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Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

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Advances in Bioscience and Biotechnology 2011年第4期2卷 304-310页

作者： Hirofumi Taki Tomoharu Gomi Bryan Knuckley Paul R. Thompson Oliver Vugrek kazuya hirata Tatsurou Miyahara Kouichiro Shinoda Hiroyuki Hounoki Eiji Sugiyama Isao Usui Masaharu Urakaze Kazuyuki Tobe Tetsuya Ishimoto Ran Inoue Ayumi Tanaka Hiroki Mano Hirofumi Ogawa Hisashi Mori 不详

The murine peptidylarginine deiminase (PAD) has five isoforms encoded by different genes and participates in a variety of cellular functions through the citrullination of target proteins. The crystal structure of human PAD4 with a dimeric form was previously solved because of the enzyme’s relevance to rheumatoid arthritis. PAD6, abundant in mouse oocytes and eggs, is believed to take part in early events of embryogenesis, but its biochemical properties are little understood. Here we have purified and characterized a recombinant PAD6. A PAD6 cDNA was cloned from mouse ovary RNA and expressed in Escherichia coli through pET29 and pGEX vectors. When benzoyl-L-arginine ethyl ester was used as a substrate, no appreciable activity was detected with a cell homogenate under conditions where a human PAD4 cDNA caused significant activity. Both proteins were affinity-purified to near homogeneity. The circular dichroism spectra of PAD6 and human PAD4 were similar in the far ultraviolet region. On molecular sieving, PAD6 was eluted faster than human PAD4. The cross-linking of PAD6 with dimethyl suberimidate clearly showed six bands on an sodium dodecyl sulfate-polyacrylamide gel. These results indicate that PAD6 can constitute a hexameric structure. The purified PAD6 still showed no enzymatic activity. This unique structure and loss in enzymatic activity is strongly suggested to favor the formation of egg cytoplasmic sheets as the architectural protein.

关键词： Peptidylarginine deiminase Isoform Dimer Hexamer Mouse Oocytes Cytoplasmic sheets.

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