An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.
Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
E
E
Tsuji
Y
Y
Misumi
T
T
Fujiwara
N
N
Takami
S
S
Ogata
Y
Y
Ikehara
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017208:Rats, Wistar; D014162:Transfection
D012333:RNA, Messenger; D004247:DNA; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4