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J A Rada, V R Achen, C A Perry, P W Fox; Proteoglycans in the human sclera. Evidence for the presence of aggrecan.. Invest. Ophthalmol. Vis. Sci. 1997;38(9):1740-1751.
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PURPOSE: The proteoglycans synthesized and accumulated within the adult human sclera (aged 50 to 80 years) were identified by their size, glycosaminoglycan side chains, and core proteins in an effort to characterize the proteoglycan content of the human sclera. METHODS: Sclerae, unlabeled, or radiolabeled in organ culture with 35SO4 or 3H-proline, were extracted in 4M guanidine-HCl and separated by Sepharose CL-2B and Superose 6 forced-pressure liquid chromatography. Peak fractions, identified by glycosaminoglycan content or radioactivity, were pooled and subjected to G-50 chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after digestion with specific glycosidases. Scleral proteoglycan core proteins were identified in Western blot analysis using specific antisera to decorin, biglycan, and aggrecan. Reverse transcription-polymerase chain reaction analyses were carried out on human scleral fibroblast RNA to confirm the transcription of one scleral proteoglycan. Proteoglycans were localized on sections of scleral tissue using specific antisera. RESULTS: After chromatography on CL-2B, scleral proteoglycans could be resolved into three major peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroitin sulfate and keratan sulfate glycosaminoglycan side chains. Results of Western blot analyses indicated that the core protein of PG-1 is the aggrecan core protein, migrating at approximately 350 kDa. Reverse transcription-polymerase chain reaction analyses confirmed that human scleral fibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycan and decorin in Western blot analyses using antibiglycan and antidecorin antibodies, respectively. Immunostaining results indicated that aggrecan, biglycan, and decorin are distributed throughout the thickness of the human sclera. CONCLUSIONS: The adult human sclera contains three major proteoglycans; aggrecan, biglycan, and decorin. It is likely that these proteoglycans contribute to the structural properties of the sclera and that the ratios of these proteoglycans will change with age, specific region, and condition of the sclera.
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