine species (mouse, frog, rat, guinea pig, rabbit, sheep, cat, pig and cow) were examined by light microscopy and six (mouse, frog, rat, guinea pig, rabbit and cow) by electron microscopy, with the use of Alcian Blue or Cupromeronic Blue in critical-electrolyte-concentration (CEC) methods to stain proteoglycans. Water (% of wet weight), hydroxyproline (mg/g dry wt.) and chondroitin sulphate (mg/g of hydroxyproline) contents were approximately constant across the species, except for mouse. Keratan sulphate contents (mg/g of hydroxyproline) increased with corneal thickness, whereas dermatan sulphate contents decreased. The oversulphated domain of keratan sulphate was absent from mouse and from corneas, increasing as percentage of total keratan with increasing corneal thickness. Sulphation of dermatan sulphate was essentially complete (i.e. one sulphate group per disaccharide unit). Chondroitin sulphate/dermatan sulphate proteoglycans were present at the d bands of the collagen fibrils of all species examined, orthogonally arrayed, with high frequency, and ocassionally at the e bands. Keratan sulphate proteoglycans were present at the a and c bands of all species examined, but with far higher frequency in the thicker corneas, where keratan sulphate contents were high. Alcian Blue CEC staining showed much higher sulphation of keratan sulphate in thick corneas, e.g., that of cow, than in thin corneas, e.g., that of mouse, in keeping with biochemical analyses. It is suggested that the constancy of interfibrillar volumes is regulated via the swelling and osmotic pressure of the interfibrillar polyanions, by adjustment of the extent of sulphation in two independent proteoglycan populations, to achieve an ''average sulphation'' of the total polyanion similar to that of fully sulphated chondroitin sulphate/dermatan sulphate. The balance of synthesis of the two kinds of proteoglycans may be determined by the O2 supply to the avascular cornea. O2 supply may also determine the conversion of chondroitin sulphate into dermatan sulphate.