Zeng Y C, Lehrman M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1990 Feb 5;265(4):2296-305.
A mutation in glycoprotein processing inhibitor-resistant (PIR) Chinese hamster ovary (CHO) cells was previously shown to result in a block at the Man5GlcNAc2 stage of the dolichol-oligosaccharide biosynthetic pathway (Lehrman, M.A., and Zeng, Y. (1989) J. Biol. Chem. 264, 1584-1593). These cells had normal mannose-P-dolichol synthase activity and were able to transfer the Man5GlcNAc2 oligosaccharides to protein. We have now characterized the mutation in greater detail. In PIR cells, biosynthesis of GDP-mannose and mannose-P-dolichol was normal, and pulse-chase analysis indicated that the rate of Man5GlcNAc2-P-P-dolichol formation in vivo was similar to that in parental CHO cells but without subsequent formation of larger intermediates. Cell fusion studies demonstrated that the PIR genotype was recessive and that PIR cells could complement the mutation in B4-2-1 cells, which fail to synthesize mannose-P-dolichol. In contrast to the results obtained with intact cells, incubation of membrane preparations of PIR cells with GDP-[3H]mannose resulted in the synthesis of intermediates containing up to 9 mannose residues, indicating that the cells contained active mannosyltransferases VI to IX. With a simplified assay for the formation of intermediates containing 6 to 9 mannoses, it was shown that physical disruption of PIR cells was able to eliminate the block at the pentamannosyl stage. Furthermore, although the temperature requirements of the reactions for the control CHO and PIR membranes were similar, Man5GlcNAc2-elongating activity in CHO membranes was inhibited by alkaline pH treatment, whereas this treatment irreversibly stimulated the activity in PIR membranes. Taken together, these results suggest that the PIR cells have a recessive defect, and that the missing gene product is required by mannosyltransferase VI in vivo for proper utilization of either mannose-P-dolichol or Man5GlcNAc2-P-P-dolichol. Since the defect was manifested in vivo but not in vitro, this requirement appears necessary for intact cells but not for disrupted cells or isolated membranes.
先前已表明，抗糖蛋白加工抑制剂（PIR）的中国仓鼠卵巢（CHO）细胞中的一种突变会导致在多萜醇寡糖生物合成途径的Man5GlcNAc2阶段出现阻断（Lehrman，M.A.和Zeng，Y.（1989年）《生物化学杂志》264，1584 - 1593）。这些细胞具有正常的甘露糖 - P - 多萜醇合成酶活性，并且能够将Man5GlcNAc2寡糖转移到蛋白质上。我们现在对该突变进行了更详细的表征。在PIR细胞中，GDP - 甘露糖和甘露糖 - P - 多萜醇的生物合成是正常的，脉冲追踪分析表明，体内Man5GlcNAc2 - P - P - 多萜醇的形成速率与亲本CHO细胞中的相似，但没有随后形成更大的中间体。细胞融合研究表明，PIR基因型是隐性的，并且PIR细胞可以互补B4 - 2 - 1细胞中的突变，B4 - 2 - 1细胞无法合成甘露糖 - P - 多萜醇。与完整细胞的结果相反，用GDP - [³H]甘露糖孵育PIR细胞的膜制剂会导致合成含有多达9个甘露糖残基的中间体，这表明细胞含有活性甘露糖基转移酶VI至IX。通过一种简化的测定法来检测含有6至9个甘露糖的中间体的形成，结果表明PIR细胞的物理破坏能够消除五甘露糖基阶段的阻断。此外，尽管对照CHO和PIR膜反应的温度要求相似，但CHO膜中的Man5GlcNAc2延长活性受到碱性pH处理的抑制，而这种处理不可逆地刺激了PIR膜中的活性。综上所述，这些结果表明PIR细胞存在隐性缺陷，并且甘露糖基转移酶VI在体内正确利用甘露糖 - P - 多萜醇或Man5GlcNAc2 - P - P - 多萜醇需要缺失的基因产物。由于该缺陷在体内而非体外表现出来，这种需求对于完整细胞似乎是必要的，但对于破碎的细胞或分离的膜则不是。
