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1.
Proc Natl Acad Sci U S A ; 116(8): 2837-2842, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718416

RESUMO

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.


Assuntos
Galectina 1/química , Galectina 3/química , Glicoconjugados/química , Polissacarídeos/química , Amino Açúcares/química , Amino Açúcares/metabolismo , Sítios de Ligação , Proteínas Sanguíneas , Adesão Celular/genética , Proliferação de Células/genética , Galectina 1/genética , Galectina 3/genética , Galectinas , Humanos , Lactose/química , Ligantes , Nanopartículas/química , Polissacarídeos/genética
2.
Int J Mol Sci ; 23(12)2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35742860

RESUMO

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.


Assuntos
Neoplasias Colorretais , Proteômica , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA , Fatores de Transcrição Forkhead , Galectina 4 , Humanos , Espaço Intracelular/metabolismo , Proteômica/métodos , Recombinases , Fatores de Transcrição
3.
Biochemistry ; 60(7): 547-558, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33560106

RESUMO

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.


Assuntos
Lectinas Tipo C/química , Mucina-1/química , Sequência de Aminoácidos , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Calorimetria/métodos , Epitopos/metabolismo , Galactose , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Mucina-1/metabolismo , Ligação Proteica
4.
Histochem Cell Biol ; 156(3): 253-272, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34152508

RESUMO

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.


Assuntos
Galectinas/metabolismo , Engenharia de Proteínas , Linhagem Celular Tumoral , Galectinas/análise , Galectinas/isolamento & purificação , Humanos , Espectrometria de Massas
5.
Biochem J ; 477(17): 3147-3165, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32766716

RESUMO

Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.


Assuntos
Galectinas/química , Multimerização Proteica , Sítios de Ligação , Linhagem Celular Tumoral , Galectinas/genética , Humanos , Isomerismo , Espectroscopia de Ressonância Magnética
6.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200965

RESUMO

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose-a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.


Assuntos
Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Glucose/metabolismo , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Proteômica/métodos
7.
Cell Tissue Res ; 379(1): 13-35, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31773304

RESUMO

The emerging multifunctionality of galectins by specific protein-glycan/protein interactions explains the interest to determine their expression during embryogenesis. Complete network analysis of all seven chicken galectins (CGs) is presented in the course of differentiation of eye lens that originates from a single type of progenitor cell. It answers the questions on levels of expression and individual patterns of distribution. A qualitative difference occurs in the CG-1A/B paralogue pair, underscoring conspicuous divergence. Considering different cell phenotypes, lens fiber and also epithelial cells can both express the same CG, with developmental upregulation for CG-3 and CG-8. Except for expression of the lens-specific CG (C-GRIFIN), no other CG appeared to be controlled by the transcription factors L-Maf and Pax6. Studying presence and nature of binding partners for CGs, we tested labeled galectins in histochemistry and in ligand blotting. Mass spectrometric (glyco)protein identification after affinity chromatography prominently yielded four types of crystallins, N-CAM, and, in the cases of CG-3 and CG-8, N-cadherin. Should such pairing be functional in situ, it may be involved in tightly packing intracellular lens proteins and forming membrane contact as well as in gaining plasticity and stability of adhesion processes. The expression of CGs throughout embryogenesis is postulated to give meaning to spatiotemporal alterations in the local glycome.


Assuntos
Cristalinas/metabolismo , Galectinas/metabolismo , Cristalino/embriologia , Animais , Western Blotting , Embrião de Galinha , Cromatografia de Afinidade , Galectinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/metabolismo , Ligantes , Fatores de Transcrição Maf/metabolismo , Microscopia de Fluorescência , Fator de Transcrição PAX6/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/metabolismo
8.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32718059

RESUMO

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.


Assuntos
Neoplasias Colorretais , Mutação da Fase de Leitura , Instabilidade Genômica , Repetições de Microssatélites , Proteínas de Neoplasias , Degradação do RNAm Mediada por Códon sem Sentido , Fosfoproteínas , Proteínas de Ligação a RNA , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteômica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
IUBMB Life ; 71(3): 364-375, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30550624

RESUMO

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.


Assuntos
Antineoplásicos/farmacologia , Galectina 4/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Glutamina/metabolismo , Células HCT116 , Humanos , Marcação por Isótopo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/genética , Isótopos de Nitrogênio , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteoma/genética , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes/farmacologia
10.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454892

RESUMO

Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.


Assuntos
Bioensaio , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Vesículas Extracelulares/metabolismo , Instabilidade de Microssatélites , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Aminoácidos/metabolismo , Bioensaio/métodos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Vesículas Extracelulares/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Humanos , Marcação por Isótopo , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes
11.
Cell Commun Signal ; 15(1): 14, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28376875

RESUMO

BACKGROUND: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. METHODS: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. RESULTS: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. CONCLUSION: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.


Assuntos
Neoplasias Colorretais/metabolismo , Reparo de Erro de Pareamento de DNA , Exossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Quimiocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Exossomos/ultraestrutura , Mutação da Fase de Leitura/genética , Células HCT116 , Células Hep G2 , Humanos , Instabilidade de Microssatélites , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteoma/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Reprodutibilidade dos Testes
12.
Molecules ; 22(9)2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28925965

RESUMO

Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated ß-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) ß-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.


Assuntos
Galectinas/química , Glicoconjugados/química , Animais , Sítios de Ligação , Células CHO , Cricetulus , Galactosídeos/química , Humanos , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequências de Repetição em Tandem , Termodinâmica , Aderências Teciduais
13.
Angew Chem Int Ed Engl ; 56(46): 14677-14681, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-28940633

RESUMO

Chemical and biological tools are harnessed to investigate the impact of spatial factors for functional pairing of human lectins with counterreceptors. The homodimeric adhesion/growth-regulatory galectin-1 and a set of covalently linked homo-oligomers from di- to tetramers serve as proof-of-principle test cases. Glycodendrimersomes provide a versatile and sensitive diagnostic platform to reveal thresholds for ligand density and protein concentration in aggregation assays (trans-activity), irrespective of linker length between lectin domains. Monitoring the affinity of cell binding and ensuing tumor growth inhibition reveal the linker length to be a bidirectional switch for cis-activity. The discovery that two aspects of lectin functionality (trans- versus cis-activity) respond non-uniformly to a structural change underscores the power of combining synthetic and biological tools to advance understanding of the sugar functionality of the cell surface.


Assuntos
Lectinas/química , Polissacarídeos/química , Açúcares/química , Membrana Celular/química , Humanos
14.
J Proteome Res ; 15(12): 4412-4422, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27801591

RESUMO

Endogenous lectins have the capacity to translate glycan-encoded information on the cell surface into effects on cell growth. As test cases to examine changes in protein presence associated with tumor growth inhibition, we applied SILAC-based proteomics on human colon carcinoma cells treated with galectin-4 (Gal-4). The five tested lines-LS 180, Vaco 432, Colo 205, CX 1, and HCT 116-responded with differentiation and reduced proliferation to Gal-4 binding. In proteomic analysis (mass spectral data deposited with PRIDE, PXD003489), 2654 proteins were quantified, of which 190 were down-regulated and 115 were up-regulated (>2-fold). 1D annotation analysis of the results indicated down-regulation of DNA replication-associated processes, while protein presence for secretory and transport functions appeared increased. The strongest induction was found for CALB2 (calretinin; ∼24-fold), TGM2 (protein-glutamine γ-glutamyltransferase 2; ∼11-fold), S100A3 (∼10-fold), and GSN (gelsolin; 9.5-fold), and the most pronounced decreases were seen for CDKN2A (tumor suppressor ARF; ∼6-fold), EPCAM (epithelial cell adhesion molecule; ∼6-fold), UBE2C (ubiquitin-conjugating enzyme E2 C; ∼5-fold), KIF2C (kinesin-like protein KIF2C; 5-fold), and LMNB1 (lamin-B1; ∼5-fold). The presence of the common proliferation marker Ki-67 was diminished about 4-fold. By tracing significant alterations of protein expression likely relevant for the observed phenotypic effects, the capacity of a galectin to affect the proteome of human colon cancer cells at multiple sites is revealed.


Assuntos
Membrana Celular/metabolismo , Neoplasias do Colo/metabolismo , Galectina 4/farmacologia , Proteoma/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Galectina 4/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Proteoma/análise , Proteômica/métodos
15.
Artigo em Inglês | MEDLINE | ID: mdl-23908026

RESUMO

A novel Emericella nidulans endo-ß-1,4-galactanase (EnGAL) demonstrates a strong capacity to generate high levels of very potent prebiotic oligosaccharides from potato pulp, a by-product of the agricultural potato-starch industry. EnGAL belongs to glycoside hydrolase family 53 and shows high (72.5%) sequence identity to an endo-ß-1,4-galactanase from Aspergillus aculeatus. Diffraction data extending to 2.0 Å resolution were collected from a crystal of EnGAL grown from conditions containing 0.2 M zinc acetate. The crystal structure showed a high similarity between EnGAL and other endo-ß-1,4-galactanases belonging to GH53. It also revealed 15 zinc ions bound to the protein, one of which is located in the active site, where it is coordinated by residues Glu136 and Glu246 which comprise the catalytic machinery. The majority of the zinc ions are located on the surface of the enzyme, in some cases with side chains from two different molecules as ligands, thus explaining why the presence of zinc ions was essential for crystallization.


Assuntos
Emericella , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Emericella/enzimologia , Emericella/genética , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Difração de Raios X , Zinco/química
17.
Protein Pept Lett ; 23(11): 1003-1012, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27697034

RESUMO

The potent multifunctionality of human galectins is based on their modular structure in a not yet fully understood manner. A strategy to dissect the contributions of individual sequence stretches to lectin activity is based on engineering variants of the natural proteins, which are composed of novel combinations of distinct parts. On proof-of-principle level, we here describe the design of a hybrid constituted by the N-terminal tail of chimera-type galectin-3 and the Nterminal carbohydrate recognition domain of tandem-repeat-type galectin-8, its production, purification and its serine phosphorylation characteristic for galectin- 3's tail. As measured for the respective parental proteins, its binding to (neo)glycoproteins is specific for ß-galactosides and inhibitable by lactose, with KD-value closer to galectin-8 than galectin-3. Cell surface staining indicated similarity of the hybrid's reactivity to O-glycans and sensitivity for sialylation to respective properties of tandem-repeattype galectin-8 and its N-terminal domain. Applied as histochemical tool on tissue sections of murine jejunum and epididymis, intense lactose-inhibitable signals were recorded intracellularly, with a distribution profile akin to that of galectin-3. Tested as agglutinin, the hybrid was potent, excelling wild-type control galectins. The chimera-type design can thus serve as platform for tuning crosslinking activity.


Assuntos
Epididimo/metabolismo , Galactosídeos/metabolismo , Galectina 3/metabolismo , Galectinas/metabolismo , Jejuno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Aglutininas/metabolismo , Animais , Sítios de Ligação/genética , Galectina 3/genética , Galectinas/genética , Humanos , Lactose/química , Masculino , Camundongos , Fosforilação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Coloração e Rotulagem , Relação Estrutura-Atividade
18.
Biochimie ; 128-129: 34-47, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27296808

RESUMO

Occurrence of the adhesion/growth-regulatory galectins as family sets the challenge to achieve a complete network analysis. Along this route taken for a well-suited model organism (chicken), we fill the remaining gap to characterize its seventh member known from rat as galectin-related inter-fiber protein (GRIFIN) in the lens. Its single-copy gene is common to vertebrates, with one or more deviations from the so-called signature sequence for ligand (lactose) contact. The chicken protein is a homodimeric agglutinin with capacity to bind ß-galactosides, especially the histo-blood group B tetrasaccharide, shown by solid-phase/cell assays and a glycan microarray. Mass spectrometric identification of two lactose-binding peptides after tryptic on-bead fragmentation suggests an interaction at the canonical region despite a sequence change from Arg to Val at the site, which impairs reactivity of human galectin-1. RT-PCR and Western blot analyses of specimen from adult chicken organs reveal restriction of expression to the lens, here immunohistochemically throughout its main body. This report sets the stage for detailed structure-activity studies to define factors relevant for affinity beyond the signature sequence and to perform the first complete network analysis of the galectin family in developing and adult organs of a vertebrate.


Assuntos
Proteínas Aviárias/genética , Proteínas do Olho/genética , Galectinas/genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Sítios de Ligação/genética , Western Blotting , Galinhas , Proteínas do Olho/classificação , Proteínas do Olho/metabolismo , Galectinas/classificação , Galectinas/metabolismo , Humanos , Imuno-Histoquímica , Lactose/metabolismo , Cristalino/metabolismo , Filogenia , Ligação Proteica , Multimerização Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
19.
Protein Eng Des Sel ; 28(7): 199-210, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25796447

RESUMO

Lectins translate information encoded in glycan chains of cellular glycoconjugates into bioeffects. The topological presentation of contact sites for cognate sugar binding is a crucial factor toward this end. To dissect the significance of such phylogenetically conserved properties, the design and engineering of non-natural variants are attractive approaches. Here, a homodimeric human lectin, i.e. adhesion/growth-regulatory galectin-1, is converted into a tandem-repeat display by introducing the 33-amino-acid linker of another family member (i.e. galectin-8). The yield of variant was reduced by about a third. This protein had ∼10-fold higher activity in hemagglutination. Nearly complete sequence determination by mass-spectrometric in-source decay and fingerprinting excluded the presence of any modifications. When (1)H-(15)N heteronuclear single-quantum coherence data on the (15)N-labeled variant and wild-type protein were compared, changes in chemical shifts, signal intensities and resonance multiplicities revealed reduction of stability of interfacial contacts between the lectin domains and an increase in inter-domain flexibility. When both binding sites in the variant were loaded with ligand, association of the two carbohydrate recognition domains was enhanced, corroborated by gel filtration. Dynamic changes in the spatial presentation of the two lectin domains in the context of a tandem-repeat display can alter counterreceptor targeting relative to the fixed positions found in the proto-type galectin homodimer.


Assuntos
Galectina 1/química , Galectina 1/genética , Engenharia de Proteínas/métodos , Multimerização Proteica , Sequência de Aminoácidos , Galectina 1/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Sequências de Repetição em Tandem
20.
PLoS One ; 9(1): e83902, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24404142

RESUMO

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production.


Assuntos
Neuraminidase/metabolismo , Polissacarídeos/metabolismo , Trypanosoma rangeli/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Ativação Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neuraminidase/química , Neuraminidase/genética , Polissacarídeos/química , Conformação Proteica , Engenharia de Proteínas , Alinhamento de Sequência , Especificidade por Substrato , Trypanosoma rangeli/genética
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