BINDING, INTERNALIZATION, AND DEGRADATION OF ANTIPROLIFERATIVE HEPARAN-SULFATE BY HUMAN EMBRYONIC LUNG FIBROBLASTS

Binding, internalization, and degradation of I-125-labeled, antiprolif erative, or nonantiproliferative heparan sulfate by human embryonic lu ng fibroblasts was investigated. Both L-iduronate-rich, antiproliferat ive heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan su lfate types were bound with approximately the same affinity to one hig h-affinity site (K-d approximately 10(-8) M) and to one low-affinity s ite (K-d approximately 10(-6) M), respectively. Results of Hill-plot a nalysis suggested that the two sites are independent. Competition expe riments with unlabeled glycosaminoglycans indicated that the binding s ites had a selective specificity for sulfated, L-iduronate-rich hepara n sulfate. Dermatan sulfate, which is also antiproliferative, was weak ly bound to the cells. The antiproliferative effects of heparan and de rmatan sulfate appeared to be additive. Hence, the two glycosaminoglyc ans probably exert their effect through different mechanisms. At conce ntrations above 5 mu g/ml (approximately 10(-7) M), heparan sulfate wa s taken up by human embryonic lung fibroblasts, suggesting that the lo w-affinity site represents an endocytosis receptor. The antiproliferat ive effect of L-iduronate-rich heparan sulfate species was also exerte d at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran su lfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural an alysis of the inactive and active heparan sulphate preparations indica ted that although sulphated L-iduronate appears essential for antiprol iferative activity, it is not absolutely required for binding to the c ells. Degradation of internalized heparan sulfate was analyzed by poly acrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiprolifera tive one was only marginally affected. (C) 1997 Wiley-Liss, Inc.