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The NALP3 protein, known structurally associated with inflammasomes functions as activator of procaspases and mediates an inflammatory response. The genetic alterations arising from mutations in NALP3 gene ultimately lead to rapid activation of the caspase 1 and induce the overexpression of IL-1β resulting in autoinflammatory disorders named Familial Cold Autoinflammatory Syndrome, Muckle-Wells syndrome, and Chronic Infantile Neurological Cutaneous and Articular Syndrome. The experimental studies for governing the altered functional aspects of V262A mutation are on cutting edge, necessitating the scientific knowledge in this area to be enlightened. Site directed mutagenesis has revolutionized in generating site specific variability in the DNA sequences for incorporating the desired mutations with the rationale of correcting the acquired mutations or generating mutants for experimental studies. A specific point mutation at the 785 position of the third exon of NALP3 DNA sequence (V262A) was introduced via PCR assisted site directed mutagenesis. The mutated amplification products were treated with Dpn I endonuclease and cloned into E .coli cells. The plasmid DNA carrying the mutated NALP3 sequences was harvested from the transformed cells grown on ampicillin medium and purified. Due to low concentration and purity of the plasmid with V262A mutation, plasmid DNA carrying T436P mutation provided by lab supervisors was used for DNA sequencing. Since the sequence of the mutated DNA was only up to 131 nucleotides long, the mutation induced at the 1306 position was not located by nucleotide alignment tools though it showed similarity with library sequences.
The innate immune responses despite being non specific with respect to antigenic epitopes, involve various cellular receptors to recognize specific structural components or conformations present on pathogens called pathogen associated molecular patterns (PAMPs). The PAMPs are recognized and acted upon by the innate immune system by specific receptors present on the cytological effectors of innate immune response, collectively known as pathogen recognition receptors (PRRs). One of the major classes of PRRs belongs to a group of intracellular caterpillar proteins called Nod like receptors abbreviated as NLRs (Gonzalez-Ben´Ä±tez et al., 2008). NLRs in humans are coded by a set of 22 genes and composed of a carboxyl terminal domain, a middle domain called NACHT and an amino terminal effector domain. The human NLRs structured with a pyrin domain at the N terminus is referred as NLR family pyrin domain containing and abbreviated as NALPs.
Even though a large number of NALPs receptors have been characterized, the most important NALP known to play a major role in the cellular defense events is the pyrin like protein NALP3. The NALP3 protein coded by the CIAS1 gene of the NLR set of genes, also called as Apaf1-like protein 1 or cryopyrin consists of a pyrin domain (PYD), a site for binding nucleotides (NBS) and a motif formed of leucine rich repeats (LRR). The interactions between the PYD domains of NALPs and apoptosis associated speck like protein (ASC) along with caspases trigger the formation of a cellular signaling complex inside the cellular compartment called inflammasome. NALP3 inflammasomes mediate the cleavage and synthesis of cytokines IL-1β, IL-18 and IL-33, which are renowned for their ability to mediate powerful inflammatory response (Sutterwala et al., 2006). The NALP3 also functions as an activator of NF-kβ cellular signaling pathways and regulate apoptotic pathways. Moreover, NALPs when associated with inflammasomes sense dangerous signals such as uric acid and ATP produced by damaged cells. However it was also found that the genetic alterations arising from mutations in NALP3 rapidly activate the caspase 1 and induce the overexpression of IL-1β resulting in the development of different autoinflammatory disorders named familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome which is also clinically termed as neonatal-onset multisystem inflammatory disease (NOMID) (Hoffman et al., 2001).
CINCA is a rare inflammatory disorder of hereditary origin seen in infants and clinically characterized by skin rashes with the deposition of polymorphonuclear cells at the perivascular sites, fever, swelling and inflammation of joints as well as inflammatory lesions of meninges. The molecular basis underlying the cause of CINCA arises from different types of mutations including mis sense mutations within the CIAS1 gene present on human chromosome 1 q44. These mutations usually occur as single mutations or as cluster of genetic alterations ultimately resulting in the modification of NBS. The catalytic properties of the NBS are conferred within the molecule by the presence of ATP/GTPase P loops confined within a highly conserved motif and therefore a single amino acid change can dramatically alter its function. Most of the mutations causing CINCA including V262A are found to occur in the third exon of the CIAS1 gene. In the case of V262A mutation, the thymine base at the 785 position in the third exon of CIAS1 gene is replaced by a cytosine base. As a result of this mutation leading to a change in genetic code, valine at the 262 position of NBS peptide is replaced by alanine and thus alter the normal structure and functions of nucleotide binding site causing the over expression of inflammatory mediators (Feldmann et al.,2002).
FCAS and MWS are also hereditary disorders transmitted to the offspring via somatic chromosomes bearing mutations on the cryopyrin gene CIAS1. The genotypic expression of both FCAS and MWS follow the pattern of autosomal dominance. As seen in CINCA, the genetic alterations resulting from mutations are most likely to cluster within the NBS coding region of exon 3 of CIAS1 gene and these mutations, for example A439T and G569R, affect the oligomerization of the NBS. The structural and functional alteration of the peptide caused by the post mutational codon changes and defective oligomerization enhance the ability of altered NBS to interact very rapidly with the ASC and activate the caspase to mediate the conversion of pro-IL-1β to active IL-1β .The overexpression of IL-1β further triggers the biochemical and signaling pathways involved in generating an acute phase response (Stehlik and Reed, 2004). The symptoms facilitating the clinical characterization of both MWS and FCAS include fever, inflammation of conjunctiva, shivering, and severe skin rashes resembling urticaria. The differential diagnosis of MWS from FCAS is made by the fact that FCAS is mainly triggered by exposure to cold whereas the MWS can arise without any such exposures (Kanazawa and Furukawa, 2007).
The classical genetics dealt with studies governing the nature, inheritance and effects of mutation had encountered difficulties in generating mutant phenotypes. These problems faced in generating mutants for conducting genetic and clinical studies have been solved by the advent of genetic engineering techniques. A recent method called site directed muatgenesis is renowned for its simplicity and accuracy in introducing mutations at a particular base present in a cloned fragment of DNA. The method involves the amplification of the DNA in vitro in the presence of a short synthetic oligonucleotide fragment containing the desired mutation or mismatch base with a known sequence facilitating complementary binding. The amplification products carrying the desired mutations can then be cloned into suitable bacterial cells and harvested out as unmethylated polynucleotide chains containing mutated sequences (Primrose and Twyman, 2006). The purified DNA sample carrying the mutated sequences is sequenced via thermal cycle sequencing methods. The effects of the mutation on the structural and functional aspects at the organism level can be exploited by gene expression studies.
The experimental studies for governing the effects and altered functional aspects of V262A mutation are on cutting edge e and data available for specifying its exact role in CINCA is still inadequate (Infevers database, 2007). The objective of this laboration was to introduce a specific point mutation at the 785 position of NALP3 DNA sequence (V262A) via site directed mutagenesis and elucidate the exact sequence of the mutated sequence to locate the mutation and analyze its similarity with the subject sequence of human genome.
The mutagenesis primers used for introducing the V262A mutation was designed by the web based QuikChange® Primer Design Program (Stratagene, 2009) (Appendix I). The sense and antisense primers designed were 29 nucleotides long and had an optimum melting temperature or Tm of 78.81°C. The primer was designed in such a way that it would introduce the mutation at the 785 position of exon three of the NALP3 gene resulting in amino acid change at 262 positions in the peptide from valine to alanine. The mutagenesis reaction coupled with in vitro amplification of DNA was then carried out using the T3 thermocycler (Biometra, Germany) for 18 cycles. The mutagenesis PCR was performed strictly in accordance with the QuickChange®II Site-Directed Mutagenesis Kit (Stratagene, 2005). The resultant products of the mutagenesis reaction was then cleaved with Dpn I restriction enzyme. The total amount of DpnI used in the reaction mixture for the cleavage of the DNA was 10U and the reaction was set at 37°C for one hour. This process of restriction endonuclease cleavage was performed strictly in accordance with the QuickChange®II Site-Directed Mutagenesis Kit (Stratagene, 2009). The digested DNA sample was then transformed into XL10- Gold Ultracompetent Cells which were then cultured overnight in LB broth containing ampicillin instead of NZY broth. The LB broth was additionally supplemented with 1 M MgCl2, MgSO4 and 20% glucose.The subsequent steps for the culturing of transformed cells were performed in accordance with the QuickChange®II Site-Directed Mutagenesis Kit (Stratagene, 2005). The culture plates were observed for the presence of transformed cells; a separate individual bacterial colony was carefully picked up from the transformed colonies and inoculated into LB broth added with ampicillin in a concentration of 0.1 mg/ml. The culture was incubated at 37°C in a shaking incubator for 16 hours. Isolation and purification of mutant plasmid DNA from the overnight culture was done by using Qiaprep Spin Miniprep Kit (Qiagen, 2006). The measurement of the concentration of purified mutant plasmid DNA was thereafter conducted by the Nanodrop ND 1000 (Sareen Werner, USA). The thermal cycle sequencing was then conducted with a sense primer of 20 nucleotides length. The primer had a Tm of 59.90°C with a GC content of 55%. The primer used for sequencing PCR is given below;
Sense primer 5'-ACACACGACTGCGTCTCATC-3'
The total amount of template DNA contained within the sequencing reaction mix was 25 ng and the sequencing PCR protocol was strictly followed in accordance with DNA Sequencing BigDye Terminator V 3.1 cycle sequencing kit (Applied Biosystems, 2002). The purification of amplified sequencing PCR product was then carried out according to the DyeEx 2.0 Spin Kit (Qiagen, 2002) to eliminate the unincorporated dye from sequencing reaction. The purified sample was dried by incubating at 90°C for 20 minutes and added with 15 µl of template suppression reagent. The purified mutated plasmid DNA was then sequenced by using ABI 310 Genetic Analyzer (GMI, USA).
Results and Discussions
A site directed mutagenesis was performed at the 785 position of human NALP3 DNA sequence using the Quick change II site directed mutagenesis kit. The mutated NALP3 strand carrying cytosine base at the aforesaid position instead of thymine was then digested with the restriction endonuclease Dpn I and introduced into the competent host bacterial cells. The recombinant plasmid carrying the mutations were then isolated and purified from the transformed cells. The Nanodrop method employed to determine the concentration and purity of the isolated plasmid DNA revealed a plasmid DNA concentration of 13.7 ng/µl with a relative purity of 4.2, as shown in Table 1.
Table 1. Concentration and purity of the plasmid DNA carrying mutated NALP3 sequence
Type of nucleic acid
Purity ( A 260/ 280 )
The quantification of the plasmid DNA sample therefore drew the conclusion that a very low concentration of DNA was present in the isolated sample. This could have resulted from weak and inadequate elution leading to a very less concentration of DNA in the final elute. A relatively less purity was suggestive of the isolated sample with a high contamination of proteins and the reason for this could be the inadequate treatments in the plasmid isolation steps to remove proteins. Since the isolated DNA sample was of very less concentration and purity, the isolated and purified plasmid DNA sample borrowed from other authors (Bandaru and Kamble, 2009) was used for further experiments. The plasmid DNA received was carrying L264A mutation within the NALP3 sequence and had a concentration and purity of 31.2 ng/µl and 1.93 respectively. Thermal cycle sequencing was then performed with the purified and quantified plasmid DNA carrying the L264A mutation within the NALP3 sequence followed by subsequent purification of the amplification products. The exact sequence of purified plasmid carrying the NALP3 mutated sequences obtained from ABI 301 Genetic analyzer is given below;
The total number of nucleotides in the resultant sequence was found to be 613 with a large amount of nucleotides containing unknown base (N). A possible reason for this might be the display of similar peaks generated by more than two nucleotides which in turn made the distinction of separate and specific nucleotides difficult by the sequencer (Eton Bioscience Inc., 2003).
The sequencing data obtained for the plasmid DNA carrying mutated NALP3 sequences was analyzed for its similarity with the human genome using BLAST. The algorithmic analysis conducted through BLAST could not reveal any sequence similarity between the sequence of the sample and any part of human chromosomal DNA sequences. A wide variety of causes might account for the lack of any similarity or homology between the test and target sequences. This may result from the development of any conformational changes characteristic of DNA secondary structure by owing a high concentration of AT rich repeats. It may also have arisen as a consequence of the contamination of the sequencing sample leading to the generation of sequencing data with overlapping peaks. Finally, the most important but a very usual cause for this phenomenon might result from picking up two bacterial colonies unintentionally or by chance for the plasmid isolation (Eton Bioscience Inc., 2003).
A comparative analysis was further performed with the sequencing results provided by Lab supervisors for DNA sample carrying T436P mutation. The sequence is given below;
The algorithmic analysis was performed by employing BLAST and the results of which are given in Appendix II indicated 93% similarity of the query sequence with the library sequence, that is, human genomic sequences contained within the database. This was supported by the fact that the data obtained from BLAST showed that a total number of 122 out of 133 nucleotides were identical or homologous. However the mutated sequences failed to show any nucleotides after the 133 position for including the sequences for DNA strand carrying mutation at the 1306 base. Therefore the results obtained for nucleotide BLAST alignment between the sequence carrying the T436P mutation and the query sequence could not locate the mutation at the desired region (the replacement of adenine by cytosine at 1306 position) in the mutated sequence. The results were confirmed with and same results were showed by the sequence alignment using the Clustalw2.
The main causes involved in the generation of obscure sequencing result relate to the premature cessation of the sequencing reaction. This could result from either due to incorrect primer selection or the presence of DNA molecules containing highly repetitive sequences. The primer used for setting the thermal cycle sequencing for the T436P mutated strand was reverse primer. This primer would be able to get extended from the 1179 base position in the mutated DNA sequence at least until it covered the mutated region at the 1306 base. Since only 133 nucleotides were read by the sequencer, the primer selected could have failed in generating the complete sequence in the presence of the DNA polymerase enzyme. Moreover, the presence of repetitive sequences in the DNA strand might have changed the conformation of the DNA molecules to attain a secondary structure. The conformational changes also result from the exhaustion of the dNTPs during the sequencing reaction (Iowa State University, 2007).
The sequencing data was insufficient to locate the position of mutation and could not align completely with the wild type sequences. The secondary structure for both mutated and wild type sequences were predicted using Pole Bioinformatics Lyonnais, Network Protein Sequence Analysis. The representative data regarding the prediction of secondary structure of normal wild type and mutated NALP3 protein are given in Appendix III and IV respectively. Since the T436P mutation changed the amino acid at the 436 position from threonine to proline, it would affect the structural conformation and functions of the protein. Threonine is basically an essential amino acid possessing CHO group and considered an α- amino acid. The polar nature of this amino acid makes it easily susceptible to a number of post translational modifications soon after its synthesis like glycosylation and phosphorylation. Proline on the other hand, though it is considered as α amino acid, possess different properties to the polypeptide chains. The presence of proline in the mutated NALP3 may disrupt the normal folding and secondary structure of the peptide.
Though the sequencing data was insufficient to locate the position of mutation, it was suggested that site directed mutagenesis could be used an effective method to introduce site specific mutations in the desired DNA sequences. The effects of the mutation on the structural features of the protein can then be further studied by expression of the mutated genes in microbial cells. Alternatively cell culture based gene expression strategies could be a better choice for analyzing the effects of mutation on living system.