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凝集素全能小帮手 — 5种方式助力糖生物学研究

2022-04-25
浏览次数: 138

糖蛋白(聚糖)以及生物结合物通过介导细胞与细胞间的相互作用,对生物机体发挥至关重要的作用。糖链结构的变化在许多疾病,包括肿瘤、糖尿病以及自身免疫性疾病等中发挥核心作用。但是,目前只有很小一部分的生物标志物是与细胞表面的糖蛋白相关。主要原因是单糖形成聚糖的方式多样性和复杂性,导致对复杂糖蛋白(例如,凝集素)结构的研究进展很慢。


凝集素(Lectin)一词源于拉丁语”legere”,意思是“选择”,指它们具有识别并结合不同碳水化合物结构的能力。但是,为什么这种特性使其在糖生物学上有如此应用前景呢?


随着生物标志物研究的不断发展,利用凝集素进行研究的新方法也逐渐崭露头角。本文总结了凝集素作为糖生物学研究工具的5种主要应用。


1.凝集素可灵活应用于组织/细胞免疫组化实验

To elicit biologically-relevant information about aberrant glycosylation, you need to visualize cell glycan distribution and its impact on morphology. Fortunately, lectins are compatible tools for molecular imaging protocols, such as immunohistochemistry (IHC) and immunofluorescence (IF), as the tissue staining protocols for antibodies are mostly valid for lectins. Both workflows are simple to implement, producing rapid results. 


In your IHC workflow, you can conjugate a lectin to enzymes (e.g., horseradish peroxidase) or haptens (e.g., biotin). The resulting lectin conjugate and appropriate substrate can be viewed under light microscopy or electron microscopy.  


Your other option for your IF workflow, label lectins with a fluorescent dye (e.g., fluorescein isothiocyanate (FITC)), which allows you to view glycan distribution with a fluorescent microscope. The use of lectins in IF has the added advantage of multiplexing, which allows you to use multiple fluorescent dyes to visualize multiple types of glycan chains in the same sample.


2.可实现使用最小的样本量进行高效的聚糖分析

Scanning a sample for multiple glycan sequences can be time consuming without high-throughput analysis methods. Lectins can provide time-saving and cost-effective analysis with the help of very small solid surfaces, such as microchips.  

Because you want a comprehensive glycan profiling of your sample, you need to detect as many different glycan structures as possible. Compared to antiglycan antibodies which have stringent specificity towards glycans, making them more time-consuming to use, lectins are more versatile in their binding patterns, meaning that one lectin can bind multiple glycans (1). This makes lectins more effective than antibodies as microarray tools.


3.无需糖链释放——直接进行聚糖检测

One of the advantages of lectins is their ability to detect glycans without the need to release them from their bioconjugates. Lectin blotting, based on western blotting, is a method that provides insight into glycan structures while they are still attached to their glycoproteins. Intact glycoproteins or glycolipids can be probed with lectins and there is no requirement for cleaving the whole complex. This also makes lectin blotting a promising tool for comparing glycosylation networks between control and test samples. For instance, researchers could demonstrate altered sialylation and fucosylation of N-glycans in colorectal cancer by comparing biofluid samples from cancer patients to those from healthy subjects (2).  

Lectin microarray is another method for rapid glycan detection that does not require glycan release. This allows you to detect and differentiate all possible disease-associated glycan isomers, aberrant sialic acid linkages, and terminal glycan structures within glycoproteins and glycolipids. For example, lectin microarray was used extensively to help researchers discover predictive biomarkers in several cancer types, such as colorectal (3) and gastric cancer (4).


4.可用于活细胞分析(Flow-Cytometry流式实验应用)

Understanding the correlation between specific glycan structures and cell characteristics is necessary for elucidating cell size, proliferation, and differentiation. To obtain biologically-relevant data, you should be able to work with live cells. The great news is that you can integrate fluorescently labelled lectins into flow cytometry protocols to perform live-cell imaging.  

The use of lectins in flow cytometry is advantageous because it offers an opportunity for quantitative analysis of glycan profiles in different cell subtypes. Lectins can help characterize cellular subpopulations even when a specific cell type is scarce in the overall cell population.  

The combination of lectins and flow cytometry led to many breakthroughs in stem cell research. Using lectins in flow cytometry, researchers were able to successfully characterize human embryonic stem cells (hESCs) and their differentiated progeny based on lectin-binding profiles. This also allowed them to isolate neural progenitor cells to analyze their role in brain development in detail (5).


5.可有效分离糖蛋白

Imagine wanting to analyze a specific aberrant glycoprotein in a sample, but the concentration of that particular glycoprotein is low. The solution is to run affinity chromatography to obtain eluates enriched with the glycoprotein of your interest.  

In lectin affinity chromatography, you can utilize the carbohydrate specificity of lectins by immobilizing them on your chromatography surface or matrix. Upon washing, the immobilized lectin binds the corresponding glycoprotein while the rest of the sample is washed away. The resulting elution is now suitable for mass spectroscopy and proteomics, generating further insight into the glycosylation mechanism of your protein. For example, lectin affinity chromatography played a significant role in the enrichment of core fucosylated peptides that are potential biomarkers in pancreatic cancer (6). 


结论


凝集素因其独特的理化特性使其成为研究细胞膜表面糖蛋白结构、功能及其对复杂疾病影响的理想工具。搭配糖样本制备平台和分析技术,可快速实现糖样本的分析,获得高度一致和可靠的数据结果。

目前,利用凝集素识别和分析糖蛋白样本的方法有很多,因此需要根据具体的实验需求选择最合适的产品。Vector Laboratories公司旨在通过对凝集素工作流程的详细阐述,为广大用于提供尽可能多的信息和支持。想要了解更多的凝集素产品的应用,可访问https://vectorlabs.com/browse/lectins 或咨询联系Vector Laboratories中国独家代理商欣博盛生物科技有限公司 http://www.neobioscience.com/


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