Inhibition of chondroitin sulfate incorporation into human thyroglobulin by p-nitrophenyl-beta-D-xylopyranoside.

Human thyroglobulin (TG) is unique among glycoproteins and TGs of other species in having a chondroitin sulfate chain. We studied the effects of p-nitrophenol-beta-D-xylopyranoside (PNXP), an inhibitor of chondroitin sulfate incorporation into core protein of proteoglycans, on the synthesis of human TG. Fragments of normal thyroid tissue from two patients were preincubated for 1 h with PNXP before adding [35S] sulfate and [3H]leucine. TG was purified by ammonium sulfate precipitation, sucrose gradient centrifugation, and CsCl equilibrium density gradient centrifugation. Chondroitin ABC lyase released 38-42% of the [35S]sulfate from control TG, synthesized in the absence of inhibitor, demonstrating the presence of chondroitin sulfate units. In the samples incubated with increasing concentrations of PNXP (0.2 and 1 mM), the fraction of [35S] sulfate released by ABC lyase decreased progressively (14-23% and 8-11%, respectively). This confirms that, as in chondroitin sulfate proteoglycans, chondroitin sulfate synthesis in TG is initiated by the transfer of a galactose unit, by galactosyltransferase, to a xylosyl-serine in the TG peptide backbone. This step is prevented by PNXP, which is a competitive acceptor of galactose. The density of TG synthesized in the presence of PNXP was determined by CsCl equilibrium density centrifugation. [3H] leucine-labeled TG from the samples incubated with PNXP was less dense than [3H]leucine-labeled TG from the control specimen. The newly synthesized TG was less dense either because of the lack of the chondroitin sulfate chain or because further processing of the TG was impaired. To differentiate these possibilities, we treated control, [3H]leucine-labeled TG with chondroitin ABC lyase and compared its density to that of [3H] leucine-labeled TG synthesized in the presence of 1 mM PNXP. The decrease in density produced by PNXP was greater than the decrease brought about by chondroitin ABC lyase (4.3-5.2 x 10(-3) and 2.4-2.8 x 10(-3) g/cm3, respectively). Therefore, the density contributed by the chondroitin chain was insufficient to account for the entire density shift seen with PNXP. We conclude that 1) chondroitin sulfate synthesis is initiated by the transfer of a galactosyl unit to xylosyl-serine in the TG peptide backbone; and 2) inhibition of this step decreases the density of TG by preventing the addition of the chondroitin chain and interfering with the further processing of TG.