The aim of this work was the molecular characterization of gene PRB1 in three subjects with different salivary proteome profile. The PRB1 gene encodes a member of the heterogeneous family of Basic Proline rich Proteins (PRB), produced by the parotid gland and secreted in human saliva. The protein polymorphism resulting from post-translational modifications of the pre-protein PRB1, are further complicated by the gene allelic variants. Those variants involves the third exon of the gene, and are related to the presence or absence of a tandem repeat sequence 183 bp long. That tandem repeat may originate three different allelic forms, defined small, medium or large depending on the number of repeats inside the third exon. The information reported in the literature about the class of PRP genes, and in particular PRB1 are, from the molecular point of view, fragmentary and difficult to reconstruct. In fact, the literature on this topic covers a time span ranging from the seventies (Azen & Oppenheim, 1973), when the associated peptides have been characterized for the first time, up to the end of the nineties (Stubbs et al., 1998). Since the eighties, there have been various attempts to characterize the PRB1 allelic variants, based on the resulting peptides. This approach has greatly complicated the classification of the gene, but also of his allelic variants and the deriving proteins (Maeda et al., 1985 ; Lyons et al., 1988a, b; Azen et al., 1993). Today we know that from PRB1, considering its allelic and splicing variants, are expressed six different proteins (Marconi et al., 2015). However, amongst the three allelic variants of this gene, the only of which we have the complete nucleotide sequence is the medium. Conversely, for the small and large we have so far only partial sequences, referring only to the third exon. Among the objectives of this study, there was the reconstruction of the complete sequences of the small and large variants, of which there have been previously characterized two putative bearers from the proteomic point of view. At the same time, we also sequenced the PRB1 gene from a putative homozygotic bearer of the medium variant. While for the subject bearer of the small variant, we were able to identify and sequence the entire gene, the same was not possible for the subject bearer of the large variant. In fact, from our analysis this last subject has shown to be homozygous carrier of the medium variant. This result is incompatible with the presence in his saliva of Ps2 protein, which is characteristic of the large variant. In the absence of further mass spectrometric analysis, we cannot explain the reason for this discrepancy between genomic and proteomic data. The analysis of the complete sequences did not allow us to understand why it has not been identified to the mass spectrometer the peptide relative to the splice variant classified by Maeda with the acronym cP5 (Maeda et al., 1985), while in all the subject has been identified the peptide and the relative cP4 splice variant. Pk-o protein has so far been reported to be expressed from the large variant only, although the first classification of splice variants had been made from an individual carrying the medium variant. Since in none of the three sequenced subjects the splice acceptor site in 3' of the cP5 variant results to be altered, it is possible that the relative peptide has not been identified to the mass spectrometer. Otherwise, specific splicing factors, that recognize Splicing Regulatory Elements (SRES; Hernandez-Imaz et al., 2015) in the neighborhood of the two molecular acceptor sites, prevented the transcription of the cP5 in favor of cP4. In order to shed light on this aspect, there are some possible strategies in perspective. Recently, in fact, studies have been published in which extensive in vivo screening for the detection of SRES is coupled with RNA affinity purification and mass spectrometry, which allow also the identification of the splicing factors which bound to the SRES (Wang Y & Z Wang, 2014; Wang et al., 2013). This strategy could be effectively integrated with the analysis of minigenes (Hernandez-Imaz et al., 2015), specifically designed on the model of the third exon of PRB1, where alternative splicing takes place and where are probably placed the SRES. Molecular analysis has also enabled us to identify a set of Single Nucleotide Polymorphims (SNPs), most of which have never been described so far, localized specially inside introns and some even within exons. In particular, for two of these, we have detected a non-synonymous substitution at the amino acid level, the role of which can only be clarified by means of appropriate functional studies. Although translationally silent, even synonymous SNPs may have an important function, especially in alternative splicing. In fact, these substitutions can determine the genesis or destruction of SRES, or strengthen cryptic donor /acceptor sites. Furthermore, they can result in the alteration of the secondary structure of the mRNA, important for the exons definition and the pause sites of RNA II. This could, in turn, alter the processivity of the polymerase, with possible consequences on the choice of splicing sites. The reconstruction of the complete sequences and the comparison with the data available in the literature (Lyons et al., 1988) allowed us to produce an updated model, which allows to explain the generation of allelic variants small and large from the medium variant. Hopefully, the reconstruction also of the large allelic variation, and possibly the very large, will lay the basis for the validation of our model and possibly also its extension to other allelic variants of the PRB gene class. The genotypic characterization of allelic variants of PRB1 may be, in the future, an important predictive tool about the subjective susceptibility towards a series of oral pathogens. In fact, polymorphic variations in the PRB1 peptides can build the basis for the prediction of individual differences at the level of the oral microflora, with repercussions on the susceptibility to infections and diseases in this body part (Newman et al., 1993 and 1996; O'Sullivan et al., 2000).

Sequenziamento e Analisi Molecolare di Varianti Alleliche del Gene PRB1

ORRU', ROBERTO
2016

Abstract

The aim of this work was the molecular characterization of gene PRB1 in three subjects with different salivary proteome profile. The PRB1 gene encodes a member of the heterogeneous family of Basic Proline rich Proteins (PRB), produced by the parotid gland and secreted in human saliva. The protein polymorphism resulting from post-translational modifications of the pre-protein PRB1, are further complicated by the gene allelic variants. Those variants involves the third exon of the gene, and are related to the presence or absence of a tandem repeat sequence 183 bp long. That tandem repeat may originate three different allelic forms, defined small, medium or large depending on the number of repeats inside the third exon. The information reported in the literature about the class of PRP genes, and in particular PRB1 are, from the molecular point of view, fragmentary and difficult to reconstruct. In fact, the literature on this topic covers a time span ranging from the seventies (Azen & Oppenheim, 1973), when the associated peptides have been characterized for the first time, up to the end of the nineties (Stubbs et al., 1998). Since the eighties, there have been various attempts to characterize the PRB1 allelic variants, based on the resulting peptides. This approach has greatly complicated the classification of the gene, but also of his allelic variants and the deriving proteins (Maeda et al., 1985 ; Lyons et al., 1988a, b; Azen et al., 1993). Today we know that from PRB1, considering its allelic and splicing variants, are expressed six different proteins (Marconi et al., 2015). However, amongst the three allelic variants of this gene, the only of which we have the complete nucleotide sequence is the medium. Conversely, for the small and large we have so far only partial sequences, referring only to the third exon. Among the objectives of this study, there was the reconstruction of the complete sequences of the small and large variants, of which there have been previously characterized two putative bearers from the proteomic point of view. At the same time, we also sequenced the PRB1 gene from a putative homozygotic bearer of the medium variant. While for the subject bearer of the small variant, we were able to identify and sequence the entire gene, the same was not possible for the subject bearer of the large variant. In fact, from our analysis this last subject has shown to be homozygous carrier of the medium variant. This result is incompatible with the presence in his saliva of Ps2 protein, which is characteristic of the large variant. In the absence of further mass spectrometric analysis, we cannot explain the reason for this discrepancy between genomic and proteomic data. The analysis of the complete sequences did not allow us to understand why it has not been identified to the mass spectrometer the peptide relative to the splice variant classified by Maeda with the acronym cP5 (Maeda et al., 1985), while in all the subject has been identified the peptide and the relative cP4 splice variant. Pk-o protein has so far been reported to be expressed from the large variant only, although the first classification of splice variants had been made from an individual carrying the medium variant. Since in none of the three sequenced subjects the splice acceptor site in 3' of the cP5 variant results to be altered, it is possible that the relative peptide has not been identified to the mass spectrometer. Otherwise, specific splicing factors, that recognize Splicing Regulatory Elements (SRES; Hernandez-Imaz et al., 2015) in the neighborhood of the two molecular acceptor sites, prevented the transcription of the cP5 in favor of cP4. In order to shed light on this aspect, there are some possible strategies in perspective. Recently, in fact, studies have been published in which extensive in vivo screening for the detection of SRES is coupled with RNA affinity purification and mass spectrometry, which allow also the identification of the splicing factors which bound to the SRES (Wang Y & Z Wang, 2014; Wang et al., 2013). This strategy could be effectively integrated with the analysis of minigenes (Hernandez-Imaz et al., 2015), specifically designed on the model of the third exon of PRB1, where alternative splicing takes place and where are probably placed the SRES. Molecular analysis has also enabled us to identify a set of Single Nucleotide Polymorphims (SNPs), most of which have never been described so far, localized specially inside introns and some even within exons. In particular, for two of these, we have detected a non-synonymous substitution at the amino acid level, the role of which can only be clarified by means of appropriate functional studies. Although translationally silent, even synonymous SNPs may have an important function, especially in alternative splicing. In fact, these substitutions can determine the genesis or destruction of SRES, or strengthen cryptic donor /acceptor sites. Furthermore, they can result in the alteration of the secondary structure of the mRNA, important for the exons definition and the pause sites of RNA II. This could, in turn, alter the processivity of the polymerase, with possible consequences on the choice of splicing sites. The reconstruction of the complete sequences and the comparison with the data available in the literature (Lyons et al., 1988) allowed us to produce an updated model, which allows to explain the generation of allelic variants small and large from the medium variant. Hopefully, the reconstruction also of the large allelic variation, and possibly the very large, will lay the basis for the validation of our model and possibly also its extension to other allelic variants of the PRB gene class. The genotypic characterization of allelic variants of PRB1 may be, in the future, an important predictive tool about the subjective susceptibility towards a series of oral pathogens. In fact, polymorphic variations in the PRB1 peptides can build the basis for the prediction of individual differences at the level of the oral microflora, with repercussions on the susceptibility to infections and diseases in this body part (Newman et al., 1993 and 1996; O'Sullivan et al., 2000).
DNA
PCR
PRB1
primer walking
proline-rich proteins
proteoma salivare
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11584/266626
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