Detection of Specific Glycan Moieties on the Cell Surface

在目前的工作中，我们使用我们实验室以前开发的突变的半乳糖基转移酶来检测人宫颈癌细胞(HeLa)表面的GlcNAc和LacNAc。突变酶在供体部位有一个空腔，以适应在C2处有化学句柄的UDP-Gal，如叠氮(GalNAz)或酮基(C2-keto-Gal)。所述化学手柄用于与荧光探针或携带生物素的生物正交基偶联以检测受体GlcNAc或LacNAc。我们使用β-1-4-半乳糖基转移酶-1(β-1，4GalT-1)的双突变体Y289L-M344H-β-1，4Gal-T1，在镁离子存在的情况下将GalNAz转移到GlcNAc，用于检测活细胞表面的GlcNAc残基。Tyr289Leu(Y289L)突变允许雕刻空洞以容纳UDP-GalNAz，而第二个突变Met344His(M344H)位于酶的金属结合部位，将金属辅因子需求从Mn2+改变为Mg2+。由于与镁离子相比，锰离子对细胞有毒性，因此镁离子依赖酶对活细胞的标记是非常有用的。使用共聚焦显微镜和流式细胞术进行检测。只有在唾液酸酶和半乳糖苷酶处理的HeLa细胞上才能检测到绿膜荧光信号(对应于Dibo-Alexa 488)，这表明HeLa细胞表面没有丰富的含有游离GlcNAc残基的多糖。相反，未成熟糖链上的末端游离bGlcNAc残基存在于血小板受体的糖蛋白Ib(GPIB)上，通过这里描述的方法检测到该受体聚集在4oC。我们进一步证明，双突变酶还能够将修饰的UDP-keto Gal转移到O-GlcNAc修饰的蛋白质α-晶体蛋白。这表明，这种依赖镁的Y289L-M344H-b4Gal-T1突变酶也可以用于检测最近报道的细胞表面蛋白胞外结构域的O-GlcNAc翻译后修饰。因此，突变酶Y289L-M344H-b4Gal-T1可能成为检测N-糖链和O-GlcN酰化胞外蛋白中GlcNAc残基的有用工具。活细胞和组织表面糖链中游离GlcNAc残基的特异性检测可能会对检测细胞糖基化的变化产生重大影响，并最终被用作检测与疾病相关的糖链异常的工具。用α-1，3-半乳糖基转移酶(α-1，3GalT)突变酶α-1，3GalT-280AGG282检测细胞表面的LacNAc部分，该酶将GalNAz或C2-keto-Gal转移到N-乙酰乳糖胺(LacNAc)。GalNAz或C2-keto-Gal标记的多糖分别与炔-生物素或氨氧基-生物素偶联。在固定细胞上，用链霉亲和素-Alexa荧光素488检测偶联生物素。在提取物上，用链霉亲和素-辣根过氧化物酶检测偶联生物素。在细胞膜上检测到荧光信号，而不是对照样品中不存在UDP-GalNAz。用唾液酸酶预处理的细胞，信号强度增加，表明暴露的LacNAc残基的密度通过唾液酸的去除而增加。Western Blot分析也得到了类似的结果。总之，Y289L-M344H-β-1，4Gal-T1和α-1，3GalT-280AGG282酶与本实验室目前正在生产的其他糖基转移酶相结合，可以作为研究细胞高特异性糖表型的有力工具。

In the present work, we have used mutant galactosyltransferases previously developed in our lab to detect GlcNAc and LacNAc on the surface of human cervical cancer cells (HeLa). The mutant enzymes have a cavity that has been carved in the donor site to accommodate UDP-Gal with a chemical handle at C2, such as azide (GalNAz) or keto group (C2-keto-Gal). The chemical handles are used for conjugation with fluoroprobes or biotin carrying bio-orthogonal group to detect the acceptor GlcNAc or LacNAc. We are using a double mutant of beta-1-4-galactosyltransferase-1 (beta-1,4GalT-1), Y289L-M344H-beta-1,4Gal-T1, that transfers GalNAz to GlcNAc in the presence of Mg2+, for detecting GlcNAc residue on the surface of live cells. The Tyr289Leu (Y289L) mutation allows the carving of the cavity to accommodate UDP-GalNAz, whereas the second mutation, Met344His (M344H), located in the enzyme's metal binding site, changes the metal cofactor requirement from Mn2+ to Mg2+. Since Mn2+, in contrast to Mg2+, is toxic to the cells, the Mg2+ dependent enzyme is very useful for labeling of live cells. Detection is investigated using confocal microscopy and flow cytometry. Green membrane fluorescent signal (corresponding to DIBO-Alexa 488) is detected on the HeLa cells only when cells are pre-treated with sialidase and galactosidase enzymes, indicating that glycans with free GlcNAc residues are not abundant on the surface of HeLa cells. In contrast, terminal free bGlcNAc residue on immature glycans present on glycoprotein Ib (GPIb) of platelet receptors, which cluster at 4o C, where detected by the method described here. We have furthermore shown, that the double mutant enzyme is also able to transfer modified UDP-keto Gal to O-GlcNAc modified protein alpha-crystallin. This indicates that this magnesium-dependent Y289L-M344H-b4Gal-T1 mutant enzyme can also be used for the detection of O-GlcNAc post-translational modification of the extracellular domain of cell surface proteins that has recently been reported. For this reason, the mutant enzyme Y289L-M344H-b4Gal-T1 could potentially be a useful tool for the detection of GlcNAc residues from N-glycans as well as from O-GlcNAcylated extracellular proteins. The application of the specific detection of free GlcNAc residue in the glycans on the surface of live cells and tissues could potentially have a great impact in the detection of alterations of cell glycosylation and eventually be utilized as a tool for the detection of glycan aberrations linked to disease. The LacNAc moiety on the cell surface is detected using alpha-1,3-galactosyltransferase (alpha-1,3GalT) mutant enzyme, alpha-1,3GalT-280AGG282 , which transfers GalNAz or C2-keto-Gal to N-acetyl-lactosamine (LacNAc). The GalNAz or C2-keto-Gal labeled glycans are coupled with alkyne- or aminooxy-biotin, respectively. On fixed cells, coupled biotin is detected with streptavidin-Alexa Fluor 488. On extracts, coupled biotin is detected with streptavidin-HRP. Fluorescent signal is detected on cell membranes, as opposed to control samples where no UDP-GalNAz is present. Cells that are pre-treated with sialidase, increased the signal intensity, indicating that the density of exposed LacNAc residues is augmented by the removal of sialic acid. Similar results are obtained by Western Blot analysis. In conclusion, the use of Y289L-M344H-beta-1,4Gal-T1 and alpha-1,3GalT-280AGG282 enzymes, together with other glycosyltransferases currently being produced in our lab, could be powerful tools to investigate the cells glycophenotype with high specificity.