糖蛋白（聚糖）以及生物結合物通過介導細胞與細胞間的相互作用，對生物機體發(fā)揮至關重要的作用。糖鏈結構的變化在許多疾病，包括腫瘤、糖尿病以及自身免疫性疾病等中發(fā)揮核心作用。但是，目前只有很小一部分的生物標志物是與細胞表面的糖蛋白相關。主要原因是單糖形成聚糖的方式多樣性和復雜性，導致對復雜糖蛋白（例如，凝集素）結構的研究進展很慢。

凝集素（Lectin）一詞源于拉丁語”legere”，意思是“選擇”，指它們具有識別并結合不同碳水化合物結構的能力。但是，為什么這種特性使其在糖生物學上有如此應用前景呢?

隨著生物標志物研究的不斷發(fā)展，利用凝集素進行研究的新方法也逐漸嶄露頭角。本文總結了凝集素作為糖生物學研究工具的5種主要應用。

1.凝集素可靈活應用于組織/細胞免疫組化實驗

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.可實現(xiàn)使用最小的樣本量進行高效的聚糖分析

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.無需糖鏈釋放——直接進行聚糖檢測

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.可用于活細胞分析（Flow-Cytometry流式實驗應用）

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.可有效分離糖蛋白

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).?

結論

凝集素因其獨特的理化特性使其成為研究細胞膜表面糖蛋白結構、功能及其對復雜疾病影響的理想工具。搭配糖樣本制備平臺和分析技術，可快速實現(xiàn)糖樣本的分析，獲得高度一致和可靠的數據結果。

目前，利用凝集素識別和分析糖蛋白樣本的方法有很多，因此需要根據具體的實驗需求選擇最合適的產品。Vector Laboratories公司旨在通過對凝集素工作流程的詳細闡述，為廣大用于提供盡可能多的信息和支持。想要了解更多的凝集素產品的應用，可訪問https://vectorlabs.com/browse/lectins 或咨詢聯(lián)系Vector Laboratories中國獨家代理商欣博盛生物科技有限公司 http://www.yuebanme.com/

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