Detection of Specific Glycan Moieties on the Cell Surface

在目前的工作中，我们已经使用突变的半乳糖基转移酶先前在我们的实验室开发的检测GlcNAc和LacNAc的人宫颈癌细胞（HeLa）的表面上。突变酶具有在供体位点中雕刻的空腔，以容纳在C2处具有化学手柄的UDP-Gal，例如叠氮化物（GalNAz）或酮基（C2-酮基-Gal）。化学手柄用于与携带生物正交基团的荧光探针或生物素缀合以检测受体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Y289 L-M344 H-beta-1，4Gal-T1，其在Mg 2+存在下将GalNAz转移至GlcNAc，用于检测活细胞表面上的GlcNAc残基。Tyr 289 Leu（Y289 L）突变允许切割空腔以容纳UDP-GalNAz，而位于酶的金属结合位点的第二个突变Met 344 His（M344 H）将金属辅因子的需求从Mn 2+改变为Mg 2+。由于与Mg 2+相反，Mn 2+对细胞有毒，因此Mg 2+依赖性酶对于标记活细胞非常有用。使用共聚焦显微镜和流式细胞术研究检测。仅当用唾液酸酶和半乳糖苷酶预处理细胞时，在HeLa细胞上检测到绿色膜荧光信号（对应于DIBO-Alexa 488），表明具有游离GlcNAc残基的聚糖在HeLa细胞表面上不丰富。 使用α-1，3-半乳糖基转移酶（α-1，3GalT）突变酶α-1，3GalT-280 AGG 282检测细胞表面上的LacNAc部分，该突变酶将GalNAz或C2-酮-Gal转移至N-乙酰基-乳糖胺（LacNAc）。GalNAz或C2-酮基-Gal标记的聚糖分别与炔-或氨氧基-生物素偶联。在固定的细胞上，用链霉亲和素-Alexa Fluor 488检测偶联的生物素。在提取物上，用链霉亲和素-HRP检测偶联生物素。与不存在UDP-GalNAz的对照样品相反，在细胞膜上检测到荧光信号。用唾液酸酶预处理的细胞增加了信号强度，表明暴露的LacNAc残基的密度通过去除唾液酸而增加。通过Western印迹分析获得类似的结果。总之，使用Y289 L-M344 H-beta-1，4Gal-T1和alpha-1，3GalT-280 AGG 282酶，以及我们实验室目前正在生产的其他糖基转移酶，可以成为以高特异性研究细胞糖表型的有力工具。

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. 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.