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  • The P H activity and the large number of Hyp

    2020-02-25

    The P4H activity and the large number of 4-Hyp residues that are formed, amounting to half of all the Pro residues or 10% of all residues, have been identified as having a clear role in the structure and chemistry of the triple helix [3]. The role of the rare 3-Hyp residues is less clear and is still emerging [5]. Without any 4-hydroxylation, the triple helix has a stability of around 23 °C, whereas essentially full hydroxylation leads to a thermal stability around body temperature of 37 °C in humans [6]. In poikilotherms, such as fish, the collagen melting temperature matches their environmental temperature, so varies from low values for deep sea animals to much higher for fish in tropical waters [7], [8]. In these cases, the Hyp content is lower, but sufficient to give adequate stability [9]. The presence of Hyp residues also assists in the folding of the triple helix, prior to secretion from the cell [10], but is not essential for folding to occur. Thus, in recombinant human collagen produced in plant cetylpyridinium sale without any of the specific P4H activity introduced, a non-hydroxylated collagen is formed that, nevertheless, still folds into a triple helical structure if higher temperatures are avoided, albeit at a reduced rate [10]. The study using recombinant plant production also indicated the importance of Hyp for the interactions of collagens. In particular, it showed that the characteristic lateral aggregation of the major, interstitial collagens into fibrillar structures did not occur when Hyp was absent [10]. The role of Hyp in producing a significant and stable hydration structure [11] facilitates the lateral aggregation. At the level of individual Gly-Xaa-Yaa triplets, the presence or not of Hyp can affect collagen binding to other, non-collagenous molecules. For example, if proline hydroxylation is absent, binding to α2β1 integrin is reduced [12] as is also the case for discoidin domain receptor, where a native triple helical conformation is required for binding, but receptor activation is absent when Hyp is absent [13]. In the last decade, significant interest has emerged in a new group of collagen-like structures that are present in bacteria, and which have significant potential as biomedical materials [14], [15]. The proteins typically contain uninterrupted (Gly-Xaa Yaa)n domains that are stable at around 37 °C [15]. Initially this stability requirement was not surprising as those from pathogens would need stability at human body temperature. Subsequently, however, bacterial, non-animal collagens of comparable stability have also been observed in non-pathogenic species [16]. What was surprising was that this high stability was achieved in the absence of Hyp modifications [17]. However, examination of the sequence showed that in addition to a high Pro content, there were several sequences that could provide significant stability, including Arg residues in Yaa positions [18] and ‘charge pairs’, where oppositely charged residues are proximal in the tertiary structure [19], such as Lys-Gly-Glu, Lys-Gly-Asp and Asp-Gly-Lys sequences, that add significantly to stability [19]. Indeed, in an E. coli phage a stability of 42 °C has been observed [20], while selective modifications to a sequence, using information of the relative stabilising effects of different triplets [21] has increased the stability of the S. pyogenes Scl2 product from 37 °C to 42 °C [22]. The lack of hydroxylation means that these proteins are readily produced by fermentation in E. coli[23]. Introduction of the mammalian P4H activity into this host has proved problematic [24] and yeast systems have proved necessary for effective production of recombinant human collagens, but do not give as good yields [25]. Human collagens expressed in E. coli systems, without P4H, do not give such good yields either compared to those for the smaller bacterial collagens [25], [26]. Thus, if supplemental hydroxylation were required, fermentation in E. coli does not readily allow the introduction of an effective mammalian enzyme system. In E coli, effective synthesis and/or transport of the L-ascorbate required for the mammalian enzyme activity is absent [24].