Chawla D, Hughes R C
National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K.
Biochem J. 1991 Oct 1;279 ( Pt 1)(Pt 1):159-65. doi: 10.1042/bj2790159.
Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1----2 on both alpha 1----3- and alpha 1----6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.
布雷菲德菌素A（BFA）是一种能诱导高尔基体蛋白重新分布到内质网中的药物，被用于确定亚细胞区室化在天冬酰胺连接寡糖加工过程中的作用。用[3H]甘露糖对幼仓鼠肾细胞进行30 - 60分钟的脉冲标记，然后在有或没有BFA的情况下追踪长达数小时，或者在有或没有该药物的情况下连续标记数小时。用链霉蛋白酶将细胞糖蛋白消化成糖肽，然后通过凝集素亲和色谱法将其分离成聚糖类别，或者用内切糖苷酶H和内切糖苷酶D进一步消化。通过纸色谱法将后一过程中获得的释放寡糖彼此分离，并与抗内切糖苷酶糖肽分离。结果表明，BFA能诱导蛋白连接的Glc3Man9GlcNAc2寡糖非常快速地加工成Man5GlcNAc2，并转化为复合型和杂合型聚糖。未处理细胞和BFA处理细胞之间的主要差异在于，后者细胞中双触角型和杂合型聚糖大幅增加。这些结果表明，单触角型GlcNAcMan5GlcNAc2杂合聚糖的半乳糖基化会阻断甘露糖苷酶II和N - 乙酰葡糖胺基转移酶II的后续作用，从而产生半乳糖基化的杂合型聚糖。同样，N - 乙酰葡糖胺基转移酶I和II产物的半乳糖基化，即在α1----3 - 和α1----6连接的甘露糖残基上都被GlcNAcβ1----2取代的Man3GlcNAc2核心，会阻断分支N - 乙酰葡糖胺基转移酶IV和V，从而导致双触角型聚糖增加，三触角型和四触角型聚糖减少。
