Collagen is the main constituent of human tissues and organs. This complex polymeric fibrous protein is located in the extracellular space, where it confers resistance and elasticity to the tissues by connecting cells to each other through formation of fibers and intricate networks with adhesion proteins and proteoglycans, the so-called extracellular matrix. The process of collagen biosynthesis starts with the production of tissue-specific "pro-α" collagen chains, which undergo numerous post-translational modifications that eventually lead to formation of trimeric right-handed helices, called procollagens. These trimeric helices are delivered through secretory pathways to the extra cellular space, where they form more intricate collagen structures such as fibrils and fibers. Procollagen post-translational modifications are key to correct collagen maturation. These events occur inside the endoplasmic reticulum (ER). In particular, hydroxylations are fundamental for the proper association between helices. Whilst prolyl hydroxylases have been extensively studied, lysyl hydroxylases (LH) are less characterized from a functional and biochemical point of view because of their structural complexity. LHs are multidomain, glycosylated enzymes of approximately 90 kDa. In humans, three main LH isoforms (LH1, LH2 and LH3) have been described. All LH isoforms are able to catalyze the hydroxylation of collagen lysines in the ER, however LH3 is the most peculiar since it is also capable of glycosylating hydroxylysines. Furthermore, LH3 sub-cellular localization is also peculiar, since this isoform localizes not only in the ER, but also in the extracellular space. The process of LH3 trafficking and secretion has been recently discovered and at present very little is known at the molecular level. Deficiency or mutations in LHs enzymes dramatically affect the assembly and secretion of multiple collagen types, leading to development of mild to severe invalidating syndromes including the Ehlers-Danlos syndrome (LH1), the Bruck syndrome (LH2) and various forms of osteogenesis imperfecta (LH3). Despite several studies have described the biochemical mechanisms of lysine hydroxylation, very little is known about the molecular architecture of LH enzymes. Indeed, numerous questions are still unsolved: how do LH enzymes recognize their substrates and cofactors? Why is LH3 secreted, despite sharing 70% sequence identity with other LH isoforms? Why LH3 does have additional glycosyltranferase activities? To gain biochemical and structural knowledge on LH enzymes, the C-terminal domain carrying the predicted hydroxylation active site of the three LH isoforms was cloned, expressed in E. coli and purified. Unfortunately, during purification these fragments showed aggregation, therefore could not be used for structural studies. Attempts to produce the full-length form of LH3 using mammalian cells allowed instead to obtain pure, soluble and stable recombinant protein, which was used for activity assays and crystallization trials. Taken together, the results obtained in this thesis work constitute a solid starting point for further biochemical, biophysical and structural analysis to address the outstanding questions on LH biology, and possibly for a deeper understanding of the diseases involving this important enzyme class.
Il collagene è il principale costituente di tessuti e organi umani. Questa complessa proteina polimerica fibrosa si trova nello spazio extracellulare, dove conferisce resistenza ed elasticità ai tessuti connettendo le cellule tra loro attraverso la formazione di fibre ed intricati networks con proteine di adesione e proteoglicani, la cosiddetta matrice extracellulare. Il processo di biosintesi del collagene inizia con la produzione di catene “pro-α” collageniche tessuto specifiche, che vanno incontro a numerose modificazioni post-traduzionali e che infine portano alla formazione di triple eliche destrorse, chiamate procollageni. Queste triple eliche sono trasportate attraverso il pathway secretorio allo spazio extracellulare, dove formano strutture più complesse di collagene come fibrille e fibre. Le modificazioni post-traduzionali del procollagene sono la chiave della corretta maturazione del collagene. Questi eventi di modifica avvengono all’interno del reticolo endoplasmatico (ER). In particolare, gli eventi di idrossilazione sono fondamentali per la corretta associazione tra eliche. Mentre le prolil idrossilasi sono state ampiamente studiate, le lisil idrossilasi (LH) sono meno caratterizzate dal punto di vista funzionale e biochimico per via della loro complessità strutturale. Le LH sono enzimi multidominio, glicosilati, di approssimativamente 90kDa. Nell’uomo sono state descritte tre principali isoforme di LH (LH1, LH2 e LH3). Tutte le isoforme sono capaci di catalizzare l’idrossilazione delle lisine del collagene nell’ER, tuttavia LH3 è l’isoforma più particolare dato che è anche capace di glicosilare le idrossilisine. Inoltre, anche la localizzazione subcellulare di LH3 è particolare, dato che questa isoforma si localizza non solo nel ER, ma anche nello spazio extracellulare. Il processo di trafficking e secrezione di LH3 è stato recentemente scoperto e ad oggi davvero poco si conosce a livello molecolare. Mancanza o mutazione degli enzimi LH influenza notevolmente l’assemblaggio e la secrezione di molteplici tipi di collagene, portando allo sviluppo di lievi o gravi sindromi invalidanti tra cui la sindrome di Ehlers-Danlos (LH1), la sindrome di Bruck (LH2) e varie forme di osteogenesi imperfetta (LH3). Non ostante molti studi hanno descritto il meccanismo biochimico dell’idrossilazione delle lisine, veramente poco si sa riguardo l’architettura molecolare degli enzimi LH. Infatti, molte questioni sono ancora irrisolte: come fanno gli enzimi LH a riconoscere i loro substrati e cofattori? Perché LH3 è secreta, non ostante condivida il 70% di identità di sequenza con le altre isoforme di LH? Perché LH3 presenta addizionali attività glicosiltransferasiche? Per guadagnare conoscenze biochimiche e strutturali sugli enzimi LH, il dominio C-terminale previsto come il sito lisil idrossilasico delle tre isoforme di LH è stato clonato, espresso in E. coli e purificato. Purtroppo, durante la purificazione questi frammenti hanno mostrato aggregazione, quindi non potevano essere usati per studi strutturali. Tentativi di produrre l’intero enzima LH3 usando cellule di mammifero hanno permesso invece di ottenere una proteina ricombinante pura, solubile e stabile, che è stata utilizzata per saggi di attività e trials di cristallizzazione. Messi insieme, i risultati ottenuti in questo lavoro di tesi costituiscono un solido punto di partenza per future analisi di tipo biochimico, biofisico e strutturale per affrontare le questioni in sospeso sulla biologia delle LH, e possibilmente per una profonda comprensione delle malattie in cui è coinvolta questa importante classe di enzimi.
Espressione in forma ricombinante e purificazione dell’enzima umano di rimodellamento del collagene lisil idrossilasi 3 (LH3)
MELPIGNANO, FRANCESCO
2015/2016
Abstract
Collagen is the main constituent of human tissues and organs. This complex polymeric fibrous protein is located in the extracellular space, where it confers resistance and elasticity to the tissues by connecting cells to each other through formation of fibers and intricate networks with adhesion proteins and proteoglycans, the so-called extracellular matrix. The process of collagen biosynthesis starts with the production of tissue-specific "pro-α" collagen chains, which undergo numerous post-translational modifications that eventually lead to formation of trimeric right-handed helices, called procollagens. These trimeric helices are delivered through secretory pathways to the extra cellular space, where they form more intricate collagen structures such as fibrils and fibers. Procollagen post-translational modifications are key to correct collagen maturation. These events occur inside the endoplasmic reticulum (ER). In particular, hydroxylations are fundamental for the proper association between helices. Whilst prolyl hydroxylases have been extensively studied, lysyl hydroxylases (LH) are less characterized from a functional and biochemical point of view because of their structural complexity. LHs are multidomain, glycosylated enzymes of approximately 90 kDa. In humans, three main LH isoforms (LH1, LH2 and LH3) have been described. All LH isoforms are able to catalyze the hydroxylation of collagen lysines in the ER, however LH3 is the most peculiar since it is also capable of glycosylating hydroxylysines. Furthermore, LH3 sub-cellular localization is also peculiar, since this isoform localizes not only in the ER, but also in the extracellular space. The process of LH3 trafficking and secretion has been recently discovered and at present very little is known at the molecular level. Deficiency or mutations in LHs enzymes dramatically affect the assembly and secretion of multiple collagen types, leading to development of mild to severe invalidating syndromes including the Ehlers-Danlos syndrome (LH1), the Bruck syndrome (LH2) and various forms of osteogenesis imperfecta (LH3). Despite several studies have described the biochemical mechanisms of lysine hydroxylation, very little is known about the molecular architecture of LH enzymes. Indeed, numerous questions are still unsolved: how do LH enzymes recognize their substrates and cofactors? Why is LH3 secreted, despite sharing 70% sequence identity with other LH isoforms? Why LH3 does have additional glycosyltranferase activities? To gain biochemical and structural knowledge on LH enzymes, the C-terminal domain carrying the predicted hydroxylation active site of the three LH isoforms was cloned, expressed in E. coli and purified. Unfortunately, during purification these fragments showed aggregation, therefore could not be used for structural studies. Attempts to produce the full-length form of LH3 using mammalian cells allowed instead to obtain pure, soluble and stable recombinant protein, which was used for activity assays and crystallization trials. Taken together, the results obtained in this thesis work constitute a solid starting point for further biochemical, biophysical and structural analysis to address the outstanding questions on LH biology, and possibly for a deeper understanding of the diseases involving this important enzyme class.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/18170