Integrated analysis of glycan and RNA in single cells

Minoshima, Fumi; Ozaki, Haruka; Tateno, Hiroaki

doi:10.1101/2020.06.15.153536

Cited by 6 publications

(12 citation statements)

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“…We aimed to develop a strategy to profile the glycome of the intact gut microbiota without prior fluorescence labeling using Glycan-seq [15]. Cultured bacterial cells were incubated with DNA-barcoded lectins, which, upon binding, released their DNA barcodes after UV exposure, because lectins were conjugated with DNA barcodes via a photocleavable linker (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Lectins were conjugated to the DNA oligonucleotide as previously described [15]. Briefly, 100 μg of each lectin was dissolved in 100 μl of PBS mixed with dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS) (Funakoshi Co., Ltd., Tokyo, Japan) at 10 times the molar amount and then incubated in the dark for 1 hour at 20°C.…”

Section: Methodsmentioning

confidence: 99%

“…The DNA barcodes derived from lectins were directly extracted from the reads in FASTQ format. The number of DNA barcodes bound to each cell was counted using a barcode DNA counting system (Mizuho Information & Research Institute, Inc., Tokyo, Japan) [15]. The first three bases in each read were removed to better match the DNA barcode sequence.…”

Section: Methodsmentioning

confidence: 99%

“…We recently developed a highly multiplexed glycan profiling method called Glycan-seq, which analyzes bulk and single cells using DNA-barcoded lectins and next-generation sequencing [15]. In this study, we applied Glycan-seq to analyze the gut microbiota of mice without performing any prior bacterial culturing and fluorescence labeling.…”

Section: Introductionmentioning

confidence: 99%

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Glycomic profiling of the gut microbiota by Glycan-seq

Oinam

Minoshima

Tateno

2021

Preprint

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Background: There has been immense interest in studying the relationship between the gut microbiota and human health. Bacterial glycans modulate the cross talk between the gut microbiota and its host. However, little is known about these glycans because of the lack of appropriate technology to study them. Methods: We previously developed a sequencing-based glycan profiling method called Glycan-seq, which is based on the use of 39 DNA-barcoded lectins. In this study, we applied this technology to analyze the glycome of the intact gut microbiota of mice. Fecal microbiota was incubated with 39 DNA-barcoded lectins exposed to UV, and the number of released DNA barcodes were counted by next-generation sequencing to obtain a signal for each lectin bound to the microbiota. In parallel, the bacterial composition of the gut microbiota was analyzed by 16S rRNA gene sequencing. Finally, we performed a lectin pull-down experiment followed by 16S rRNA gene sequencing to identify lectin-reactive bacteria. Results: The evaluation of cultured gram-positive (Deinococcus radiodurans) and gram-negative (Escherichia coli) bacteria showed significantly distinct glycan profiles between these bacteria, which were selected and further analyzed by flow cytometry. The results of flow cytometry agreed well with those obtained by Glycan-seq, indicating that Glycan-seq can be used for bacterial glycomic analysis. We thus applied Glycan-seq to comparatively analyze the glycomes of young and old mice gut microbiotas. The glycomes of the young and old microbiotas had significantly distinct glycan profiles, which reflect the different bacterial compositions of young and old gut microbiotas based on 16S rRNA gene sequencing. Therefore, the difference in the glycomic profiles between young and old microbiotas may be due to their differing bacterial compositions. α2-6Sia-binders bound specifically to the young microbiota. Lectin pull-down followed by 16S rRNA gene sequencing of the young microbiota identified Lactobacillaceae as the most abundant bacterial family with glycans reacting with α2-6Sia-binders. Conclusion: The Glycan-seq system can, without any prior culturing and fluorescence labeling, reveal the glycomic profile of the intact bacterial gut microbiota. A combination of lectin pull-down and 16S rRNA gene sequencing can identify lectin-reactive bacteria.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Glycomic profiling of the gut microbiota by Glycan-seq

Oinam

Minoshima

Tateno

2021

Preprint

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“…Injection of the "cassette" of ~100 barcoded conjugates in vivo can be used to decrease the number of animals required for experiments and introduce critical internal controls. In vivo injection of multivalent constructs that display glycan-binding proteins (lectins) would be a powerful tool to study in vivo glycobiology; such approach has not yet been exploited yet but closely related DNA-encoded lectin array 54 or phage-displayed glycan array 18 technologies have been reported recently. Many natural lectins function only as homodimers, trimers or tetramers.…”

Section: Discussionmentioning

confidence: 99%

DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

Lima

Atrazhev

Sarkar

et al. 2021

Preprint

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Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ~200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo.

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Quantitative evaluation of glycan‐binding specificity of recombinant concanavalin A produced in lettuce (Lactuca sativa)

Oinam

Hayashi

Hiemori

et al. 2022

Biotech & Bioengineering

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Concanavalin A (ConA), a mannose (Man)‐specific leguminous lectin isolated from the jack bean (Canavalia ensiformis) seed extracts, was discovered over a century ago. Although ConA has been extensively applied in various life science research, recombinant mature ConA expression has not been fully established. Here, we aimed to produce recombinant ConA (rConA) in lettuce (Lactuca sativa) using an Agrobacterium tumefaciens‐mediated transient expression system. rConA could be produced as a fully active form from soluble fractions of lettuce leaves and purified by affinity chromatography. From 12 g wet weight of lettuce leaves, 0.9 mg rConA could be purified. The glycan‐binding properties of rConA were then compared with that of the native ConA isolated from jack bean using glycoconjugate microarray and frontal affinity chromatography. rConA demonstrated a glycan‐binding specificity similar to nConA. Both molecules bound to N‐glycans containing a terminal Man residue. Consistent with previous reports, terminal Manα1‐6Man was found to be an essential unit for the high‐affinity binding of rConA and nConA, while bisecting GlcNAc diminished the binding of rConA and nConA to Manα1‐6Man‐terminated N‐glycans. These results demonstrate that the fully active rConA could be produced using the A. tumefaciens‐mediated transient expression system and used as a recombinant substitute for nConA.

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Integrated analysis of glycan and RNA in single cells

Cited by 6 publications

References 50 publications

Glycomic profiling of the gut microbiota by Glycan-seq

Glycomic profiling of the gut microbiota by Glycan-seq

DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

Quantitative evaluation of glycan‐binding specificity of recombinant concanavalin A produced in lettuce (Lactuca sativa)

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