HYALURONAN AND CHONDROITIN SULFATE PROTEOGLYCANS ARE COLOCALIZED TO THE CILIARY ZONULE OF THE RAT EYE - A HISTOCHEMICAL AND IMMUNOCYTOCHEMICAL STUDY

In previous studies, chondroitin sulfate proteoglycans have been local ized to the periphery of the zonular fibers and the individual zonular fibrils (or microfibrils) after Cuprolinic blue staining in conjuncti on with chondroitinase digestions and immunogold labelling with 2-B-6 antibody. In the present study, we wished to determine if these proteo glycans are linked to hyaluronan to form a large multimolecular aggreg ate. To accomplish this, we localized the hyaluronan using a biotinyla ted hyaluronan-binding protein fragment of chondroitin sulfate proteog lycan, containing also the link protein, pu rifled from bovine nasal c artilage. The results showed that the ciliary zonule of the rat eye wa s reactive with the biotinylated hyaluronan-binding probe as demonstra ted by streptavidin-peroxidase-diaminobenzidine staining and streptavi din-gold labelling. Hyaluronan-gold labelling showed that the gold par ticles were mostly localized on the periphery of the zonular fibers, w hich was similar to the localization pattern of the zonule associated- proteoglycans. This hyaluronan-binding probe also strongly labelled th e sites of zonule insertion over the basement membrane of the inner ci liary epithelium at the pars plana and the lens capsule at the equator ial region, which suggests its probable role in the attachment of cili ary zonule to the basement membranes. To demonstrate whether these two molecules are linked to one another, ultrastructural colocalization o f both hyaluronan and chondroitin sulfate proteoglycans was performed on the same sections by double-gold labelling, and combined Cuprolinic blue staining and hyaluronan-gold labelling. Gold particles of 15 and 10 nm in sizes labelling both hyaluronan and chondroitin 4-sulfate, w ere colocalized to the surface of the zonular fibers. The combined Cup rolinic blue staining and hyaluronan-gold labelling showed that the go ld particles were localized towards the ends of the Cuprolinic blue-st ained rodlets, which strongly suggests that these chondroitin sulfate proteoglycans are linked to the hyaluronan chain to form a large aggre gate surrounding the periphery of the zonular fibers. These ciliary zo nule-associated proteoglycan-hy aluronan aggregates may play a;role in organizing the individual zonular fibrils (microfibrils) into bundles of zonular fibers.