Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.
Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.
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小鼠支持细胞分泌含有糖蛋白的 6-磷酸甘露糖，这些糖蛋白被生精细胞内吞。
DOI：
10.1095/biolreprod49.5.1055
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发表时间：
1993
影响因子：
3.6
通讯作者：
Eddy,EM
中科院分区：
文献类型：
--
作者：
O'Brien,DA;Gabel,CA;Eddy,EM
从青春期前小鼠分离支持细胞，并在无血清培养基中培养，以确定它们是否分泌含有甘露糖6-磷酸（M6 P）的糖蛋白。溶酶体酶前体（通常带有M6 P识别标志物）的条件培养基分析表明，支持细胞选择性分泌β-N-乙酰氨基己糖苷酶和α-甘露糖苷酶，但不分泌β-葡萄糖醛酸酶或β-半乳糖苷酶。用[35 S]甲硫氨酸标记支持细胞代谢，并在阳离子非依赖性M6 P受体亲和柱上分离条件培养基。大多数分泌蛋白不与色谱柱结合（峰A）;然而，约10%的放射性以低亲和力部分洗脱（峰B），5 - 11%的回收cpm与色谱柱结合，并用2.5 mM M6 P洗脱（峰C）。通过一维和二维电泳和荧光分析法分析了每个馏分中的放射性标记蛋白质。峰B中存在分子量为30，000和35，000的两条蛋白质条带。峰C含有至少10种分子量在30，000和135，000之间且等电点< 6.5的含M6 P的糖蛋白。通过用特异性抗体进行免疫沉淀，将峰B和C中突出的35，000分子量组分鉴定为组织蛋白酶原L。当粗线期精母细胞和圆形精子细胞培养过夜的存在下，峰C糖蛋白放射性标记的125 I，这两种生殖细胞类型积累这些Sertoli M6 P-糖蛋白的受体介导的过程，特异性抑制M6 P。由生殖细胞摄取的Sertoli M6 P-糖蛋白被加工成较低分子量的形式。这些结果提供的证据表明，生精细胞表面上的M6 P受体内吞分泌的糖蛋白，可能存在于生精上皮。
Sertoli cells were isolated from prepubertal mice and cultured in serum-free medium to determine whether they secrete glycoproteins containing mannose 6-phosphate (M6P). Assays of the conditioned medium for lysosomal enzyme precursors, which typically bear the M6P recognition marker, indicated that Sertoli cells selectively secreted β-N-acetylhexosaminidase and alpha-mannosidase, but not β-glucuronidase or β-galactosidase. Sertoli cells were labeled metabolically with [35S]methionine and the conditioned medium was fractionated on a cation-independent M6P receptor affinity column. Most of the secreted proteins did not bind to the column (peak A); however, ~ 10% of the radioactivity eluted as a low-affinity fraction (peak B), and 5−11% of the recovered cpm bound to the column and were eluted with 2.5 mM M6P (peak C). The radiolabeled proteins in each fraction were analyzed by one- and two-dimensional electrophoresis and fluorography. Two protein bands with molecular weights of 30,000 and 35,000 were present in peak B. Peak C contained at least ten M6P-containing glycoproteins with molecular weights between 30,000 and 135,000 and isoelectric points < 6.5. The 35,000-molecular-weight constituent prominent both in peaks B and C was identified as procathepsin L by immunoprecipitation with a specific antibody. When pachytene spermatocytes and round spermatids were cultured overnight in the presence of peak C glycoproteins radiolabeled with125I, both germ cell types accumulated these Sertoli M6P-glycoproteins by a receptor-mediated process that was specifically inhibited by M6P. The Sertoli M6P-glycoproteins taken up by germ cells were processed to lower molecular weight forms. These results provide evidence that M6P receptors on the surface of spermatogenic cells endocytose secrete glycoproteins that are likely to be present in the seminiferous epithelium.