Primary Studies On The Substitution Of SFX Sequences In B.Mori Silk Fibroin Gene For GAV Motif Of Human α-synuclein Gene

The aggregation principle of the Bombyx mori silk fibroin in the crystallizeddomains is similar to that of α-Synuclein which is a pathogeneic protein in humanParkinson's disease. In the certain conditions, a very conservative sequence composedof hydrophobic amino acids and some random coil sequences would induce theconservation of the whole protein structure and fibrillization. It has been found thatthe hydrophobic region in these two kinds of proteins is the key factor to theformation of β sheet. Experiment in vitro has indicated that α-Syn, in which the66VGGAVVTGV74 sequence (abbreviated to GAV motif) is decisivly important, iseasy to be accumulated together to form the fiber-like. In order to surpport it, twotruncated proteins of α-Syn65 (amino acid residues 1 to 65 of of α-Syn) and α-Syn74(amino acid residues 1 to 74 of α-Syn) were respectively expressed and studied;theresults indicated that partial districts within protein might regulate the aggregationbehavior of the whole protein. The GAV motif is necessary and can cause theconversion of its whole structure in the host protein with random coil, to form thefibrillization. GAV motif is mainly composed of amino acids Gly, Ala, and Val,which may take on the different function in the process of protein accumulation. Indetails, the amino acid Gly is most flexible and helpful to maintain the random coilstate of amino acid sequence in the solution; the amino acid Ala can induce theformation of protein secondary structure; the strong hydrophobicity of amino acid Valhas the inclination to form β-sheet. Several repetitive sequences mainly composed ofamino acids Gly, Ala and Val, such as"VGYGAGV","VGAGYGV","YGAGVGAGY","GAGAGSGAGA", and "VGAGYGAGAG" (named as SFX), appears in the crystallized domain of silk fibroin. Thesequence SFX is one of the important compositions to constitute β-layer. Animportant mechanism of nucleation-dependent aggregation has been found in theconversion from random coil to β-sheet of Bombyx mori silk fibroin and in theprocess of spinning silk by silkworm. In order to elucidate whether α-Synuclein couldbe fibrillized after the replacement of its accumulating core by another, we replacedthe core region of α-Syn1-74 with the core region of B.mori silk fibroin via PCR. Therecombiant expression plasmids α-Syn74SFX were successfully constructed. Theα-Syn74SFX proteins were respectively pured by the ion exchange pufication systemand the FPLC pufication system, then incubated for 6 days, and examined thestructure with the THT fluorescence and the atomic force microscope.The resultsindicated that the GAV motif of human α-Syn colud not be functionally replaced bythe SFX sequences of silkworm silk fibroin in the crystallized doamins, which was tobe another evidence to elucidate that the GAV motif was especially important to theaggregation of human α-Syn.This research would be helpful to the molecularmechanism of protein fibrillization and be significant for artificial spinning of silkfibroin.