| Abstract: | The metabolism of endogenously labeled proteoglycans was studied in rat ovarian granulosa cell cultures by a series of pulse-chase experiments using 35S]sulfate as a precursor. More than 90% of the newly synthesized proteoglycans are transported to the cell surface (trypsin-accessible compartment) with a median transit time of 13 min. The membrane-bound heparan sulfate-proteoglycan (HS-PG) is lost from the cell surface either by release into the medium (30%, with t1/2 of 4 h) or by internalization (70%, with t1/2 of 4 h). Internalized HS-PG, which does not recycle to the cell surface, is degraded by two major pathways. In pathway 1, 60% of the internalized HS-PG migrates to lysosomes with a relatively short t1/2 of 30 min, where it is rapidly degraded, releasing free 35S]sulfate without detectable intermediate products. Chloroquine treatment inhibited degradation, resulting in the accumulation of intact proteoglycans inside the cell. In pathway 2, 40% of the internalized HS-PG is first subjected to extensive proteolysis and limited endoglycosidic degradation yielding single HS chains about 1/3 of their original size (t1/2 of 30 min). Chloroquine did not inhibit this step. The partially degraded HS is then degraded further by limited endoglycosidic activity to about 1/4-1/5 the original size (t1/2 of 30-60 min). This step is inhibited by chloroquine. These smaller fragments have a relatively long t1/2 of 3-4 h before rapid degradation in the lysosomes, releasing free 35S]sulfate. Approximately 7% of the newly synthesized HS-PG that is not transported to the cell surface is degraded directly by pathway 2. The larger dermatan sulfate proteoglycan (DS-I) is transported to the cell surface from which it is quantitatively released into the medium with a t1/2 of 4-6 h. The smaller DS-PG (DS-II) is metabolized similarly to the HS-PG. Most (greater than 90%) is transported to the cell surface from which it is lost either by release into the medium (40%) or by internalization (60%). About 60% of the internalized DS-II is degraded by pathway 1 (t1/2 of 30 min), while the remainder appears to be degraded by pathway 2 with an overall t1/2 of 4 h. However, in contrast to the degradation of HS-PG by pathway 2, no endoglycosidic degradation of the DS chains occurred. |
