Isolation and molecular cloning of epidermal- and hair follicle-specific peptidylarginine deiminase (type III) from rat.
Peptidylarginine deiminase (PAD) is a post-translational modification enzyme that catalyzes deimination of arginine residues of proteins in the presence of calcium ions. There are three types of PAD in rodent tissues: PAD types I, II, and III [Terakawa et al. (1991) J. Biochem. 110, 661-666]. Type III enzyme was detected only in the epidermis and in hair follicles. In this study, we have purified PAD type III from 2-day-old rat epidermis by a four-step procedure that included soybean trypsin inhibitor-affinity chromatography. The enzyme was purified about 600-fold from the crude extract and the recovery was 23%. The final preparation of the enzyme gave only a single protein band on SDS-PAGE and showed an apparent molecular weight of 76,000. Subsequently, we cloned and sequenced the full-length cDNA encoding rat PAD type III by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed from the internal amino acid sequences and by the rapid amplification of the cDNA ends method. The composite cDNA sequence contained a 5' untranslated region of 42 bp, an open reading frame of 1,995 bases that encoded 664 amino acids (Mr=75,036), a 3' untranslated region of 1,063 bp, and part of a poly(A)+ tail. The entire reading frame sequence of rat PAD type III showed 51% homology with that of rat PAD type II, and the C-terminal region is highly conserved between the two types. The cloned gene was expressed in Escherichia coli cells to produce PAD type III, which had not only enzymatic activity, but also immunoreactivity against specific antibodies toward PAD type II. Furthermore, the specific expression of the enzyme in the epidermis and hair follicles was confirmed by RT-PCR assays of mRNAs from several tissues.
Nishijyo
T
T
Laboratory of Biochemistry, School of Agriculture, Ibaraki University, Inashiki-gun.
Kawada
A
A
Kanno
T
T
Shiraiwa
M
M
Takahara
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D004817:Epidermis / Q000201:enzymology; D018859:Hair Follicle / Q000201:enzymology; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D006867:Hydrolases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases