Detection of Specific Glycan Moieties on the Cell Surface

细胞表面特定聚糖部分的检测

• 批准号: 8349512
• 负责人: Pradman K Qasba
• 金额: $ 18.95万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349512
• 关键词: Alkynes; Antibodies; Azides; Biotin; Catalytic Domain; Cell membrane; Cell physiology; Cell surface; Cells; Chemicals; Complex; Confocal Microscopy; Coupled; Detection; Development; Environment; Enzymes; Excision; Family; Flow Cytometry; Galactosidase; Galactosyltransferases; Glycoconjugates; Hela Cells; Human; Inflammation; Label; Lectin; Life; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Membrane; Metal Binding Site; Metals; Methods; Monosaccharides; Mutate; Mutation; Neuraminidase; Pathology; Polysaccharides; Process; Sampling; Sialic Acids; Signal Transduction; Site; Specificity; Streptavidin; Structure; Surface; Western Blotting; Work; base; cancer cell; cell fixing; cofactor; density; glycosylation; glycosyltransferase; lactosamine; mutant; novel; sugar; tool

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.

在目前的工作中，我们已经使用了先前在实验室中开发的突变体半乳糖基转移酶来检测人宫颈癌细胞（HELA）表面上的GlcNAC和LACNAC。突变酶的腔体已在供体部位雕刻，以适应C2处于C2的化学手柄的UDP-GAL，例如叠氮化物（Galnaz）或Keto组（C2-Keto-Gal）。该化学手柄用于与含有生物正交组织的氟检测或生物素结合，以检测受体GlcNAC或LACNAC。我们使用的是β-1-4-半乳糖基转移酶-1（β-1,4GALT-1），Y289L-M344H-BETA-1,4GAL-T1的双突变体，该突变体在MG2+的情况下将Galnaz转移到GlcNAC上，以检测GLCNAC，用于检测GLCNAC在活细胞表面上的glcnac残基。 Tyr289Leu（Y289L）突变允许雕刻腔体可容纳UDP-GALNAZ，而位于酶的金属结合位点中的第二个突变MET344HI（M344H）将金属辅因子的需求从MN2+到MG2+。由于MN2+与MG2+相反，对细胞有毒，因此MG2+依赖性酶对标记活细胞非常有用。使用共聚焦显微镜和流式细胞仪研究检测。仅当细胞与唾液酸酶和半乳糖苷酶预先治疗时，才在HeLa细胞上检测到绿色膜荧光信号（对应于Dibo-alexa 488），这表明具有游离GlcNAC残基的Glycans在HELA细胞表面上不丰富。 使用alpha-1,3-乳糖基转移酶（alpha-1,3galt）突变酶检测到细胞表面的LACNAC部分，Alpha-1,3Galt-280AGG282，将其转移到galnaz或C2-Keto-Gal或N-乙酰基合素（lacnac）。 galnaz或C2-keto-gal标记的聚糖分别与炔烃或氨基氧基 - 生物素耦合。在固定细胞上，用链霉亲和素 - 埃拉克萨荧光488检测到偶联的生物素。在提取物上，用链霉亲蛋白-HRP检测到偶联的生物素。在细胞膜上检测到荧光信号，而不是在没有UDP-galnaz的对照样品上检测到。用唾液酸酶预处理的细胞增加了信号强度，表明暴露于LACNAC残基的密度通过去除唾液酸而增强。通过Western印迹分析获得了类似的结果。总之，使用Y289L-M344H-BETA-1,4GAL-T1和Alpha-1,3GALT-280AGG282酶以及我们实验室中目前正在生产的其他糖基转移酶的酶的使用，可以是研究具有高特异性的糖脂型的强大工具。