PROTEOGLYCANS IN THE HUMAN SCLERA - EVIDENCE FOR THE PRESENCE OF AGGRECAN

Purpose. The proteoglycans synthesized and accumulated within the adul t human sclera (aged 50 to 80 years) were identified by their size, gl ycosaminoglycan side chains, and core proteins in an effort to charact erize the proteoglycan content of the human sclera. Methods. Sclerae, unlabeled, or radiolabeled in organ culture with (SO4)-S-35 or H-3-pro line, were extracted in 4M guanidine-HCl and separated by Sepharose CL -2B and Superose 6 forced-pressure liquid chromatography. Peak fractio ns, identified by glycosaminoglycan Content or radioactivity, were poo led and subjected to G-50 chromatography or sodium dodecyl sulfate-pol yacrylamide gel electrophoresis before and after digestion with specif ic glycosidases. Scleral proteoglycan core proteins were identified in Western blot analysis using specific antisera to decorin, biglycan, a nd aggrecan. Reverse transcription-polymerase chain reaction analyses were carried out on human scleral fibroblast RNA to confirm the transc ription of one scleral proteoglycan. Proteoglycans were localized on s ections of scleral tissue using specific antisera. Results. After chro matography on CL-2B, scleral proteoglycans could be resolved into thre e major peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroitin sulfate and keratan sulfate glycosaminogl ycan side chains. Results. of Western blot analyses indicated that the core protein of PG-1 is the aggrecan core protein, migrating at appro ximate to 350 kDa. Reverse transcription-polymerase chain reaction ana lyses confirmed that human scleral fibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycan and decor in in Western blot analyses using antibiglycan and antidecorin antibod ies, respectively. Immunostaining results indicated that aggrecan, big lycan, and decorin are distributed throughout the thickness of the hum an sclera. Conclusions. The adult human sclera contains three major pr oteoglycans; aggrecan, biglycan, and decorin. It is likely that these proteoglycans contribute to the structural properties of the sclera an d that the ratios of these proteoglycans will change with age, specifi c region, and condition of the sclera.