Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling

Gao, Ziyuan; Chen, Sufeng; Du, Jing; Wu, Zhen; Ge, Wei; Gao, Song; Zhou, Zeyang; Yang, Xiaodong; Xing, Yufei; Shi, Minhua; Hu, Yueyun; Tang, Wen; Xia, Jun; Zhang, Xumin; Jiang, Jian‐Hua; Yang, Shuang

doi:10.1039/d3ra00072a

Cited by 7 publications

(3 citation statements)

References 51 publications

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“…Free fucose is usually quantified by HPLC [ 22 , 23 , 24 , 25 ] and enzymatic assays with L-fucose dehydrogenase [ 19 , 26 ]. Recently, lectin-based microfluidic detection assays have been developed [ 27 , 28 ], as well as fluorescence-based assays with probes and electrochemical sensors [ 20 , 29 ]. Shin et al [ 30 ] developed a molecular biosensor for quantifying 2-fucosyllactose (2FL) in breast milk samples.…”

Section: Introductionmentioning

confidence: 99%

Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli

2023

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L-Fucose is a monosaccharide abundant in mammalian glycoconjugates. In humans, fucose can be found in human milk oligosaccharides (HMOs), mucins, and glycoproteins in the intestinal epithelium. The bacterial consumption of fucose and fucosylated HMOs is critical in the gut microbiome assembly of infants, dominated by Bifidobacterium. Fucose metabolism is important for the production of short-chain fatty acids and is involved in cross-feeding microbial interactions. Methods for assessing fucose concentrations in complex media are lacking. Here we designed and developed a molecular quantification method of free fucose using fluorescent Escherichia coli. For this, low- and high-copy plasmids were evaluated with and without the transcription factor fucR and its respective fucose-inducible promoter controlling the reporter gene sfGFP. E. coli BL21 transformed with a high copy plasmid containing pFuc and fucR displayed a high resolution across increasing fucose concentrations and high fluorescence/OD values after 18 h. The molecular circuit was specific against other monosaccharides and showed a linear response in the 0–45 mM range. Adjusting data to the Hill equation suggested non-cooperative, simple regulation of FucR to its promoter. Finally, the biosensor was tested on different concentrations of free fucose and the supernatant of Bifidobacterium bifidum JCM 1254 supplemented with 2-fucosyl lactose, indicating the applicability of the method in detecting free fucose. In conclusion, a bacterial biosensor of fucose was validated with good sensitivity and precision. A biological method for quantifying fucose could be useful for nutraceutical and microbiological applications, as well as molecular diagnostics.

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Section: Introductionmentioning

confidence: 99%

Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli

2023

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“…Advancements in glycoproteomics-mass spectrometry (MS) are key to exploring subtle changes in disease biomarkers, especially altered glycomes in disease. A solid-phase chemoenzymatic approach allows the identification of both N-linked and O-linked glycans using various glycosidases or O-glycoproteases. − To improve specificity and selectivity, these glycoproteins must be comprehensively characterized on their glycosylation, through which their abnormal changes may provide a correlation between tumor stages and disease-specific glycosylation. , …”

Section: Introductionmentioning

confidence: 99%

“…24−28 To improve specificity and selectivity, these glycoproteins must be comprehensively characterized on their glycosylation, through which their abnormal changes may provide a correlation between tumor stages and disease-specific glycosylation. 29,30 Protein glycosylation, the nontemplate biosynthesis of glycans on proteins, is involved in the occurrence and development of lung cancer. Abnormal glycosylation defines tumor malignancy and progression, and is dynamically formed through the altered expression of glycoenzymes in tumor cells.…”

Section: ■ Introductionmentioning

confidence: 99%

Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study

Hu,

Ge,

Xia

et al. 2023

J. Proteome Res.

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Lung cancer is the leading cause of cancer-related death, with high morbidity and mortality rates due to the lack of reliable methods for diagnosing lung cancer at an early stage. Lowdose computed tomography can help detect abnormal areas in the lungs, but only 16% of cases are diagnosed early. Tests for lung cancer markers are often employed to determine genetic expression or mutations in lung carcinogenesis. Serum glycome analysis is a promising new method for early lung cancer diagnosis as glycopatterns exhibit significant differences in lung cancer patients. In this study, we employed a solid-phase chemoenzymatic method to systematically compare glycopatterns in benign cases, adenocarcinoma before and after surgery, and advanced stages of adenocarcinoma. Our findings indicate that serum high-mannose levels are elevated in both benign cases and adenocarcinoma, while complex N-glycans, including fucose and 2,6-linked sialic acid, are downregulated in the serum. Subsequently, we developed an algorithm that utilizes 16 altered N-glycans, 7 upregulated and 9 downregulated, to generate a score based on their intensity. This score can predict the stages of cancer progression in patients through glycan characterization. This methodology offers a potential means of diagnosing lung cancer through serum glycome analysis.

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Lectins and their applications in biomedical research

Gerlach,

Flannery,

Kilcoyne

2024

Translational Glycobiology in Human Health and Disease

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Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling

Abstract: The LAFLQ method quantifies glycoproteins by fluorophore labeling and lectin affinity. On-plate fluorescence detection enables simultaneous analysis of multiple samples. Glycosylations in human biofluids can be achieved using different lectins.

Cited by 7 publications

References 51 publications

Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli

Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli

Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study

Lectins and their applications in biomedical research

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