Jacobsson K G, Lindahl U
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Biochem J. 1987 Sep 1;246(2):409-15. doi: 10.1042/bj2460409.
Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.
用无机[35S]硫酸盐对由腹水建立的小鼠肥大细胞瘤细胞培养物进行1小时的脉冲标记，产生了含有分子量为60,000 - 100,000多糖链的标记肝素蛋白聚糖。经过24小时的追踪孵育后，如凝胶色谱所示，大部分35S出现在细胞内多糖片段中，其大小与市售肝素相似，分子量为5000 - 25,000。追踪孵育6小时后从培养物中分离出的产物包括部分降解的游离多糖链，此外还有残留的蛋白聚糖，其大小比最初脉冲标记的蛋白聚糖小。经碱处理从残留的追踪孵育蛋白聚糖中释放的多糖链与脉冲标记1小时后蛋白聚糖衍生的链大小相同。这些结果表明，肝素蛋白聚糖向多糖片段的细胞内降解是由完整多糖链的释放引发的，可能是通过肽酶的作用，并且通过内切糖苷酶对这些链的切割来进行。具有严格底物特异性的内切葡糖醛酸酶[Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278 - 10282]先前已被认为参与了后一步骤。更纯化的肥大细胞瘤细胞（基本上不含巨噬细胞）培养物不会将[35S]肝素蛋白聚糖代谢为多糖片段，而是积累游离的完整多糖链，即假定的完全降解途径的中间体。当这种纯化的细胞与贴壁的小鼠腹膜细胞（可能是巨噬细胞）共培养时，观察到了多糖片段的形成。初步推测肥大细胞内切葡糖醛酸酶活性的表达取决于这些细胞与巨噬细胞之间的协作。
