An optimized protocol for isolation of S-nitrosylated proteins from C. elegans.

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants.

EPIC: software toolkit for elution profile-based inference of protein complexes.

Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container (https://hub.docker.com/r/baderlab/bio-epic/).

Biochemical Approaches to Studying Caenorhabditis elegans ESCRT Functions In Vitro.

Our fundamental understanding of the roles played by the endosomal sorting complex required for transport (ESCRT) machinery in cells comes from interdisciplinary approaches that combine numerous in vivo and in vitro techniques. Here, we focus on methods used to biochemically characterize Caenorhabditis elegans ESCRT components in vitro, including the production and characterization of recombinant ESCRT complexes and their use in membrane interaction studies. Key methodologies used include gel filtration chromatography, glycerol density gradient analysis, multi-angle light scattering, liposome co-flotation, and single-liposome fluorescence microscopy. Collectively, these studies have enabled us to define subunit stoichiometry of soluble C. elegans ESCRT complexes and to demonstrate that the late-acting ESCRT-III complex facilitates membrane bending and remodeling, at least in part by virtue of its ability to sense the curvature of lipid bilayers.

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein. The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.).

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms.

Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11-amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α-helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2 O2 ), suggesting a protective role in a chaperone-like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress-response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.

A complex containing the O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 regulates GABA neuron function.

Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.

Size of Protein is a Major Factor that Affects Retention on Preparative IMAC Columns.

Immobilized metal affinity chromatography (IMAC) is a specific high-capacity technique used in large-scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6× histidine, (His)6-tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow-rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine-containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow-through (output). Data obtained suggest that regardless of the flow-rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium-sized proteins (~ 50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.

Expression and purification of the SNX1/SNX6 complex.

The sorting nexin (SNX) family proteins play an essential role in vesicular transport, cell signaling, and membrane remodeling. The SNX members SNX1/2 and SNX5/6 form dimers, and mediate endosome-to-trans Golgi network (TGN) transport through coordinating cargo selection and membrane remodeling. It is well-known how a SNX-BAR protein forms a homodimer; however, it is less clear how a heterodimer is formed. Here a detailed expression and purification protocol of the SNX1/SNX6 complex, from both worm and human, is described. Keys to the successful protein production include co-expression of both genes, and inclusion of glycerol in the protein buffer. Solution studies suggest that SNX1 and SNX6 form a 1:1 heterodimer. The production of a large amount, high quality of the SNX1/SNX6 complex provides a basis for future biochemical and structural studies of the complex, and in vitro reconstitution of SNX1/SNX6-mediated transport.

Cell type-specific transcriptome profiling in C. elegans using the Translating Ribosome Affinity Purification technique.

Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods. Here, we describe the use of the Translating Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression patterns at the level of translating mRNAs. In TRAP, a ribosomal protein is fused to a tag (GFP) and is expressed under cell type-specific promoters to mark genetically defined cell types in vivo. Affinity purification of lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNAs are used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide profiles of transcripts from the targeted cell type. The ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to enable the discovery of molecular components in response to external or genetic perturbations.

PRMT-5 converts monomethylarginines into symmetrical dimethylarginines in Caenorhabditis elegans.

The transmethylation to arginine residues of proteins is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric (ADMA) and symmetric dimethylarginines (SDMA). Although we previously demonstrated that the generation of ADMA residues in whole proteins is driven by PRMT-1 in Caenorhabditis elegans, much less is known about MMA and SDMA in vivo. In this study, we measured the amounts of different methylarginines in whole protein extracts made from wild-type (N2) C. elegans and from prmt-1 and prmt-5 null mutants using liquid chromatography-tandem mass spectrometry. Interestingly, we found that the amounts of MMA and SDMA are about fourfold higher than those of ADMA in N2 protein lysates using acid hydrolysis. We were unable to detect SDMA residues in the prmt-5 null mutant. In comparison with N2, an increase in SDMA and decrease in MMA were observed in prmt-1 mutant worms with no ADMA, but ADMA and MMA levels were unchanged in prmt-5 mutant worms. These results suggest that PRMT-1 contributes, at least in part, to MMA production, but that PRMT-5 catalyzes the symmetric dimethylation of substrates containing MMA residues in vivo.

The C. elegans Taste Receptor Homolog LITE-1 Is a Photoreceptor.

Many animal tissues/cells are photosensitive, yet only two types of photoreceptors (i.e., opsins and cryptochromes) have been discovered in metazoans. The question arises as to whether unknown types of photoreceptors exist in the animal kingdom. LITE-1, a seven-transmembrane gustatory receptor (GR) homolog, mediates UV-light-induced avoidance behavior in C. elegans. However, it is not known whether LITE-1 functions as a chemoreceptor or photoreceptor. Here, we show that LITE-1 directly absorbs both UVA and UVB light with an extinction coefficient 10-100 times that of opsins and cryptochromes, indicating that LITE-1 is highly efficient in capturing photons. Unlike typical photoreceptors employing a prosthetic chromophore to capture photons, LITE-1 strictly depends on its protein conformation for photon absorption. We have further identified two tryptophan residues critical for LITE-1 function. Interestingly, unlike GPCRs, LITE-1 adopts a reversed membrane topology. Thus, LITE-1, a taste receptor homolog, represents a distinct type of photoreceptor in the animal kingdom.

Benchmarking of protein carbonylation analysis in Caenorhabditis elegans: specific considerations and general advice.

Oxidative stress has been extensively studied due to its correlation with cellular disorders and aging. In proteins, one biomarker of oxidative stress is the presence of carbonyl groups, such as aldehyde and ketone, in specific amino acid side chains such as lysine, proline, arginine and threonine, so-called protein carbonylation (PC). PC study is now a growing field in general and medical science since PC accumulation is associated with various pathologies and disorders. At present, enzyme-linked immunosorbent assays (ELISA) seem to be the most robust method of quantifying the presence of carbonyl groups in proteins, despite having some recognised caveats. In parallel, gel-based approaches present cross-comparison difficulties, along with other technical problems. As generic PC analyses still suffer from poor homogeneity, leading to cross-data analysis difficulties and poor results overlap, the need for harmonisation in the field of carbonyl detection is now widely accepted. This study aims to highlight some of the technical challenges in proteomic gel-based multiplexing experiments when dealing with PC in difficult samples like those from Caenorhabditis elegans, from protein extraction to carbonyl detection. We demonstrate that some critical technical parameters, such as labelling time, probe concentration, and total and carbonylated protein recovery rates, should be re-addressed in a sample-specific way. We also defined a procedure to cost-effectively adapt CyDye™-hydrazide-based protocols to specific samples, especially when the experimental interest is focused on studying differences between stimulating conditions with a maximised signal-to-noise ratio. Moreover, we have improved an already-existing powerful solubilisation buffer, making it potentially useful for hard-to-solubilise protein pellets. Lastly, the depicted methodology exemplifies a simple way of normalising carbonyl-related signal to total protein in SDS-PAGE multiplexing experiments. Within that scope, we also proposed a simple way to quantify carbonyl groups by on-gel spotting diluted dye-containing labelling buffer. Proof of the robustness of the procedure was also highlighted by the high linear correlation between the level of carbonyls and the ultraviolet exposure duration of whole worms (R2=0.993). Altogether, these results will help to standardise existing protocols in the growing field of proteomic carbonylation studies.

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans.

To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 µm in diameter. From a saturated forward genetic screen of 6.7 × 10(5) mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 µm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.

Purification, crystallization and initial crystallographic analysis of the α-catenin homologue HMP-1 from Caenorhabditis elegans.

Adherens junctions transmit mechanical force between cells. In these junctions, ß-catenin binds to cadherins and to the N-terminal domain of α-catenin, which in turn binds to actin filaments via its C-terminal domain. The middle (M) domain of α-catenin plays an important role in responding to mechanical tension. The nematode Caenorhabditis elegans contains α- and ß-catenin homologues called HMP-1 and HMP-2, respectively, but HMP-1 behaves differently from its mammalian homologue. Thus, structural and biochemical studies of HMP-1 have been initiated to understand the mechanism of HMP-1 and the evolution of α-catenin. The N-terminal domain of HMP-1 in complex with the minimal HMP-1-binding region of HMP-2 was purified and crystallized. These crystals diffracted to 1.6 Å resolution and belonged to space group P3(1)21, with unit-cell parameters a = b = 57.1, c = 155.4 Å. The M domain of HMP-1 was also purified and crystallized. The M-domain crystals diffracted to 2.4 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 72.8, b = 81.5, c = 151.4 Å. Diffraction data were collected and processed from each crystal, and the structures were solved by molecular replacement.

NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

Method: In vitro analysis of pericentriolar material assembly.

Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.

Purification of galectin-1 mutants using an immobilized Galactoseß1-4Fucose affinity adsorbent.

Galectins are a family of lectins characterized by their carbohydrate recognition domains containing eight conserved amino acid residues, which allows the binding of galectin to ß-galactoside sugars such as Galß1-4GlcNAc. Since galectin-glycan interactions occur extracellularly, recombinant galectins are often used for the functional analysis of these interactions. Although it is relatively easy to purify galectins via affinity to Galß1-4GlcNAc using affinity adsorbents such as asialofetuin-Sepharose, it could be difficult to do so with mutated galectins, which may have reduced affinity towards their endogenous ligands. However, this is not the case with Caenorhabditis elegans galectin LEC-6; binding to its endogenous recognition unit Galß1-4Fuc, a unique disaccharide found only in invertebrates, is not necessarily affected by point mutations of the eight well-conserved amino acids. In this study, we constructed mutants of mouse galectin-1 carrying substitutions of each of the eight conserved amino acid residues (H44F, N46D, R48H, V59A, N61D, W68F, E71Q, and R73H) and examined their affinity for Galß1-4GlcNAc and Galß1-4Fuc. These mutants, except W68F, had very low affinity for asialofetuin-Sepharose; however, most of them (with the exception of H44F and R48H) could be purified using Galß1-4Fuc-Sepharose. The affinity of the purified mutant galectins for glycans containing Galß1-4Fuc or Galß1-4GlcNAc moieties was quantitatively examined by frontal affinity chromatography, and the results indicated that the mutants retained the affinity only for Galß1-4Fuc. Given that other mammalian galectins are known to bind Galß1-4Fuc, our data suggest that immobilized Galß1-4Fuc ligands could be generally used for easy one-step affinity purification of mutant galectins.