Study On Single Nucleotide Polymorphism Of PAF-AH And The Relationship Between The Enzymatic Activity And Psoriasis

Platelet-activating factor (PAF, 1-O-alkyl-2-acetylsn-glycero-3-phosphor- choline) is a biologically active phospholipid with diverse potent biological effects synthesized by inflammatory cells, platelet and vascular endothelial cells. It is associated with the pathology of several human diseases, particularly inflammation. PAF acetylhydrolase (PAF-AH), a calcium independent phospholipase A2, catalysis the deacetylation and blockades the activity of PAF. The mutation of PAF-AH gene may contribute to the occurrence of inflammatory disease, especially atopic disease and autoimmune disease.Researchers have studied the relationship between PAF-AH and diseases, and they found there are kinds of single nucleotide polymorphism(SNP) in the gene of PAF-AH, such as Arg92His,Ile198Thr,Val279Phe ,Gln281Arg and Val379Ala, which have correlation with asthma, atopic dermatitis, septemia, cerbral infarction and cardiovascular disease and so on. Val279Phe and Gln281Arg are located in the enzyme active center, between Ser273 and Asp296, so that be significant of the correct fold of the protein. The polymorphism Ile198Thr in human plasmatic PAF-AH is located near tyrosine 205, which has been proposed to be important for LDL binding, and the LDL-associated form of PAF-AH is responsible for all of the PAF hydrolysis, so the mutation will influence the degradation of PAF. Val379Ala is situated in the vicinity of His351, one of the amino acids-along with Ser273 and Asp296-belonging to the catalytic triad characteristic of phospholipases, so the variant might influence enzymatic activities. The identification of genetic lesions that abolish plasma PAF-AH activity was an important step towards the investigation of the role of this enzyme in diseases.Psoriasis is a common, chronic, inflammatory disease of the skin. The pathogenesis of psoriasis is complicated, in which inflammatory mediators and inflammatory cells play pivotal roles. Izaki et al. found scale from lesional psoriatic skin contained substantial amounts of PAF, and PAF level was elevated in patients with psoriasis than healthy. PAF in patients with psoriasis significantly decreased after treatment and was correlated moderately with clinical improvement as indicated by the psoriasis area and severity index (PASI). In our previous study, we found the activity of plasma PAF-AH in patients with psoriasis was significantly lower than that in the health control. The activity of plasma PAF-AH in patients of progress period was significant lower than that in quiescent period and extinction period, and the activity of the latter two periods are similar.Since the activity of enzyme is influenced by multiple factors, which restricts the research, it is important to study the activity of PAF-AH from molecular biology aspect. We planed to construct prokaryotic expression vector of PAF-AH, express and purify functional proteins. Then we detected the enzymatic activity of SNPs of PAF-AH, and analyzed the correlation between SNPs of PAF-AH and psoriasis pathogenesis.PartⅠConstruction of Prokaryotic Expression Vector of PAF-AH In this part, we constructed prokaryotic expression vector of PAF-AH and got the SNPs of PAF-AH.At first, the full-length gene of PAF-AH was amplified from peripheral blood mononuclear cells (PBMC) by RT-PCR and cloned into the T vector. Then, the DNA sequence was subcloned into the expression vector pGEX-4T-3 after digestion with restriction enzymes. The SNPs of PAF-AH (Arg92His, Ile198Thr, Val279Phe, Gln281Arg, Val379Ala) were cloned by overlap PCR and the full-length gene of PAF-AH was utilized as template.The restriction endonuclease cutting and sequence analysis indicated that the sequence of wild-type and SNPs type of PAF-AH gene was identical with that in the GenBank.PartⅡExpression and Purification of PAF-AH ProteinIn this part, the PAF-AH proteins were expressed and purified. PAF-AH-GST fusion protein was produced in E. coli cells which induced by the lactose analog isopropylβ-D-thiogalactoside (IPTG). After that, the PAF-AH-GST fusion protein was purified by bound to Glutathione Sepharose 4B and then cleaved by thrombin protease.The 72 KD fusion proteins were identified by SDS-PAGE and Western blots in the bacterial lysate and supernatant. After purification, Western blot analysis results showed the purified protein was recognized by specific antibody, and the 72 KD GST-PAF-AH and 44 KD PAF-AH proteins were also identified.PartⅢEnzymatic Kinetic Analysis of SNPs of PAF-AHIn this part, we detected the enzymatic activity of SNPs of PAF-AH and analyzed the correlation between SNPs of PAF-AH and psoriasis pathogenesis. PAF-AH proteins were quantitated by Bradford assay at first. Then, the activity of PAF-AH proteins were measured by PAF-AH Assay kit. The assay uses 2-thio PAF which serves as a substrate. Upon hydrolysis of the acetyl thioester bond at the sn-2 position by PAF-SH free thiols are detected using 5,5-dithio-bis-(2-nitrobenzonic acid) (DNTB). Equal amount of PAF-AH proteins were added to the different concentration substrates (10/20/40/60/80/100uM), and then the absorbance was read every minute at 405nm. The reaction rate for PAF-AH proteins was determined using the formula provided by the assay kit. Km value and Vmax of different SNPs of PAF-AH and wide type PAF-AH were calculated by Lineweaver-Burk graphic method.Our kinetic experiments revealed a Km of 23uM for the wild-type enzyme. Compared to the wild-type enzyme, the Val279Phe are inactivity, Ile198Thr and Val379Ala showed 4 and 2 fold higher Km, which means the activity of enzyme are significantly reduced. Arg92His and Gln281Arg showed 1.3 and 1.5 fold higher Km, only a slightly reduce of activity.In Conclusion, Val279Phe, Ile198Thr and Val379Ala, the three common variants of the plasmatic PAF-AH have been showed to be of functional importance. These finding might explain the lower activity of PAF-AH in psoriasis patients, which prolong the activities of PAF, and associated with the pathogenesis and development of psoriasis. Thus, the SNPs of PAF-AH seems to play a key role in psoriasis.On the basis of our study, the activity of PAF-AH may be used as a valuable mark for the diagnosis and the assessment of pathogenetic condition of psoriasis. And may be approached of the treatment of psoriasis with recombinant PAF-AH and so on.