Extracellular Matrix (ECM) Proteoglycans Produced by Cultured Bovine Corneal Endothelial Cells

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Abstract
The nature of the proteoglycan(s) (PG) found in the extracellular matrix (ECM) layer produced by cultured bovine comeal endothelial (BCE) cells is analyzed. The PG(s) account for approximately 5 to 6% of the dry weight of the ECM, regardless of the amount of extracellular soluble PG available in the medium. A 4 M guanidinium chloride (GuCl) extract of ECM was separated on a dissociative cesium chloride (CsCl) gradient (1.25 g/cm3 starting density). Results showed one main peak of PG substance(s) comprising 91% of the total labelled substance and uronic acid, banding at a specific buoyant density of 1.29 g/cm3. The molecular weight of this major PG(s) as estimated by gel filtration on Sepharose CL-4B ranged from 0.5 to 0.7 × 106. Further chemical analysis of the main PG(s) band revealed a protein moiety accounting for 45% of the weight and carbohydrates-glycosaminoglycans (GAG) accounting for the remaining 55%. Analysis of the GAG chains (over the entire gradient) showed a composition, based on the susceptibility of the PG substance(s) to degrading enzymes, of 50% heparan sulfate, 43.5% dermatan sulfate, and 6.5% chondroitin 4- and 6-sulfate chains. BCE cell cultures grown in the presence of β-D-xyloside produced an ECM lacking more than 90% of the GAG content found in the control ECM. The medium-soluble GAG chains, produced in vast excess in cultures grown in the presence of β-D-xyloside, are composed mainly of chondroitin 4- and 6-sulfates.