Methods of in vitro study of galectin-glycomaterial interaction

Heine, Viktoria; Dey, Carina; Bojarová, Pavla; Křen, Vladimı́r; Elling, Lothar

doi:10.1016/j.biotechadv.2022.107928

Cited by 14 publications

(12 citation statements)

References 167 publications

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“…The processes involving Gal-3 in vivo are complex, therefore fine-tuning in which interactions are possible to observe come down to versatile in vitro methods. Sophisticated analytical methods need to be applied, both from an instrumental perspective considering sensitivity and selectivity, and from a theoretical modeling/molecular simulations perspective ( Heine et al, 2022 ). The refinement of perspective galectin antagonists needs to incorporate detailed insight into molecular interactions supported by various instrumental methods.…”

Section: Galectin-3 Targeted Strategies For Preserving Cognitionmentioning

confidence: 99%

Galectin-3 Involvement in Cognitive Processes for New Therapeutic Considerations

Mijailović

Vesić

Arsenijević

et al. 2022

Front. Cell. Neurosci.

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Cognitive impairment may be a consequence of the normal aging process, but it may also be the hallmark of various neurodegenerative and psychiatric diseases. Early identification of individuals at particular risk for cognitive decline is critical, as it is imperative to maintain a cognitive reserve in these neuropsychiatric entities. In recent years, galectin-3 (Gal-3), a member of the galectin family, has received considerable attention with respect to aspects of neuroinflammation and neurodegeneration. The mechanisms behind the putative relationship between Gal-3 and cognitive impairment are not yet clear. Intrigued by this versatile molecule and its unique modular architecture, the latest data on this relationship are presented here. This mini-review summarizes recent findings on the mechanisms by which Gal-3 affects cognitive functioning in both animal and human models. Particular emphasis is placed on the role of Gal-3 in modulating the inflammatory response as a fine-tuner of microglia morphology and phenotype. A review of recent literature on the utility of Gal-3 as a biomarker is provided, and approaches to strategically exploit Gal-3 activities with therapeutic intentions in neuropsychiatric diseases are outlined.

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Section: Galectin-3 Targeted Strategies For Preserving Cognitionmentioning

confidence: 99%

Galectin-3 Involvement in Cognitive Processes for New Therapeutic Considerations

Mijailović

Vesić

Arsenijević

et al. 2022

Front. Cell. Neurosci.

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“…In recent years, the multivalent presentation of ligands became crucial for increasing the galectin affinities to glycan ligands [ 14 , 15 , 50 ]. Since ASF and fetuin were identified as suitable multivalent binders for galectins [ 19 , 44 ], we established glycoprotein-decorated affinity resins based on these natural multivalent glycoproteins. We coupled ASF and fetuin to CNBr-activated Sepharose TM 4 Fast Flow with 15 mg/mL ± 0.7 mg/mL for asialofetuin and 16.4 mg/mL ± 0.9 mg/mL for fetuin, respectively ( Table S1 ).…”

Section: Discussionmentioning

confidence: 99%

“…The design and development of these galectin-specific tools and applications are usually preceded by detailed investigations of galectin’s carbohydrate specificities and binding affinities. During the last decades, various solid-phase, in-solution, and structural analytical methods emerged for studying galectin glycomaterials interactions [ 19 ]. In particular, solid-phase-based assays require the addition of functional domains for detection or immobilization issues.…”

Section: Introductionmentioning

confidence: 99%

“…Fetuin and ASF were utilized as natural glycoprotein ligands to purify mammalian lectins [ 37 , 38 , 39 , 40 , 41 , 42 ] or human antibodies [ 43 ]. Although ASF and Fetuin have been identified as suitable binders in galectin glycomaterial interactions [ 19 , 44 ], these glycoproteins are rarely used to purify galectins from bacterial crude extracts [ 33 , 45 , 46 ].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

2023

Self Cite

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Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

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“…In the past years, the multivalent presentation of ligands became crucial for increasing the galectin affinities to glycan ligands [14,15,50]. Since ASF and fetuin were identified as suitable multivalent binders for galectins [19,44], we established glycoprotein-decorated affinity resins based on these natural multivalent glycoproteins. We coupled ASF and fetuin to CNBr-activated Sepharose TM 4 Fast Flow with 15 mg/mL ± 0.7 mg/mL for asialofetuin and 16.4 mg/mL ± 0.9 mg/mL for fetuin, respectively (Table S1).…”

Section: Galectin Purification On Glycoprotein Columnsmentioning

confidence: 99%

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Dey¹,

Palm²,

Elling³

2023

Preprint

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Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

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Methods of in vitro study of galectin-glycomaterial interaction

Cited by 14 publications

References 167 publications

Galectin-3 Involvement in Cognitive Processes for New Therapeutic Considerations

Galectin-3 Involvement in Cognitive Processes for New Therapeutic Considerations

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

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