Srikrishna Geetha, Brive Lena, Freeze Hudson H
Glycobiology Program, The Burnham Institute, La Jolla, CA, USA.
Biochem Biophys Res Commun. 2005 Jul 15;332(4):1020-7. doi: 10.1016/j.bbrc.2005.05.056.
We previously reported that N-glycans from bovine lung contain novel carboxylate groups. Here, we provide evidence that the carboxylated glycans contain glutamic acid. We labeled HeLa cells with [2,3-(3)H]glutamate and used a carboxylate-specific monoclonal antibody to enrich for the desired proteins. PNGaseF digestion of these proteins released labeled N-glycans with a free amino group and 1-3 carboxylates. Mild acid hydrolysis had no effect, but strong acid hydrolysis of the glycans released >80% of the (3)H as glutamate. Reducing the carboxylates to alcohols prior to hydrolysis eliminated the [(3)H]glutamate and generated [(3)H]4-amino 5-hydroxy pentanoic acid, suggesting that [(3)H]glutamate was linked to the glycan through its gamma-carboxyl. The glutamate-containing N-glycans resisted exoglycosidase digestion and oligosaccharide processing inhibitors greatly reduced [(3)H]glutamate incorporation. These results demonstrate that mammalian cells synthesize complex-type N-glycans with glutamate linked to their antennae, further expanding their potential for covalent or ionic interactions.
我们之前报道过，牛肺中的N-聚糖含有新型羧基。在此，我们提供证据表明羧化聚糖含有谷氨酸。我们用[2,3-(³)H]谷氨酸标记HeLa细胞，并使用一种羧基特异性单克隆抗体富集所需蛋白质。这些蛋白质经PNGaseF消化后释放出带有游离氨基和1 - 3个羧基的标记N-聚糖。温和酸水解没有效果，但聚糖经强酸水解后释放出超过80%的³H作为谷氨酸。在水解前将羧基还原为醇可消除[³H]谷氨酸，并生成[³H]4-氨基5-羟基戊酸，这表明[³H]谷氨酸是通过其γ-羧基与聚糖相连的。含谷氨酸的N-聚糖抵抗外切糖苷酶消化，并且寡糖加工抑制剂大大降低了[³H]谷氨酸的掺入。这些结果表明，哺乳动物细胞合成了天线连接有谷氨酸的复合型N-聚糖，进一步扩展了它们进行共价或离子相互作用的潜力。
