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Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in 'critical-electrolyte-concentration' techniques.

ABSTRACT: Proteoglycans (PGs) in bovine corneal stroma were stained with Cupromeronic Blue in 'critical-electrolyte-concentration' (CEC) methods for electron microscopy, and were located vis-à-vis collagen fibril a-e banding patterns. Keratanase and chondroitin ABC lyase digestion showed that a + c-band- and d + e-band-associated PGs were keratan sulphate-rich and chondroitin (dermatan) sulphate-rich respectively. The CEC pattern proved that the keratan sulphate PGs at the a and c bands differed. Comparison of their CECs with their behaviour on anion-exchange chromatography confirmed previous (indirect) attempts at identification [Scott & Haigh (1985) Biosci. Rep. 5, 765-774]. Similar arguments were applied to the dermatan sulphate PGs at the d and e bands. These results strongly support the one-PG-one-binding-site hypothesis [e.g. Scott (1988) Biochem. J. 252, 313-323]. Remarkable inter-species variations in the keratan sulphate PG patterns contrast with the relatively constant picture of dermatan sulphate PG-collagen fibril interactions.

SUBMITTER: Scott JE

PROVIDER: S-EPMC1149341 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Project description:Cloned immortalized MC615 mouse chondrocytic cells were used to examine their capability to produce multiple types of matrix proteoglycans. Immunofluorescence staining indicated a uniform expression of aggrecan, biglycan and decorin by all cells. After culture with [35S]sulphate, proteo[35S]glycans secreted by the cells were found to elute in two peaks from a Sepharose CL-4B column. The first peak, at the void volume of the column, contained a large proteoglycan with an estimated average hydrodynamic mass of 10(3) kDa. The glycosaminoglycan chains of this proteoglycan had an average hydrodynamic size of 17 kDa, estimated by Sepharose CL-6B chromatography, indicating the presence of 30-70 glycosaminoglycan chains per core protein, which was consistent with the characteristics of aggrecan. Biglycan and decorin were immunoisolated from the second Sepharose CL-4B peak, and had average glycosaminoglycan hydrodynamic sizes of approx. 25 kDa and 32 kDa respectively. Glycosaminoglycan chains of the aggrecan, biglycan and decorin were treated with chondroitin ABC lyase, chondroitin AC lyase and chondroitin B lyase to determine the positions of sulphation and the degree of uronic acid epimerization. The aggrecan glycosaminoglycan chains were found to contain a 4-sulphate/6-sulphate ratio of 7:3, with no epimerization of glucuronic acid to iduronic acid. The biglycan glycosaminoglycan chains were found to contain a similar ratio of 4-sulphate/6-sulphate, but with approx. 40-45% of the glucuronic acid epimerized to iduronic acid. The decorin glycosaminoglycan chains were found to contain 4-sulphate but no detectable 6-sulphate, and approx. 30-35% epimerization of the glucuronic acid to iduronic acid. The results, using these cloned cells, indicated that a single MC615 cell is able to make all three proteoglycans with distinctive differences between the glycosaminoglycans of aggrecan, biglycan and decorin. These data indicate that a mechanism must exist for a single MC615 cell to regulate the sizes and fine structures of glycosaminoglycans on simultaneously produced, different proteoglycans in a core-protein-specific manner.

Dataset Information

Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in 'critical-electrolyte-concentration' techniques.

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