TGM 1 Gene Sequencing And Correlation Between TGase 1 Activity And Involucrin Expression In The Nail Of Patients With LI

Surface epithelial cells, such as the epidermal keratinocytes, undergo a process of terminal cell differentiation that results in the construction of a multilayered epithelium. The multilayered epithelium functions as a chemical, physical and stable barrier against environmental influences. The cornified cell envelope (CE), an extremely tough protein/lipid polymer structure that formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells, is a critical structure for barrier function at the outermost layer of the skin epidermis. It consists of two parts:a lipid envelope and a protein envelope. For CE formation in terminal keratinocyte differentiation, covalent cross-linking of constituting proteins, such as loricrin, involucrin and small proline-rich protein, is essential. This reaction requires transglutaminase 1 (TGase 1), which is a calcium-dependent enzyme catalyzing an intermolecular isopeptide bond formation between proteins.TGase 1 is expressed and activated during terminal differentiation of keratinocytes and synthesizes CE by a cross-linking reaction. TGase 1 gene (TGM 1) that encods human TGase 1 was isolated from human genomic DNA and characterized. TGM 1 spans 14.3 kilobase pairs and is composed of 15 exons. It has been identified that in humans, TGase 1 is known to participate in CE formation. It is activated by limited proteolysis during keratinocyte differentiation and contributes cooperatively in sequential cross-linking of the substrates. Consequently, disorder of the TGase 1 activity results in an irregular phenotype in the skin.Involucrin which was the first precursor discovered and cloned is an important structural component of CE. It is expressed early in the keratinocyte differentiation process and thought to be a component of the initial envelope scaffolding for subsequent cross-linking of additional precursors. in vitro, a preferred site of cross-link formation is Gln496 which is the first site to be cross-linked in vivo. The cross-linking process is progressive, additional cross-link sites are recruited with time.Lamellar ichthyosis (LI), a autosomal recessive genodermatosis, is a severe skin disorder showing widespread hyperkeratosis of the skin. TGasel that mediates cross-links in the formation of the cornified cell envelope during terminal differentiation of epidermis deficiency by TGM1 mutations is the most prevalent cause of LI. Additionally, a high expression of involucrin can be observed in the abnormal cornified cell envelope of the LI patients. Thus, disturbed function of TGasel could alter cross-linkage and the formation of CE.-But, the association between the activity of TGasel and the expression of involucrin is not clear and has not yet been found in reports.In the present study the mutation of the TGM 1 sequencing, the TGasel activity, the involucrin expression and the correlation between them in LI patients were explored.1. The nail samples were collected from eight cases of LI patients (LI group) and eight healthy volunteers (control group). Besides, the blood samples were collected from two cases of LI patients.2. The two blood samples of LI patients were sent to Ben Yuan Zheng Yang Company for TGM1 gene analysis.3. The total protein of nail was extracted by CellyticTM reagent.4. The SDS-Page was performed from the extracted protein, and the immunoblotting for involucrin was carried out on the transferred nitrocellulose membrane from the SDS-Page gel under image-analysis.5. The TGase 1 activity was detected by dot immunoblotting under image-analysis.6. The difference and correlation between the two groups were analyzed with SPSS 10.0 software.1. Sequencing of TGM1 analysis in LI cases:The mis-sense point mutations in the 3rd, 8th,12th and 13th exons were found in the two LI cases. Moreover, a pre-termination by 150T deletion in the 3rd exon was found in one LI case with sever syndrome.2. Comparison of SDS-Page from the nail total protein between two groups:There were 5 major bands,69kDa,63kDa,54kDa,48kDa,38kDa, in the LI group; while there were only the back 4 major bands in the C group. The GSM (gray scaled means) of 63kDa and the additional 69kDa in the LI group were 154.36±1.46 and 131.98±1.35 respectively; the GSM of 63kDa in the C group was 136.56±1.38, the difference in GSM between the two groups was significant, P<0.05.3. GSM comparison of involucrin expression between the two groups:In the LI group, the involucrin expressed in the major bands,69kDa and 63kDa, their GSM were 139.92±1.44 and 148.54±1.6 respectively. However, in the C group, the involucrin only expressed in the 63kDa major band, its GSM was 135.96±1.10. The difference in GSM was significant, P<0.05.4. GSM comparison of TGase activity between two groups:The GSM of TGase 1 in the C group was 169.11±1.14, that in the LI group was 122.49±1.32. The TGase 1 dot immunoblotting signals in the C group were stronger than those in the LI group, P<0.05.5. Correlation between involucrin expression and TGase 1 activity:In the C group, there was a positive correlation in 63kDa band between involucrin expression and TGase 1 activity, r=0.762, P<0.05. In the LI group there were two negative correlations in 69kDa and 63kDa bands between involucrin expression and TGase 1 activity, r=-0.837, r=-0.780, P<0.05.1. Sequencing of TGM1 analysis in LI cases showed that the mis-sense point mutations in the 3rd,8th,12th and 13th exons could be found in the two LI cases; moreover, a pre-termination by 150T deletion in the 3rd exon was only found in one LI case with sever syndrome, suggesting that abnormal TGase 1 expression may be present in the LI group.2. There were 5 major bandsin the LI group; while there were only 4 major bands in the C group, suggesting that abnormal total protein in the nails may be present in the LI group.3. In the LI group, the involucrin expressed in the major bands,69kDa and 63kDa; however, in the C group, the involucrin only expressed in the 63kDa major band, suggesting that the involucrin may be overexpressed in the LI group.4. The TGase 1 dot immunoblotting signals in the C group were stronger than those in the LI group, suggesting that the TGase 1 activity in the LI group may be weaker than that in the C group.5. In the C group, there was a positive correlation in 63kDa band between involucrin expression and TGase 1 activity; in the LI group there were two negative correlations in 69kDa and 63kDa bands between involucrin expression and TGase 1 activity. It suggests that the overexpressed involucrin may be compensative expression for defect skin barrier caused by abnormal TGase 1 activity.