Brockhausen I, Romero P A, Herscovics A
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Cancer Res. 1991 Jun 15;51(12):3136-42.
The spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture is associated with a decrease in polylactosaminoglycans, particularly those attached to the lysosomal membrane glycoprotein h-lamp-1 (Youakim et al., Cancer Res., 49:6889-6895, 1989). To elucidate the biosynthetic mechanisms leading to these alterations we have compared glycosyltransferase activities that are involved in the synthesis of polylactosaminoglycans and of the N- and O-glycan structures that provide the framework for the attachment of these chains. Glycosyltransferase activities in cell homogenates obtained from undifferentiated and differentiated CaCo-2 cells were assayed by high pressure liquid chromatography separation of enzyme products. The beta-galactosidase activities and extremely high pyrophosphatase activities in differentiated cells were effectively inhibited by 5 mM gamma-galactonolactone and 10 mM AMP, respectively. CaCo-2 cells contain most of the enzymes that are involved in N-glycan branching [N-acetylglucosamine (GlcNAc) transferases I to V] with the exception of GlcNAc transferase VI. The levels of GlcNAc transferase I activities were comparable in undifferentiated and differentiated cells, but GlcNAc transferase II to V activities were significantly increased upon differentiation. The enzyme activities that are directly involved in the synthesis of linear polylactosaminoglycans (Gal beta 4GlcNAc beta 3- repeating units), blood group i UDP-GlcNAc:Gal beta-R beta 3-GlcNAc transferase and UDP-Gal:GlcNAc beta 4-Gal transferase, were found at similar levels in undifferentiated and differentiated CaCo-2 cells. Since GlcNAc transferase III activity is known to inhibit further branching and galactosylation, these results suggest that its increased activity in differentiated CaCo-2 cells may be partly responsible for the decreased synthesis of fucosylated polylactosaminoglycans. Differentiated cells showed a 2-fold increase in O-glycan core 2 UDP-GlcNAc:Gal beta 3GalNAc alpha-R [GlcNAc to N-acetylgalactosamine (GalNAc)] beta 6-GlcNAc transferase activity. In contrast, O-glycan core 1 UDP-Gal:GalNAc alpha-R beta 3-Gal transferase activity was found decreased. Several enzymes that are found in homogenates from normal human colonic tissue are absent or barely detectable in CaCo-2 cells. These include blood group I UDP-GlcNAc:GlcNAc beta 3Gal beta-R (GlcNAc to Gal) beta 6-GlcNAc transferase, O-glycan core 3 UDP-GlcNAc:GalNAc alpha-R beta 3 GlcNAc transferase and O-glycan core 4 UDP-GlcNAc:GlcNAc beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-GlcNAc transferase.
在培养过程中，CaCo-2人结肠腺癌细胞自发分化为肠上皮细胞，这与多乳糖胺聚糖的减少有关，尤其是那些附着在溶酶体膜糖蛋白h-lamp-1上的多乳糖胺聚糖（Youakim等人，《癌症研究》，49:6889 - 6895，1989年）。为了阐明导致这些变化的生物合成机制，我们比较了参与多乳糖胺聚糖合成以及为这些链的附着提供框架的N-和O-聚糖结构合成的糖基转移酶活性。通过高压液相色谱分离酶产物来测定从未分化和分化的CaCo-2细胞获得的细胞匀浆中的糖基转移酶活性。分化细胞中的β-半乳糖苷酶活性和极高的焦磷酸酶活性分别被5 mMγ-半乳糖内酯和10 mM AMP有效抑制。CaCo-2细胞含有参与N-聚糖分支的大多数酶（N-乙酰葡糖胺（GlcNAc）转移酶I至V），但不包括GlcNAc转移酶VI。未分化和分化细胞中GlcNAc转移酶I活性水平相当，但分化后GlcNAc转移酶II至V活性显著增加。直接参与线性多乳糖胺聚糖（Galβ4GlcNAcβ3重复单元）合成的酶活性，即血型i UDP-GlcNAc:Galβ-Rβ3-GlcNAc转移酶和UDP-Gal:GlcNAcβ4-Gal转移酶，在未分化和分化的CaCo-2细胞中水平相似。由于已知GlcNAc转移酶III活性会抑制进一步的分支和半乳糖基化，这些结果表明其在分化的CaCo-2细胞中活性增加可能部分导致了岩藻糖基化多乳糖胺聚糖合成的减少。分化细胞中O-聚糖核心2 UDP-GlcNAc:Galβ3GalNAcα-R [GlcNAc至N-乙酰半乳糖胺（GalNAc）]β6-GlcNAc转移酶活性增加了2倍。相反，发现O-聚糖核心1 UDP-Gal:GalNAcα-Rβ3-Gal转移酶活性降低。在正常人结肠组织匀浆中发现的几种酶在CaCo-2细胞中不存在或几乎检测不到。这些酶包括血型I UDP-GlcNAc:GlcNAcβ3Galβ-R（GlcNAc至Gal）β6-GlcNAc转移酶、O-聚糖核心3 UDP-GlcNAc:GalNAcα-Rβ3 GlcNAc转移酶和O-聚糖核心4 UDP-GlcNAc:GlcNAcβ3GalNAc-R（GlcNAc至GalNAc）β6-GlcNAc转移酶。
