Cloning and functional expression of rat kidney dipeptidyl peptidase II.
Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.
Fukasawa
K M
KM
Department of Oral Biochemistry, Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan. kmf@po.mdu.ac.jp
Fukasawa
K
K
Higaki
K
K
Shiina
N
N
Ohno
M
M
Ito
S
S
Otogoto
J
J
Ota
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D004150:Dipeptidases; C022449:proline dipeptidase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100151:dipeptidyl peptidase II