Cipollo John F, Trimble Robert B
Glycobiology. 2002 Nov;12(11):30G-3G. doi: 10.1093/glycob/cwf083.
The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.
酿酒酵母alg12δ突变体积累带有Man(7)GlcNAc(2)寡糖的寡糖脂。为了确定alg12δ菌株中酿酒酵母糖蛋白上存在的N-聚糖结构，我们尝试纯化外切转化酶，一种高度糖基化的分泌蛋白。这些研究表明，在alg12δ背景下，外切转化酶糖基化程度轻微降低，并迅速被蛋白酶水解破坏。尽管分泌的alg9δ转化酶糖基化程度比alg12δ形式更严重降低，但矛盾的是，它在纯化过程中是稳定的。通过向细胞培养物中添加胃蛋白酶抑制剂A可防止周质转化酶的损失，这表明天冬氨酸蛋白酶具有活性。我们发现，在alg12δ酵母中外切转化酶过表达期间，足够的蛋白酶A被错误靶向到周质空间，在那里它水解转化酶。尽管与alg12δ形式相比，alg9δ转化酶糖基化不足，但它更稳定，因为在这种遗传背景下，与alg12δ细胞相比，分泌的蛋白酶A要少得多。这些观察结果可能与在表征酵母糖基化缺陷时使用其他细胞外蛋白（例如交配因子、α-葡萄糖苷酶）作为探针的研究相关。
