RESUMO
Electronic and vibrational spectroscopy in a cryogenic ion trap has been applied to protonated dopamine water clusters and assigned with the help of quantum chemistry calculations performed in the ground and electronic excited states. A dramatic hydration effect is observed when dopamine is solvated by three water molecules. The broad electronic spectra recorded for the bare and small water clusters containing protonated dopamine turn to sharp, well-resolved vibronic transitions in the 1-3 complex. This reflects the change induced by hydration in the photodynamics of protonated dopamine which is initially controlled by an excited state proton transfer (ESPT) reaction from the ammonium group toward the catechol ring. Interestingly, conformer selectivity is revealed in the 1-3 complex which shows two low lying energy conformers for which the ESPT reaction is prevented or not depending on the H-bond network formed between the dopamine and water molecules.
Assuntos
Dopamina , Prótons , Dopamina/química , Conformação Molecular , Vibração , Água/químicaRESUMO
Ultraviolet (UV) and infrared (IR) spectra of protonated dopamine (DAH+) and its hydrated clusters DAH+(H2O)1-3 are measured by cryogenic ion spectroscopy. DAH+ monomer and hydrated clusters with up to two water molecules show a broad UV spectrum, while it turns to a sharp, well-resolved one for DAH+-(H2O)3. Excited state calculations of DAH+(H2O)3 reproduce these spectral features. The conformer-selected IR spectrum of DAH+(H2O)3 is measured by IR dip spectroscopy, and its structure is assigned with the help of quantum chemical calculations. The excited state lifetime of DAH+ is much shorter than 20 ps, the cross correlation of the ps lasers, revealing a fast relaxation dynamics. The minimal energy path along the NH â π proton transfer coordinate exhibits a low energy barrier in the monomer, while this path is blocked by the high energy barrier in DAH+(H2O)3. It is concluded that the excited state proton transfer in DAH+ is inhibited by water-insertion.
Assuntos
Dopamina/química , Prótons , Teoria Quântica , Espectrofotometria/métodos , ÁguaRESUMO
Galactoseß1-4fucose (Galß1-4Fuc), a unique disaccharide unit found only on the N-glycans of Protostomia, has been intensively studied, particularly in Nematoda. Galß1-4Fuc attached to the 6-OH of the innermost GlcNAc of N-glycans has been identified as an endogenous target recognized by Caenorhabditis elegans galectin LEC-6 and might function as an endogenous ligand for other galectins as well. Interactions between galectins and N-glycans might be subject to fine-tuning through modifications of the penultimate GlcNAc and the Galß1-4Fuc unit. Similar fine-tuning is also observable in vertebrate galectins, although their major recognition unit is a Galß1-4GlcNAc. In Protostomia, it can be postulated that glycan-binding proteins and their ligands have coevolved; however, epitopes such as Galß1-4Fuc were then hijacked as targets by other organisms. Fungal (Coprinopsis cinerea) galectin 2, CGL2, binds the Galß1-4Fuc on C. elegans glycans to exert its nematotoxicity. Some human and mouse galectins bind to synthesized Galß1-4Fuc; as some parasitic nematodes express this motif, its recognition by mammalian galectins could hypothetically be involved in host defense, similar to fungal CGL2. In this review, we discuss the Galß1-4Fuc unit in Protostomia as a possible equivalent for the Galß1-4GlcNAc unit in vertebrates and a potential non-self glycomarker useful for pathogen recognition.
Assuntos
Dissacarídeos/análise , Galectinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Vias Biossintéticas , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Dissacarídeos/metabolismo , Galectinas/química , Invertebrados/química , Invertebrados/metabolismo , Simulação de Acoplamento Molecular , Alinhamento de SequênciaRESUMO
Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti-insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol-reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab-gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral-polymer coated fused silica capillary (50 µm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser-induced fluorescence under focusing conditions. The insulin-AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first-order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.
Assuntos
Marcadores de Afinidade/química , Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Fragmentos Fab das Imunoglobulinas/química , Insulina/análise , Proteínas Recombinantes/análise , Marcadores de Afinidade/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Insulina/química , Insulina/metabolismo , Focalização Isoelétrica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.
Assuntos
Assialoglicoproteínas/química , Benzofenonas/química , Reagentes de Ligações Cruzadas/química , Cisteína/química , Fetuínas/química , Galectina 1/química , Laminina/química , Maleimidas/química , Marcadores de Fotoafinidade/química , Cisteína/genética , Galectina 1/genética , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/químicaRESUMO
Galectins are a group of lectins that can bind carbohydrate chains containing ß-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-ß1,4-fucose (Galß1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galß1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galß1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galß1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.
Assuntos
Proteínas de Caenorhabditis elegans/química , Fucose/química , Galectinas/química , Oligossacarídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Galactose/química , Galectinas/genética , Galectinas/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Oligossacarídeos/metabolismo , Ligação ProteicaRESUMO
The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a specific amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, κ) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The difference of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the specific Asn residue, which had been mutated. Among five mutants established, three major deamidation sites-H chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering system-were identified, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identified. For the first time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10(-3) h(-1), 4.6 × 10(-3) h(-1), and 1.1 × 10(-3) h(-1) in 0.1 M phosphate buffer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.
Assuntos
Fragmentos Fab das Imunoglobulinas/metabolismo , Cadeias kappa de Imunoglobulina/metabolismo , Mutação/fisiologia , Animais , Eletroforese Capilar/métodos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/análise , Cadeias kappa de Imunoglobulina/genética , Focalização Isoelétrica/métodos , CamundongosRESUMO
Galactoseß1-4Fucose (Galß1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galß1-4Fuc compared to Galß1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galß1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galß1-4Fuc, Galß1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galß1-4Fuc is the most effective. Although the biological significance of galectin-Galß1-4Fuc interaction is obscure, it might be possible that Galß1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galß1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.
Assuntos
Dissacarídeos/metabolismo , Galectina 1/metabolismo , Glicoproteínas/metabolismo , Animais , Cromatografia de Afinidade , Galectina 3/metabolismo , Humanos , Lactose/metabolismo , Ligantes , Camundongos , Conformação Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismoRESUMO
α(1)-Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein-labeled AGP using lectins with the aid of laser-induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A-reactive or Aleuria aurantia lectin-reactive AGP. Labeled AGP was applied at the anodic end of a fused-silica capillary (50 µm id, 360 µm od, 27 cm long) coated with linear polyacryloyl-ß-alanyl-ß-alanine, and electrophoresis was carried out for about 10 min in 60 mM 3-morpholinopropane-1-sulfonic acid-NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin-reactive glycoform peaks from lectin-non-reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin-reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro-purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.
Assuntos
Concanavalina A/metabolismo , Eletroforese Capilar/métodos , Lectinas/metabolismo , Orosomucoide/análise , Biomarcadores/análise , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Orosomucoide/química , Orosomucoide/metabolismoRESUMO
LEC-1 is the first tandem repeat-type galectin isolated from an animal system; this galectin has two carbohydrate recognition domains in a single polypeptide chain. Because its two lectin domains have different sugar-binding profiles, these domains are thought to interact with different carbohydrate ligands. In our previous study, we showed that a mutant of LEC-1 in which a cysteine residue was introduced at a unique position in the N-terminal lectin domain (Nh) can be cross-linked with a model glycoprotein ligand, bovine asialofetuin, by using a bifunctional photoactivatable cross-linking reagent, benzophenone-4-maleimide. In the present work, we applied the same procedure to the C-terminal lectin domain (Ch) of LEC-1. Cross-linked products were formed in the cases of two mutants in which a cysteine residue was introduced at Lys¹77 and Ser²68, respectively. This method is very useful for capturing and assigning endogenous ligand glycoconjugates with relatively low affinities to each carbohydrate recognition domain of the whole tandem repeat-type galectin molecule.
Assuntos
Substituição de Aminoácidos , Assialoglicoproteínas/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Reagentes de Ligações Cruzadas/química , Cisteína/química , Galectinas/química , alfa-Fetoproteínas/química , Animais , Benzofenonas/química , Proteínas de Caenorhabditis elegans/genética , Bovinos , Fetuínas , Galectinas/genética , Glicoconjugados/química , Glicoconjugados/metabolismo , Ligantes , Maleimidas/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Processos Fotoquímicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/químicaRESUMO
To study the endogenous counterpart of LEC-6, a major galectin in Caenorhabditis elegans, the proteomic analysis of glycoproteins captured by an immobilized LEC-6 column was performed using the nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. A protein recovered in a significant amount was determined to be either F57F4.3 or F57F4.4, although the method used could not determine which protein was the actual counterpart. Because the knockdown of the F57F4.3/4 genes in C. elegans is reported to cause growth retardation, we performed a double knockdown of the lec-6 and F57F4.3/4 genes. Although the RNA-mediated interference (RNAi) of lec-6 led to no obvious phenotype, the RNAi of both the lec-6 and F57F4.3/4 genes led to a significant reduction in growth rate when compared to the RNAi of F57F4.3/4 alone. Furthermore, to clarify which protein, F57F4.3 or F57F4.4, was responsible for the retarded growth, the levels of the F57F4.3/4 proteins expressed in a C. elegans wild type and a mutant lacking part of the F57F4.3 gene were compared. The levels of protein expressed by the wild type and the mutant were not significantly different, suggesting that the F57F4.3 protein contributes very little to growth retardation and that the major glycoprotein that interacts with LEC-6 is the F57F4.4 protein. These results suggest that binding with LEC-6 supports the function of F57F4.4 and that their cooperative functioning regulates the growth of C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Galectinas/metabolismo , Glicoproteínas/metabolismo , Animais , Sequência de Bases , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Primers do DNA , Galectinas/genética , Glicoproteínas/genética , Proteínas de Membrana , Reação em Cadeia da Polimerase , Ligação Proteica , Interferência de RNARESUMO
We examined the sugar-cleaving abilities of ß-galactosidases from jack bean and Streptococcus towards sugars containing fucose residues, and found that jack bean ß-galactosidase has an ability to cleave the ß1-3 linkage between galactose (Gal) and fucose (Fuc) residues, but not ß1-4 linkage. On the other hand, streptococcal ß-galactosidase was found to cleave the linkage in both Galß1-4Fuc and Galß1-3Fuc disaccharide units. Such a difference in sugar-cleaving abilities between these 2 ß-galactosidases will be useful for structural analysis of glycans, especially those from species belonging to Protostomia, such as Caenorhabditis elegans.
Assuntos
Dissacarídeos/metabolismo , Fabaceae/enzimologia , Fucose/química , Galactose/química , Polissacarídeos/metabolismo , Streptococcus/enzimologia , beta-Galactosidase/metabolismo , Animais , Caenorhabditis elegans , Polissacarídeos/química , Especificidade por Substrato , beta-Galactosidase/isolamento & purificaçãoRESUMO
Galß1-4Fuc disaccharide unit was recently reported to be the endogenous structure recognized by the galectin LEC-6 isolated from the nematode Caenorhabditis elegans. LEC-1, which is another major galectin from this organism, is a tandem repeat-type galectin that contains two carbohydrate recognition domains, the N-terminal lectin domain (LEC-1Nh) and the C-terminal lectin domain (LEC-1Ch), and was also found to have an affinity for the Galß1-4Fuc disaccharide unit. In the present study, we compared the binding strengths of LEC-1, LEC-1Nh, and LEC-1Ch to Galß1-4Fuc, Galß1-3Fuc, and Galß1-4GlcNAc units as well as to LEC-6-ligand N-glycans by using frontal affinity chromatography (FAC) analysis. The two lectin domains of LEC-1 exhibited the highest affinity for Galß1-4Fuc, though sugar-binding properties differed somewhat between LEC-1Nh and LEC-1Ch. Furthermore, these two domains had significantly lower affinities for the LEC-6-binding glycans. These results suggest that the endogenous recognition unit of LEC-1 is likely to be Galß1-4Fuc, and that the endogenous ligands for LEC-1 are different from those for LEC-6.
Assuntos
Metabolismo dos Carboidratos , Dissacarídeos/metabolismo , Glicoconjugados/metabolismo , Lectinas/metabolismo , Sítios de Ligação , Sequência de Carboidratos , Cromatografia de Afinidade , Dissacarídeos/químicaRESUMO
Galectins comprise a large family of ß-galactoside-binding proteins in animals and fungi. We previously isolated cDNAs of 10 galectin and galectin-like genes (lec-1 to lec-6 and lec-8 to lec-11) from Caenorhabditis elegans and characterized the carbohydrate-binding properties of their recombinant proteins. In the present study, we isolated cDNA corresponding to an open reading frame of the DC2.3a gene from C. elegans total RNA; this cDNA encodes another potential galectin. A recombinant DC2.3a protein was expressed in Escherichia coli and used for analysis. The protein displayed hemagglutinating activity against rabbit erythrocytes, bound to an asialofetuin-Sepharose column, and was eluted with lactose. Furthermore, frontal affinity chromatography (FAC) analysis confirmed that DC2.3a recognized oligosaccharides with a non-reducing terminal galactose. According to these results, we designated DC2.3 as lec-12. The carbohydrate-binding property of the recombinant DC2.3a/LEC-12a was essentially similar to that of LEC-6. Additionally, DC2.3a/LEC-12a and LEC-6 showed higher affinities for the galactoseß1â4fucose (Galß1â4Fuc) disaccharide than for N-acetyllactosamine. This suggests that the principal recognition unit is the Galß1â4Fuc disaccharide as in the case of the C. elegans galectins. However, the recombinant DC2.3a/LEC-12a showed weak affinity for N-glycan E3, which was previously shown to be a preferential endogenous ligand for LEC-6. The DC2.3a/LEC-12a endogenous ligand structures appear to be somewhat different but contain the same galactose-fucose recognition motif.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/química , Dissacarídeos/genética , Galactosídeos/genética , Galectinas/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Dissacarídeos/química , Dissacarídeos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Galactosídeos/química , Galactosídeos/metabolismo , Galectinas/química , Galectinas/metabolismo , Ligantes , Terapia de Alvo Molecular , Plasmídeos , Ligação Proteica , CoelhosRESUMO
Useful segments (1, 2) for chemical probes embedded in a Galß1â4Fuc unit were designed and prepared for characterizing sugar-binding proteins in Caenorhabditis elegans. Segment 1 with an amino group terminus was used as a recognition unit in affinity chromatography. It was revealed that some proteins (annexins and galectins) in C. elegans have an affinity for Galß1â4Fuc.
Assuntos
Proteínas de Caenorhabditis elegans , Fucose/química , Galactose/química , Sondas Moleculares/química , Sondas Moleculares/síntese química , Oligossacarídeos/química , Animais , Sítios de Ligação , Caenorhabditis elegans , Cromatografia de Afinidade , Galectinas/química , Galectinas/metabolismo , Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Oligossacarídeos/metabolismoRESUMO
To search for the endogenous glyco-epitope in Caenorhabditis elegans, we synthesized labeled Galbeta1-3Fuc and Galbeta1-4Fuc and examined their binding affinity for C. elegans galectin LEC-6 using frontal affinity chromatography analysis. We developed a new strategy for synthesizing the labeled saccharides, in which the labeling unit, the 2-aminopyridine moiety, is coupled with a spacer unit derived from D-mannitol. Our results indicate that Galbeta1-4Fuc is the endogenous glyco-epitope present in C. elegans N-glycans.
Assuntos
Caenorhabditis elegans/química , Cromatografia de Afinidade/métodos , Dissacarídeos/síntese química , Corantes Fluorescentes/síntese química , Galectinas/isolamento & purificação , Aminopiridinas/síntese química , Aminopiridinas/química , Animais , Sítios de Ligação , Dissacarídeos/química , Corantes Fluorescentes/química , Manitol/síntese química , Manitol/químicaRESUMO
We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys(28) of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two flanking amino acid residues in the same ß-strand, Ala(27) and Ser(29), to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, confirming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.
Assuntos
Substituição de Aminoácidos , Assialoglicoproteínas , Benzofenonas , Reagentes de Ligações Cruzadas , Cisteína/química , Galectina 1 , Glicoproteínas , Maleimidas , Reagentes de Sulfidrila , alfa-Fetoproteínas , Cisteína/genética , Fetuínas , Galectina 1/genética , Humanos , Ligantes , Mutagênese Sítio-DirigidaRESUMO
Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.
Assuntos
Caenorhabditis elegans/metabolismo , Metabolismo dos Carboidratos , Galectinas/química , Galectinas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/genética , Cromatografia de Afinidade , Clonagem Molecular , Sequência Conservada , DNA Complementar , Galectinas/genética , Galectinas/isolamento & purificação , Genes de Helmintos , Cinética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Filogenia , Sinais Direcionadores de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Galbeta1-4GlcNAc is thought to be a common disaccharide unit preferentially recognized by vertebrate galectins. Eight-amino-acid residues conserved in proteins belonging to the galectin family have been suggested to be responsible for recognition. Meanwhile, we isolated and analyzed endogenous N-glycans of Caenorhabditis elegans that were captured by a C. elegans galectin LEC-6 and demonstrated that the unit of recognition for LEC-6 is a Gal-Fuc disaccharide, though the linkage between these residues was not confirmed. In the present study, we chemically synthesized Galbeta1-4Fuc and Galbeta1-3Fuc labeled with 2-aminopyridine (PA) and demonstrated that LEC-6 interacts with PA-Galbeta1-4Fuc more strongly than PA-Galbeta1-3Fuc by frontal affinity chromatography (FAC). Galbeta1-4Fuc also inhibited hemagglutination caused by LEC-6 more strongly than Galbeta1-3Fuc. FAC analysis using LEC-6 point mutants revealed that some of the conserved amino acid residues which have proven to be important for the recognition of Galbeta1-4GlcNAc are not necessary for the binding to Galbeta1-4Fuc. Another major C. elegans galectin, LEC-1, also showed preferential binding to Galbeta1-4Fuc. These results suggest that Galbeta1-4Fuc is the endogenous unit structure recognized by C. elegans galectins, which implies that C. elegans glycans and galectins may have co-evolved through an alteration in the structures of C. elegans glycans and a subsequent conversion in the sugar-binding mechanism of galectins. Furthermore, since glycans containing the Galbeta1-4Fuc disaccharide unit have been found in organisms belonging to Protostomia, this unit might be a common glyco-epitope recognized by galectins in these organisms.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Dissacarídeos/metabolismo , Galectinas/metabolismo , Glicoconjugados/química , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/metabolismo , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Sequência de Carboidratos , Dissacarídeos/síntese química , Dissacarídeos/química , Galectinas/química , Glicoconjugados/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
R-type lectins are one of the most prominent types of lectin; they exist ubiquitously in nature and mainly bind to the galactose unit of sugar chains. The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris belongs to the R-type lectin family as represented by the plant lectin ricin. It shows haemagglutination activity and is composed of a single peptide chain that includes two homologous domains: N-terminal and C-terminal domains. A truncated mutant of EW29 comprising the C-terminal domain (rC-half) has haemagglutination activity by itself. In order to clarify how rC-half recognizes ligands and shows haemagglutination activity, X-ray crystal structures of rC-half in complex with D-lactose and N-acetyl-D-galactosamine have been determined. The structure of rC-half is similar to that of the ricin B chain and consists of a beta-trefoil fold; the fold is further divided into three similar subdomains referred to as subdomains alpha, beta and gamma, which are gathered around the pseudo-threefold axis. The structures of sugar complexes demonstrated that subdomains alpha and gamma of rC-half bind terminal galactosyl and N-acetylgalactosaminyl glycans. The sugar-binding properties are common to both ligands in both subdomains and are quite similar to those of ricin B chain-lactose complexes. These results indicate that the C-terminal domain of EW29 uses these two galactose-binding sites for its function as a single-domain-type haemagglutinin.