Lemansky P, Hasilik A
Philipps-Universität Marburg, Institut für Physiologische Chemie, Karl-von-Frisch-Strasse 1, Germany.
J Cell Sci. 2001 Jan;114(Pt 2):345-52. doi: 10.1242/jcs.114.2.345.
Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [(35)S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [(35)S]sulfate in the control, 80% of the [(35)S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH(4)Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells.
人原单核细胞U937可合成溶菌酶，并将其中约三分之一保留在溶酶体内。溶菌酶未进行糖基化修饰；因此，它不能通过依赖甘露糖-6-磷酸的方式靶向溶酶体。它是一种碱性蛋白，pI为10.5，已知可与带负电荷的大分子如蛋白聚糖相互作用。因此，我们研究了后者是否参与U937细胞中溶菌酶的溶酶体靶向过程。我们通过用氯酸盐抑制其硫酸化作用，部分降低了新合成蛋白聚糖的负电荷。这增加了溶菌酶的分泌速率。用佛波酯处理U937细胞后，溶菌酶的分泌速率增加到90%以上。这与一种携带[(35)S]硫酸盐的蛋白聚糖几乎完全重新分布到分泌途径相吻合。在对照组中用[(35)S]硫酸盐短暂脉冲后，80%携带[(35)S]硫酸盐的蛋白聚糖保留在细胞内，而在处理后的细胞中这一比例降至13%。分泌的蛋白聚糖对软骨素酶ABC敏感，并与固定化的溶菌酶结合。这种相互作用被50 - 300 mM NaCl破坏。细胞内保留的蛋白聚糖以50 - 60分钟的半衰期降解，似乎被导向溶酶体，因为在NH(4)Cl存在下，降解受到强烈抑制。我们的结果表明，蛋白聚糖参与U937细胞中溶菌酶的溶酶体靶向过程。
