RESUMEN
Visualization of small micro-(20-1 µm) and nanoplastics (<1 µm) combined with chemical identification is still a challenge. To address this, we designed and manufactured easy-to-handle silicon membrane filters with a standard round filter geometry of 25 mm in diameter and a 10 mm diameter filtration area, holding hexagonal sections with periodically arranged pores of either 250 nm or 1 µm. Due to their flat and reflective surface, the filters serve as a versatile substrate for spectroscopic identification of particles. Optical markers at different levels of magnification, including the bare eye, allow for an easy transfer and repositioning of samples between instruments and methods as well as for a re-measurement of nanoscale particles. We demonstrate how nanoscale particles of weakly absorbing polymers such as polyethylene and polystyrene are analyzed on these filters by nano-FTIR, a combination of atomic force microscopy and Fourier transform infrared spectroscopy. By sequential filtration we separated the fractions of small micro and nanoplastics from a degraded polylactic acid coffee cup lid and achieved subsequent identification by Raman and nano-FTIR spectroscopy. The applications presented in this study will enable future research regarding the identification of small polymer particles difficult to access by other methods.
RESUMEN
Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-ß-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-ß-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidasa/química , Neuraminidasa/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma congolense/enzimología , Acetilgalactosamina/metabolismo , Sitios de Unión , Catálisis , Galactosa/metabolismo , Glicoproteínas/genética , Neuraminidasa/genética , Oligosacáridos/metabolismo , Proteínas Protozoarias/genética , Ácidos Siálicos/metabolismo , Trypanosoma congolense/química , Trypanosoma congolense/genéticaRESUMEN
Hepatoma-derived growth factor (HDGF) is a protein with diverse intracellular functions. Moreover, after non-conventional secretion, extracellular HDGF is able to influence different signaling pathways, leading for example to induction of processes like epithelial-mesenchymal transition (EMT) and cell migration. Intriguingly, in recent proteome studies, HDGF was also found secreted by special microvesicles called exosomes. Recently, we demonstrated the existence of two new HDGF isoforms (B and C). These isoforms are involved in different cellular processes than HDGF-A. Along this line, in the present study we discovered that full length HDGF-A clearly is located inside of exosomes, whereas the isoforms HDGF-B and HDGF-C are found exclusively on the outer surface. Furthermore, while HDGF-B and HDGF-C seem to use exosomes mediated pathway exclusively, HDGF-A was found also as unbound protein in the conditioned media. The new finding of an intra- or extra-exosomal localisation of protein splice variants opens a fascinating new perspective concerning functional diversity of HDGF isoforms. Dysregulation of HDGF expression during cancer development and tumor progression is a commonly known fact. With our new findings, unraveling the potential functional impact according to physiological versus pathophysiologically altered levels and compositions of intra- and extra-exosomal HDGF has to be addressed in future studies.
Asunto(s)
Exosomas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fragmentos de Péptidos/metabolismo , Comunicación Autocrina , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Comunicación Paracrina , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Transcripción GenéticaRESUMEN
Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Dineínas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Modelos Moleculares , Datos de Secuencia Molecular , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/metabolismoRESUMEN
Maternal infection during pregnancy increases the risk for the offspring to develop schizophrenia. However, it is still not fully understood which biochemical mechanisms are responsible for the emergence of neuropsychiatric symptoms following prenatal immune activation. The serotonin (5-hydroxytryptamine, 5-HT) and glutamate system have prominently been associated with the schizophrenia pathophysiology but also with the mechanism of antipsychotic drug actions. Here, we investigated the behavioral and cellular response to 5-HT2A and metabotropic glutamate (mGlu)2/3 receptor stimulation in male and female offspring born to lipopolysaccharide (LPS)-treated mothers. Additionally, we assessed protein expression levels of prefrontal 5-HT2A and mGlu2 receptors. Prenatally LPS-exposed male and female offspring showed locomotor hyperactivity and increased head-twitch behavior in response to the 5-HT2A receptor agonist DOI. In LPS-exposed male offspring, the mGlu2/3 receptor agonist LY379268 failed to reduce DOI-induced prepulse inhibition deficits. In LPS-males, the behavioral changes were further accompanied by enhanced DOI-induced c-Fos protein expression and an up-regulation of prefrontal 5-HT2A receptors. No changes in either 5-HT2A or mGlu2 receptor protein levels were found in female offspring. Our data support the hypothesis of an involvement of maternal infection during pregnancy contributing, at least partially, to the pathology of schizophrenia. Identifying biochemical alterations that parallel the behavioral deficits may help to improve therapeutic strategies in the treatment of this mental illness. Since most studies in rodents almost exclusively include male subjects, our data further contribute to elucidating possible gender differences in the effects of prenatal infection on 5-HT2A and mGlu2/3 receptor function.
Asunto(s)
Complicaciones Infecciosas del Embarazo , Efectos Tardíos de la Exposición Prenatal , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Caracteres Sexuales , Animales , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Lipopolisacáridos , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Corteza Prefrontal/fisiopatología , Embarazo , Inhibición Prepulso/efectos de los fármacos , Inhibición Prepulso/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Wistar , Esquizofrenia/etiología , Esquizofrenia/fisiopatologíaRESUMEN
Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.
Asunto(s)
Carbonato de Calcio/química , Gastrópodos/genética , Lectinas/genética , Nácar/metabolismo , Isoformas de Proteínas/genética , Animales , Teorema de Bayes , Western Blotting , Células COS , Chlorocebus aethiops , Biología Computacional , Cristalización , Cartilla de ADN/genética , ADN Complementario/genética , Electroforesis en Gel Bidimensional , Escherichia coli , Gastrópodos/metabolismo , Lectinas/metabolismo , Proteínas de Unión a Maltosa/metabolismo , Modelos Genéticos , Filogenia , Plásmidos/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.
Asunto(s)
Glicoproteínas/metabolismo , Neuraminidasa/metabolismo , Trypanosoma congolense/enzimología , Secuencia de Aminoácidos , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/genética , Fetuínas/metabolismo , Expresión Génica , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Neuraminidasa/genética , Neuraminidasa/aislamiento & purificación , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Trypanosoma congolense/genéticaRESUMEN
Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.
Asunto(s)
Astrocitos/citología , Astrocitos/enzimología , Catepsina K/deficiencia , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/enzimología , Encéfalo/metabolismo , Catepsina K/metabolismo , Técnicas de Cultivo de Célula , Femenino , Glucosa/metabolismo , Glutatión/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/enzimología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/enzimología , Oligodendroglía/metabolismoRESUMEN
Hepatoma-derived growth-factor-related protein 2 (HRP-2) belongs to a family with five additional members: hepatoma-derived growth factor (HDGF); lens epithelium-derived growth factor; and the HDGF-related proteins -1, -3 and -4. Very little is known regarding the function of HRP-2 in particular. This study shows for the first time heteromer formation of different members of the HRP family; HDGF and HRP-2. In addition, we discovered a previously unknown splice variant of HRP-2 mRNA encoding for a protein with a 53-amino acid deletion in its hath region. This HRP-2 isoform c interacts preferentially with a processed form of HDGF probably because of the loss of an α helix of HRP-2. Furthermore, in contrast to other isoforms of HRP-2, isoform c binds to chromatin similar to its most closely related family member lens epithelium-derived growth factor with potential consequences regarding its function in HIV integration. Interestingly, only the new HRP-2 isoform c and a processed form of HDGF are displaced from condensed mitotic metaphase chromatin. In conclusion, these observations provide a new perspective for understanding the biological functions of HDGF and related proteins.
Asunto(s)
Cromatina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Recombinantes/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , Multimerización de Proteína , ARN Mensajero/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Huso Acromático/fisiologíaRESUMEN
BACKGROUND: Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense. RESULTS: Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated. CONCLUSIONS: The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.
Asunto(s)
Variación Genética , Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidasa/química , Neuraminidasa/metabolismo , Trypanosoma congolense/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Glicopéptidos/metabolismo , Glicoproteínas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Neuraminidasa/genética , Conformación Proteica , Trypanosoma cruzi/enzimologíaRESUMEN
The glycoprotein reelin is important for embryonic neuronal migration. During adulthood reelin possibly acts as a modulator of synaptic plasticity. Several studies link reduced levels of reelin messenger RNA and protein to the pathophysiology of certain neuropsychiatric disorders. However, little is known about reelin's role for behavioral and cognitive functions in vivo. Therefore, the effect of a reelin knockdown in the medial prefrontal cortex (mPFC) of Wistar rats was examined in behavioral tasks related to neuropsychiatric disorders, such as schizophrenia. Rats treated with reelin antisense phosphothioate oligonucleotides in the mPFC during puberty or adulthood were tested for prepulse inhibition (PPI) of the acoustic startle reflex, spatial working memory, object recognition, and locomotor activity. Reelin quantification in the mPFC was assessed by Western blotting. Local reelin knockdown during puberty or adulthood induced (1) a PPI deficit as well as (2) an impairment of spatial working memory and object recognition following pubertal injections. Western blot analyses showed a distinct and highly selective reelin knockdown in the rats' mPFC. These results indicate that mPFC reelin signaling plays an important role in behavioral tasks with relevance to e.g. schizophrenia. Understanding reelin's function as a neurotrophic modulator of the extracellular matrix may help to achieve new insights into the etiology of certain neuropsychiatric diseases and foster prospective treatment strategies.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Trastornos del Conocimiento/fisiopatología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Técnicas de Silenciamiento del Gen , Trastornos del Humor/fisiopatología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Corteza Prefrontal/fisiopatología , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Factores de Edad , Animales , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen/métodos , Masculino , Trastornos del Humor/genética , Trastornos del Humor/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oligorribonucleótidos Antisentido/farmacología , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , Ratas , Ratas Wistar , Proteína Reelina , Maduración Sexual/genéticaRESUMEN
Hepatoma-derived growth factor (HDGF) was first purified as a growth factor secreted by hepatoma cells. It promotes angiogenesis and has been related to tumorigenesis. To date, little is known about the molecular mechanisms of HDGF functions and especially its routes or regulation of secretion. Here we show that secretion of HDGF requires the N-terminal 10 amino acids and that this peptide can mediate secretion of other proteins, such as enhanced green fluorescent protein, if fused to their N-terminus. Our results further demonstrate that cysteine residues at positions 12 and 108 are linked via an intramolecular disulfide bridge. Surprisingly, phosphorylation of serine 165 in the C-terminal part of HDGF plays a critical role in the secretion process. If this serine is replaced by alanine, the N-terminus is truncated, the intramolecular disulfide bridge is not formed and the protein is not secreted. In summary, these observations provide a model of how phosphorylation, a disulfide bridge and proteolytic cleavage are involved in HDGF secretion.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Cisteína/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , TransfecciónRESUMEN
Hepatoma-derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma-derived growth factor, its routes of secretion and internalization or post-translational modifications. In the present study, we show for the first time that hepatoma-derived growth factor is modified by the covalent attachment of small ubiquitin-related modifier 1 (SUMO-1), a post-translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI-TOF-MS based peptide analysis, we identified the lysine residue SUMOylated located in the N-terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma-derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma-derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma-derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma-derived growth factor.
Asunto(s)
Cromatina/metabolismo , Regulación hacia Abajo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína SUMO-1/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células COS , Chlorocebus aethiops , Secuencia de Consenso , Regulación hacia Abajo/genética , Escherichia coli , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína , Proteína SUMO-1/fisiologíaRESUMEN
Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose alpha2,3-sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional alpha2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galbeta1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.
Asunto(s)
Ciona intestinalis/enzimología , Evolución Molecular , Galactosa/metabolismo , Glucolípidos/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Sialiltransferasas/genética , Takifugu/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Ciona intestinalis/genética , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genoma/genética , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Filogenia , Ratas , Sialiltransferasas/química , Especificidad por Sustrato , Takifugu/genética , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
BACKGROUND: Hepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1-4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult rats on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures. RESULTS: HDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative. CONCLUSION: The coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells.
Asunto(s)
Química Encefálica , Péptidos y Proteínas de Señalización Intercelular/análisis , Factores de Edad , Animales , Astrocitos/química , Western Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células Cultivadas/química , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Microglía/química , Proteínas del Tejido Nervioso , Neuronas/química , Oligodendroglía/química , Especificidad de Órganos , Ratas , Ratas WistarRESUMEN
Marburg University Medical Center has been introducing a comprehensive health information system since 1999, using a single-vendor application framework with an integrated generator tool for the development of clinical applications. To find out if this architecture and our participative software engineering approach can be considered a step towards a responsive infrastructure, we compared the situation after the first deployment phase (basically a holistic approach) with the situation after the system was further developed and adapted to the users' needs using the generator tool approach. We collected system statistics and conducted user satisfaction surveys in 2001/02 and 2003 using standardized measurements. The survey results showed that user involvement as well as system content were judged significantly higher after the second deployment phase. Insofar, we could demonstrate the feasibility of our approach. However, definite statements concerning the superiority of the generator tool approach to other concepts are not yet possible. We will continue our assessment, and we strongly suggest further studies in other institutions introducing comparable clinical functionality.
Asunto(s)
Comportamiento del Consumidor , Sistemas de Información en Hospital , Centros Médicos Académicos/organización & administración , Actitud hacia los Computadores , Recolección de Datos , Alemania , Innovación Organizacional , Personal de Hospital , Programas Informáticos , Encuestas y CuestionariosRESUMEN
Siglecs are the largest family of sialic acid-recognizing lectins identified so far with 11 members in the human genome. Most of these siglecs are exclusively expressed by cells of the immune system. Comparison of different mammalian species has revealed differential and complex evolutionary paths for this protein family, even within the primate lineage. To understand the evolution of siglecs, in particular the origin of this family, we investigated the occurrence of corresponding genes in bony fish. Interestingly, only unambiguous orthologs of mammalian siglec-4, a cell adhesion molecule expressed exclusively in the nervous system, could be identified in the genomes of fugu and zebrafish, whereas no obvious orthologs of the other mammalian siglecs were found. As in mammals, fish siglec-4 expression is restricted to nervous tissues as demonstrated by northern blot. Expressed as recombinant protein, fish siglec-4 binds to sialic acids with a specificity similar to the mammalian orthologs. Relatively low sequence similarities in the cytoplasmic tail as well as an additional splice variant found in fish siglec-4 suggest alternative signaling pathways compared to mammalian species. Our observations suggest that this siglec occurs at least in the nervous system of all vertebrates.
Asunto(s)
Evolución Molecular , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Perfilación de la Expresión Génica , Lectinas/genética , Lectinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Takifugu/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Clonación Molecular , Exones/genética , Proteínas de Peces/química , Genómica , Humanos , Intrones/genética , Lectinas/química , Datos de Secuencia Molecular , Unión Proteica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Especificidad por SustratoRESUMEN
HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.
Asunto(s)
Encéfalo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Animales , Especificidad de Anticuerpos , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Química Encefálica , Proteínas de Ciclo Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Expresión Génica , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular , Ratones , Neuronas/metabolismo , Proteínas Nucleares/genética , ARN Mensajero/análisis , RatasRESUMEN
Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. We describe the functional consequences of three mis-sense mutations in the arylsulphatase A gene (Asp-335-Val, Arg-370-Trp and Arg-370-Gln), affecting an apparent intramolecular Asp-335 to Arg-370 salt bridge, and interpret the effects and clinical consequences on the basis of the three-dimensional structure of arylsulphatase A. Asp-335-Val and Arg-370-Trp substitutions each cause a complete loss of enzyme activity and are associated with the most severe form of the human disease, whereas the Arg-370-Gln-substituted enzyme retains some residual activity, being found in a patient suffering from the milder juvenile form of the disease. Detailed analysis reveals that formation of the apparent salt bridge depends critically on the presence of aspartic acid and arginine residues at positions 335 and 370, respectively. Substitution by various other amino acids, including glutamic acid and lysine, affects enzyme function severely. Biosynthesis and immunoprecipitation studies indicate that the Asp-335-Val substitution affects folding of arylsulphatase A more severely than either the Arg-370-Trp or Arg-370-Gln substitutions. In vitro mutagenesis data show that clinical severity correlates with the space occupied by residue 370. The combination with structural data suggests that the bulky tryptophan residue broadens the cleft held together by the apparent salt bridge, whereas the smaller glutamine residue still allows the cleft to close, yielding a less severely affected enzyme. The position of residue 370 in the three-dimensional structure of the enzyme provides a plausible explanation for the differing severities in loss of enzyme function caused by the mutations and thus the clinical phenotype.
Asunto(s)
Cerebrósido Sulfatasa/genética , Cerebrósido Sulfatasa/metabolismo , Leucodistrofia Metacromática/genética , Mutación Missense , Adolescente , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales , Arginina , Células Cultivadas , Cerebrósido Sulfatasa/química , Cerebrósido Sulfatasa/inmunología , Preescolar , Estabilidad de Enzimas , Aparato de Golgi/metabolismo , Humanos , Leucodistrofia Metacromática/etiología , Fosforilación , Sales (Química)/química , Transfección , Triptófano , ValinaRESUMEN
Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1 ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1 microg/ml. The expression of the fusion proteins, bHRP-4-green fluorescent protein and mHDGF-green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42 nM. Comparison of the bHRP-4 amino acid sequence with HRP-1-3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.