Etchison J R, Freeze H H
La Jolla Cancer Research Foundation, Glycobiology/Carbohydrate Chemistry Program, CA 92037, USA.
Glycobiology. 1996 Mar;6(2):177-89. doi: 10.1093/glycob/6.2.177.
We have developed a new method to co-localize multiple glycosyl transferases in different Golgi compartments. The approach relies on the proven ability of intact, sealed rat liver Golgi preparations to concentrate exogenous labeled sugar nucleotides into the lumen where they glycosylate either endogenous or artificial acceptors. The premise is that if two glycosyl transferases are co-localized within the same compartment, they will compete for the limited amount of transported donor. If the donor is consumed in glycosylating a permeable artificial glycoside within a Golgi compartment, it will be unavailable to glycosylate endogenous products within that same compartment. The greater the degree of transferase co-localization, the greater the potential decrease in glycosylation of endogenous acceptors. We provide an example consistent with these predictions. Adding 1 microM UDP[3H]Gal to Golgi preparations followed by a chase with a cocktail of unlabeled sugar nucleotides labels mostly endogenous N-linked oligosaccharides containing both beta 1,3- and beta 1,4[3H]Gal residues with and without sialic acid. Addition of increasing amounts of 4-methylumbelliferyl-beta-xyloside (Xyl beta MU) produces [3H]Gal1 beta, 4Xyl beta MU and leads to a reciprocal decrease in labeling of a restricted set of the endogenous acceptors. This decrease is preferential for [3H]Gal beta 1-->3GlcNAc beta 1-->R and, to a lesser extent, [3H]Gal beta 1-->4GlcNAc beta 1-->R structures in neutral and mono-sialylated oligosaccharides; synthesis of these structures in di- and tri-sialylated oligosaccharides was unaffected. These preferential decreases are not seen in detergent permeabilized, sugar nucleotide transport-independent Golgi incubations, and are not due to inhibition by the Gal beta 1,4Xyl beta MU product. These results argue that there is significant overlap in the functional co-localization of sialyl and galactosyltransferases in rat liver Golgi preparations and that GAG chain core specific Galactosyltransferase I is co-localized with subsets of N-glycan Gal beta 1,3 and Gal beta 1,4 transferases. This approach can be used with other glycosides and sugar nucleotides to map and co-localize other glycosyl transferases. The functional compartments defined by this approach may or may not correspond entirely with morphologically defined Golgi domains.
我们开发了一种新方法，用于在不同的高尔基体区室中共定位多种糖基转移酶。该方法依赖于完整、密封的大鼠肝脏高尔基体制剂将外源性标记糖核苷酸浓缩到腔内的已证实能力，在腔内它们将内源性或人工受体糖基化。前提是，如果两种糖基转移酶在同一区室中共定位，它们将竞争有限量的转运供体。如果供体在高尔基体区室内将可渗透的人工糖苷糖基化时被消耗，那么它将无法在同一区室内将内源性产物糖基化。转移酶共定位程度越高，内源性受体糖基化的潜在减少就越大。我们提供了一个与这些预测一致的例子。向高尔基体制剂中加入1 microM UDP[3H]Gal，然后用未标记糖核苷酸混合物进行追踪，主要标记含有和不含有唾液酸的同时带有β1,3-和β1,4[3H]Gal残基的内源性N-连接寡糖。加入越来越多的4-甲基伞形酮基-β-木糖苷（XylβMU）会产生[3H]Gal1β,4XylβMU，并导致一组受限的内源性受体的标记呈反向减少。这种减少优先发生在中性和单唾液酸化寡糖中的[3H]Galβ1→3GlcNAcβ1→R，以及程度较小的[3H]Galβ1→4GlcNAcβ1→R结构；在双唾液酸化和三唾液酸化寡糖中这些结构的合成不受影响。在去污剂通透的、与糖核苷酸转运无关的高尔基体孵育中未观察到这些优先减少，并且这不是由于Galβ1,4XylβMU产物的抑制作用。这些结果表明，大鼠肝脏高尔基体制剂中唾液酸转移酶和半乳糖基转移酶在功能共定位上有显著重叠，并且糖胺聚糖链核心特异性半乳糖基转移酶I与N-聚糖Galβ1,3和Galβ1,4转移酶的子集共定位。这种方法可与其他糖苷和糖核苷酸一起用于绘制和共定位其他糖基转移酶。通过这种方法定义的功能区室可能与形态学定义的高尔基体结构域完全对应，也可能不完全对应。
