Abstract

Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.

Highlights

  • Our previous work showed that vitaCmindeficiency collagen synthesis [4]

  • Sulfate incorporation into proteoglycans in scorbutic cartilage couldhave resulted from decreased synthesis of the entire monomer, due to core protein deficiency, or from decreased synthesis of the glycosaminoglycan chains

  • Gel filtration analysis indicated that thaeverage size of monomers from scorbutic cartilage was slightly smaller than monomers from normaltissue,butthe size of the newly synthesized glycosaminoglycan chains attached to the core ronate

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Summary

Hydrodynamic Size of Newly Synthesized Proteoglycan

Sulfate incorporation into proteoglycans in scorbutic cartilage couldhave resulted from decreased synthesis of the entire monomer, due to core protein deficiency, or from decreased synthesis of the glycosaminoglycan chains. Samples of pulse-labeled cartilage from scorbutic and ascorbate-supplemented guinea pigs were extracted with the dissociative solvent guanidine hydrochloride in the presence of protease inhibitors. The extracts were chromatographed under dissociating conditions on a Sepharose CL-2B column (Fig 1).In both cases radioactivity eluted in a broad region occupying much of the included volume, suggesting the existence of fairly heterogenous populations of polydisperse proteoglycan monomers. The size of the constituent glycosaminoglycan chains liberated by papain digestion of the proteoglycans were unchanged when examined by chro-. Chains from control and scorbutic samples eluted with a K., of 0.47 (Mr 1.3 X lo4)

Proteoglycan Biosynthesisin Scurvy
Hexuronic acid Hexose Hexosamine Protein
TABLEIV activity was measuredin homogenates of costalcartilages
Additions to basal medium
DISCUSSION
We considered the possibility that the reduction in galaca SCORBUTIC
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