Recombinant carbohydrate-binding modules for biomedical applications Biocompatibility of polysaccharide-based materials

Tese de Doutoramento em Engenharia Química e Biológica - Ramo do Conhecimento Tecnologia MicrobianaThe development of biomaterials for medical applications envisages the design of three-dimensional structures – the scaffolds. These structures, mimicking the biological structures and interacting with the surrounding tissues through biomolecular recognition, elicit cellular responses mediated by specific interactions. Among the different scaffolds used in biomedicine, the materials based on polysaccharides present promising characteristics, due to their biocompatibility, hydrophilicity, degradability and appropriate mechanical properties, allowing for a favorable controlled interaction with living systems. Recombinant proteins are widely used in the biomedical field, namely in the fuctionalization of biomaterials. It is well established that Carbohydrate-Binding Modules (CBMs) present in several glycanases are structural and functionally independent of the catalytic module; therefore, their application as fusion partners may be exploited, contributing to protein expression, solubilization, purification, and finally for the functionalization of polysaccharide-based materials. This is the main subject of this thesis: the evaluation of the potential of CBMs as tools for the improvement of the biocompatibility of polysaccharides. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins of the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. Several polysaccharide-based materials have been produced recently at the DEB-UM laboratories, namely dextrin based hydrogels and bacterial cellulose scaffolds. In this study, recombinant proteins containing a CBM with starch affinity were fused to the bioactive molecule RGD, using recombinant DNA technology, in order to functionalize dextrin-based hydrogels. The general introduction of this thesis is presented in chapter 1 and includes a bibliographic revision of: 1) the applications of polysaccharides as biomedical biomaterials (this revision is restricted to the dextrin and bacterial cellulose (BC) based materials, the ones that were used in this work); 2) the strategies available for the production of recombinant proteins, using bacterial systems; and 3) a state of the art on the CBMs and their applications. The chapter 2 describes the development of a methodology for the expression and purification of the recombinant protein CBM-RGD, which has a CBM from the human protein laforin fused to a RGD sequence. Different commercial heterologous Escherichia coli expression systems (pET 29a, pET 25b and pGEXT41) were used in order to obtain high levels of soluble protein. Despite the use of the periplasmatic secretion approach (pET25) or the fusion of CBM with enhancing solubility tag (GST), the recombinant proteins were always obtained in the insoluble fraction. The utilization of CHAPS and arginine allowed the protein solubilization and purification, but not the production of functional protein with starch binding ability. Using the pET29a vector, the recombinant proteins were obtained in inclusion bodies (IB). After solubilization and refolding, the CBM was recovered and showed starch affinity. This is the first report on the expression of the functional CBM from the human protein laforin. The chapter 3 describes the production of recombinant proteins containing a bacterial CBM, which belongs to an α- amylase from Bacillus sp. TS-23. This protein, like the laforin CBM, also has starch affinity, being designated a Starch- Binding Module (SBM). The recombinant SBM and RGD-SBM proteins were cloned, expressed, purified and tested in vitro. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel, improving the interaction with cells. The characterization of dextrin-vinyl acrylate (dextrin-VA) and dextrin-hydroxyethylmethacrylate (dextrin-HEMA) hydrogels was presented in a previous study carried out at the DEB-UM laboratories. In this work (chapter 4) the in vivo biocompatibility and degradability of these hydrogels are reported. The histological analysis of subcutaneous implants of these hydrogels, featuring inflammatory and resorption events in mice, was carried out over a period of 16 weeks. While dextrin-HEMA hydrogel was quickly and completely degraded and reabsorbed, dextrin-VA degradation occurred slowly, apparently through an erosion controlled process. A thin fibrous capsule was observed 16 weeks post-implantation, surrounding the non-degradable hydrogel. In the case of the degradable material, only a mild inflammatory reaction was observed, with few foamy macrophages being detected around the implant. This reaction was followed by complete resorption, with no signs of capsule formation or fibrosis associated with the implants. Altogether, these results strongly suggest that the dextrin hydrogels are fully biocompatible, since no toxicity on the tissues surrounding the implants was found. Moreover, it may be speculated that a controlled degradation rate of the hydrogels may be obtained, using dextrin with grafted HEMA and VA in different proportions. Chapter 5 presents the evaluation of Bacterial Cellulose – NanoFibers (BC-NFs) nanotoxicology. BC is a promising material for biomedical applications, namely due its biocompatibility. Although BC has been shown to be neither cytotoxic nor genotoxic, the properties of isolated BC-NFs on cells and tissues has never been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. The results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assay showed that NFs, produced from BC by a combination of acid and ultrasonic treatment, are not genotoxic under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no modification in the cell morphology is observed. Overall, this work reports the successful expression and isolation of the atypical human CBM, from the protein laforin. It provides a contribution to the development of a strategy based on the use of CBMs as tools for the modification of the surface properties of biomaterials, improving the interaction with cells. Finally, this work characterizes biocompatibility aspects of biomaterials currently under development at DEB-UM laboratories.O desenvolvimento de biomateriais para aplicações biomédicas centra-se no desenho de estruturas tri-dimensionais – scaffolds – capazes de mimetizar as funções biológicas e interagir com os tecidos envolventes, através do reconhecimento biomolecular. Entre os diferentes materiais usados para produzir scaffolds, os constituídos por polissacarídeos (como é o caso dos hidrogeis de dextrino e os materiais de celulose bacteriana - BC) apresentam características promissoras devido à sua biocompatibilidade, hidrofilicidade, degradabilidade e propriedades mecânicas, permitindo a sua utilização biomédica. As proteínas recombinantes são amplamente usadas em biomedicina, nomeadamente na funcionalização de diversos biomateriais. Sabe-se que os módulos de ligação a carbohidratos (CBMs), presentes em várias glicanases, são estrutural e funcionalmente independentes do domínio catalítico. Assim, a sua utilização em proteínas de fusão tem sido explorada, com o propósito de facilitar ou aumentar a expressão, solubilidade e purificação das proteínas. Uma das moléculas frequentemente usada para melhorar a adesão celular é o péptido Arg-Gly-Asp (RGD). Esta sequência, presente em diversas proteínas da matriz extra-celular, é um ligando para adesão celular mediada por integrinas, sendo reconhecido como o principal grupo funcional na adesão celular. Nos últimos anos, foram produzidos nos laboratórios do DEB-UM diversos materiais à base de polissacarídeos, nomeadamente hidrogeis de dextrino. Neste trabalho, usando tecnologia de DNA recombinante, foram produzidas proteínas bi-funcionais constituídas por um CBM (com afinidade para o amido) fundido com a molécula bio-activa RGD, com o propósito de os funcionalizar. Pretende-se assim melhorar a interacção do material com as células, favorecendo a adesão celular pela interacção com a molécula RGD que por sua vez está ligado ao material através do CBM. Na Introdução geral desta tese (capítulo 1) apresenta-se: 1) uma revisão sobre biomateriais baseados em polissacarídeos (em particular dos hidrogels de dextrino e das nanofibras (NFs) de celulose bacteriana); 2) as estratégias usadas para produzir as proteínas recombinantes em sistemas de expressão bacterianos; 3) e uma revisão sobre os CBMs e as suas aplicações. O segundo capítulo descreve a metodologia desenvolvida para a expressão e purificação da proteína de fusão CBMRGD, pertencendo este CBM à proteína humana laforina. Foram utilizados diferentes sistemas comerciais para expressão heteróloga em Escherichia coli (pET 29a, pET 25b e pGEXT41), com o intuito de obter elevados níveis de proteína solúvel. Os sistemas de expressão que permitem a secreção das proteínas para o espaço periplasmático (pET25) ou a fusão com a GST (pGEXT4 1), um tag que potencia a solubilidade das proteínas, conduziram à obtenção de proteínas insolúveis. A adição de CHAPS e arginina ao tampão de lise, embora resultando num aumento da solubilidade, não permitiu a obtenção de proteína funcional, isto é, com afinidade para o amido. Usando o vector pET29a, a proteína foi obtida em corpos de inclusão que, depois de solubilizados e submetidos ao processo de refolding, permitiram obter proteína funcional com afinidade para o amido. Este é o primeiro relato da expressão funcional deste CBM humano. No capítulo 3 descreve-se a produção de proteínas de fusão contendo um CBM bacteriano, da α-amilase do Bacillus sp. TS-23. Este CBM também apresenta afinidade para o amido, sendo por isso designado por SBM (Starch-binding module). As proteínas recombinantes SBM e RGD-SBM foram produzidas usando um sistema de expressão de E. coli. O seu efeito na adesão, spreading e proliferação celular foi avaliado in vitro, usando fibroblastos de embrião de rato 3T3. Os resultados mostraram que o tratamento do hidrogel de dextrino com RGD-SBM melhorou a adesão celular em mais de 30%. Para além disso, só na presença da proteína foi possível observar as células alongadas na sua superfície. Assim, a proteína de fusão revelou-se eficiente para funcionalizar o hidrogéis de dextrino. A caracterização dos hidrogéis de dextrino-vinil acrilato (dextrino-VA) e dextrino-hidroxietilmetacrilato (dextrino-HEMA) foi objecto de estudo em trabalhos anteriores, também desenvolvidos no DEB-UM. Neste trabalho (capítulo 4) apresentam-se os resultados da caracterização de biocompatibilidade e degradação destes hidrogéis in vivo. A análise histológica de implantes subcutâneos em ratinhos permitiu estudar os eventos de reabsorção e a resposta inflamatória. De acordo com os resultados, a degradação e reabsorção dos géis de dextrino-HEMA ocorre rapidamente; a degradação dos géis de dextrino-VA é mais lenta, devendo-se principalmente a processos de erosão. Após 16 semanas, foi observada uma fina cápsula fibrosa a rodear o implante não degradável. No caso do gel degradável, observou-se uma resposta inflamatória de baixa intensidade, sendo detectados alguns macrófagos com material fagocitado a envolver o implante. Esta reacção foi seguida pela completa reabsorção do material, não havendo sinais de formação de qualquer cápsula fibrosa. Estes resultados sugerem que os hidrogéis de dextrino são biocompatíveis, uma vez que não foram detectados sinais de toxicidade nos tecidos que envolviam o material. Os resultados sugerem também que é possível obter hidrogéis com velocidades de degradação controlada, usando dextrino substituído com HEMA e VA em diferentes proporções. O capítulo 5 apresenta o estudo da nanotoxicidade de NFs de celulose bacteriana. A BC apresenta grandes potencialidades para aplicações biomédicas, sendo descrita como um material não citotóxico ou genotóxico. No entanto, o efeito das NFs, isoladas por tratamento ácido e ultrasons, nas células e nos tecidos não foi descrito. Considerando a toxicidade associada a outros nanomateriais com forma de agulha, o estudo da nanotoxicidade destas fibras torna-se crucial. Os resultados obtidos no ensaio cometa e de reversão da Salmonella mostraram que as NFs produzidas a partir da BC, não são genotóxicas na condições utilizadas. Para além disso, os resultados obtidos nos ensaios de proliferação celular usando fibroblastos e células CHO mostraram que, apesar de uma ligeira redução na proliferação, não são detectadas diferenças morfológicas. Em resumo, este trabalho descreve, pela primeira vez, a expressão funcional do CBM atípico da proteína humana laforina. Este trabalho também contribui para o desenvolvimento de ferramentas que utilizam os CBMs recombinantes para a modificação das propriedades da superfície de materiais. Por último, são caracterizados aspectos da biocompatibilidade de materiais que estão a ser desenvolvidos nos laboratórios do DEB-UM.Fundação para a Ciência e a Tecnologia (FCT

Production of the human carbohydrate binding module from laforin protein : a comparative study between bacterial and yeast expression systems

Fundação para a Ciência e a Tecnologia (FCT) - POCTI/BIO/45356/2002 ; (SFRH/BD/18418/2004)

Cloning, expression and purification of a carbohydrate binding module in Pichia pastoris

The enzymes responsible for carbohydrate degradation are, usually, compose of two distinct modules: catalytic and a substrate binding module. Since these two modules are functionally independent, the CBMs (carbohydrate binding modules) can be fused with bioactive molecules to drive them to starch based biomaterials. In this work, the CBM cloned belongs to human phosphatase laforin, which is involved in metabolism of the glycogen. Aiming at the optimization of large scale expression, CBM peptide production was done by cloning CBM coding sequence in two different systems of Pichia pastoris: pGAPZα C which has a constitutive promoter and pPICZα C which has an inductive promoter. Both expression systems have the secretion signal α- factor. The integration of the CBM coding sequence, in yeast genome and the gene transcription were confirmed by slot-blot and northern-blot, respectively. The fermentation conditions for different P. pastoris clones were optimized and recombinant protein was purified from fermentation medium by affinity chromatography. Purified protein was analysed by western-blot. Fictionalization studies on starch based biomaterials are being performed

Expression of the functional carbohydrate-binding module (CBM) of human laforin

Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.Fundação para a Ciência e a Tecnologia (FCT, Portugal)

Escherichia coli expression, refolding and characterization of human laforin

Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5–7 mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI

Characterization of dextrin-based hydrogels : rheology, biocompatibility, and degradation

A new class of degradable dextrin-based hydrogels (dextrin-HEMA) was developed. The hydroxyethyl methacrylate ester (HEMA) hydroxyl groups were activated with N,N' carbonyldiimidazole (CDI), followed by their coupling to dextrin, yielding a derivatized material that can be polymerized in aqueous solution to form hydrogels. A comparative study of the stability of the dextrin-HEMA hydrogels and dextrin-vinyl acrylate (dextrin-VA, produced in previous work) revealed that only the firsts are effectively hydrolyzed under physiological conditions. A severe mass loss of dextrin-HEMA gels occurs over time, culminating in the complete dissolution of the gels. Rheologic analysis confirmed that physical structuring is less pronounced when dextrin is modified with methacrylate side groups. The biocompatibility results revealed that the dextrin hydrogels have negligible cell toxicity, irrespective of the hydrogel type (HEMA and VA), allowing cell adhesion and proliferation. Gathering the biocompatibility and the ability to tailor the release profiles, we consider dextrin a promising biomaterial for biomedical applications, namely for controlled release

Studies on the cellulose-binding domains adsorption to cellulose

Cellulose-binding domains (CBD) are modular peptides, present in many glycanases, which anchor these enzymes to the substrate. In this work, the effect of CBD adsorption on the surface properties of a model cellulose, Whatman CF11, was studied. The methods applied include inverse gas chromatography (IGC), ESCA, X-ray diffraction, and scanning electron microscopy (SEM). The CBD partition affinity (0.85 L/g) was calculated from adsorption isotherms. However, true adsorption equilibrium does not exist, since CBDs are apparently irreversibly adsorbed to the fibers. Both IGC and ESCA showed that fibers with adsorbed CBD have a lower acidic character and also a slightly higher affinity toward aliphatic molecules. This may however be a consequence of an increased surface area, a hypothesis that is supported by microscopic observations. The crystallinity index was not affected by CBD treatment.Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/6934/2001

Production of recombinant carbohydrate-binding modules fused to RGD : functional studies using bacterial cellulose

The attachment of cells to biomedical materials can be improved by using adhesion molecules, present in the extracellular matrix substances, such as fibronectin, vitronectin, or laminin. In many cases, Arg-Gly-Asp (RGD) was found to be the major functional amino acid sequence responsible for cellular adhesion. In the present study, a method for producing chimerics proteins, RGDCBM, with functions similar to fibronectin, which contains a cellulose-binding module (CBM), was developed. The CBM used was from the cellulosoma of the bacteria Clostridium thermocellum. The genes encoding these CBM-containing chimeric proteins were cloned, and the protein expressed and purified. Bacterial cellulose (BC) secreted by Gluconacetobacter xylinus was produced. Polystyrene surfaces and bacterial cellulose sheets where ‘‘coated’’ with these RGD-containing proteins, and then used in adhesion/biocompatibility tests, using a mouse embryo fibroblasts culture. The results showed that the proteins containing the RGD or GRGDY sequence were able to improve the adhesion of the fibroblast on the polystyrene plate, furthermore proteins containing the RGD sequence were more effective than the proteins containing the GRGDY sequence. Preliminary adhesion studies of fibroblast cultures on cellulose sheets, functionalized with the recombinant proteins, showed positive effects on the adhesion and proliferation of the cells. The results demonstrated that the proteins containing the RGD sequence were able to increase significantly the adhesion of fibroblast to BC surfaces when compared with the controls (cellulose treated with the CBM or buffer). The results also demonstrated that the protein containing one RGD sequence have a stronger effect than the protein containing two RGDs

Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence

Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. Results The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. Conclusion The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil)Fundação para a Ciência e a Tecnologia (FCT

Cloning and expression of carbohydrate binding module in Pichia pastoris

The main goal of this work is the production of recombinant biologically active peptides fused with a Carbohydrate Binding Module (CBM). Aiming at the optimization of large scale expression, CBM peptide production was done by cloning CBM coding sequence in two different systems of Pichia pastoris: pGAPZαC which has a constitutive promoter and pPICZαC which has an inductive promoter. The integration of the CBM coding sequence in the yeast genome was confirmed by slot-blot for both expression systems. Transcription was analysed by northern-blot and SDS-PAGE. The results obtained with these two expression systems were different. Apparently, there were no clones of P. pastoris transformed with pGAPZαC-CBM that had produced any protein with starch affinity, under the batch and fed-batch conditions tested in this work. On the other hand, only one studied clone of P. pastoris transformed with pPICZαC-CBM vector had produced, in batch conditions, a protein with affinity for starch. However, under fed-batch conditions, the results obtained with this clone were not conclusive, suggesting that conditions for large scale production must be optimized