Collagen is a fibrous protein whose tertiary structure is a triple-helical spiral. Its high structural stability depends on the post-translational modifications (PTMs) - hydroxylations and glycosylations - which are operated by enzymes localized in the endoplasmic reticulum (ER) during the collagen maturation phase. The recently solved structure of collagen-lysyl hydroxylase 3 (LH3) enabled the understanding of key catalytic features of this class of enzymes. Nevertheless, the complex intrinsic nature of the collagen substrate impaired, so far, the characterization of the substrate recognition properties of these enzymes. To date, catalytic activities and binding affinity characterization has been performed using synthetic collagen peptides, which are far from being the lysyl-hydroxylase physiological substrate. Therefore, the recombinant production and biophysical characterization of fully folded triple-helical collagens constitutes an immediate need to further broaden the understanding of the lysyl-hydroxylase mode of action. A sequence coding for human collagen type III was designed to be expressed in E. coli together with a viral collagen prolyl-hydroxylase (L593 from Acanthamoeba polyphaga mimivirus). The recombinant collagen was purified through affinity and size-exclusion chromatography (SEC) allowing to obtain pure triple-helical collagen in milligram amount. Biophysical assays using circular dichroism (CD) and differential scanning fluorimetry (DSF) allowed to characterize the collagen product demonstrating its proper triple-helical folding. The recombinant collagen was also tested for binding with LH3 using surface plasmon resonance (SPR) showing nanomolar affinities. Binding data were corroborated by SEC coelution experiments which showed the formation of the LH3-collagen complex. These results constitute an excellent starting point for a further structural characterization of the complex.
Caratterizzazione di collagene umano ricombinante e la sua interazione con lisil-idrossilasi. Il collagene è una proteina di natura fibrosa, la cui struttura terziaria è una spirale a tripla elica. La sua elevata stabilità strutturale dipende da modificazioni post-traduzionali (PTMs) - idrossilazione e glicosilazione – operate da enzimi localizzati nel reticolo endoplasmatico (ER) durante la fase di maturazione del collagene. La struttura recentemente risolta della lisil-idrossilasi 3 (LH3) ha permesso di comprendere le caratteristiche catalitiche chiave di questa classe di enzimi. Tuttavia, la natura intrinsecamente complessa del collagene ha ostacolato, fino ad oggi, la caratterizzazione delle proprietà di riconoscimento del substrato di questi enzimi. Ad oggi, le attività catalitiche e la caratterizzazione dell'affinità di legame sono state eseguite, utilizzando peptidi di collagene sintetico, molto diversi rispetto al substrato fisiologico della lisil-idrossilasi. Pertanto, la produzione ricombinante e la caratterizzazione biofisica di collageni a tripla elica completamente piegati costituiscono una necessità immediata di ampliare ulteriormente la comprensione della modalità di azione della lisil-idrossilasi. Una sequenza codificante per collagene umano di tipo III è stata progettata per essere espressa in E. coli insieme a una prolil-idrossilasi di collagene virale (L593 da Acanthamoeba polyphaga mimivirus). Il collagene ricombinante è stato purificato mediante cromatografia di affinità e di esclusione dimensionale (SEC) che consente di ottenere collagene a tripla elica puro in quantità di milligrammi. Saggi biofisici che utilizzano dicroismo circolare (CD) e fluorimetria a scansione differenziale (DSF) hanno permesso di caratterizzare il collagene prodotto, dimostrando il suo corretto folding a tripla elica. Il collagene ricombinante è stato anche testato per il legame con LH3, utilizzando la risonanza plasmonica di superficie (SPR) mostrando affinità nanomolari. I dati vincolanti sono stati corroborati da esperimenti di coeluzione del SEC che hanno dimostrato la formazione del complesso LH3-collagene. Questi risultati costituiscono un ottimo punto di partenza per un'ulteriore caratterizzazione strutturale del complesso.
Characterization of recombinat human collagen and its interaction with lysyl-hydroxylases enzymes
BARBALINARDO, GIANSALVO
2017/2018
Abstract
Collagen is a fibrous protein whose tertiary structure is a triple-helical spiral. Its high structural stability depends on the post-translational modifications (PTMs) - hydroxylations and glycosylations - which are operated by enzymes localized in the endoplasmic reticulum (ER) during the collagen maturation phase. The recently solved structure of collagen-lysyl hydroxylase 3 (LH3) enabled the understanding of key catalytic features of this class of enzymes. Nevertheless, the complex intrinsic nature of the collagen substrate impaired, so far, the characterization of the substrate recognition properties of these enzymes. To date, catalytic activities and binding affinity characterization has been performed using synthetic collagen peptides, which are far from being the lysyl-hydroxylase physiological substrate. Therefore, the recombinant production and biophysical characterization of fully folded triple-helical collagens constitutes an immediate need to further broaden the understanding of the lysyl-hydroxylase mode of action. A sequence coding for human collagen type III was designed to be expressed in E. coli together with a viral collagen prolyl-hydroxylase (L593 from Acanthamoeba polyphaga mimivirus). The recombinant collagen was purified through affinity and size-exclusion chromatography (SEC) allowing to obtain pure triple-helical collagen in milligram amount. Biophysical assays using circular dichroism (CD) and differential scanning fluorimetry (DSF) allowed to characterize the collagen product demonstrating its proper triple-helical folding. The recombinant collagen was also tested for binding with LH3 using surface plasmon resonance (SPR) showing nanomolar affinities. Binding data were corroborated by SEC coelution experiments which showed the formation of the LH3-collagen complex. These results constitute an excellent starting point for a further structural characterization of the complex.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/20.500.14239/19300