An active-site mutation (Gly633 .fwdarw. Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum
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説明
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)
収録刊行物
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- Biochemistry
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Biochemistry 31 11921-11927, 1992-12-01
American Chemical Society (ACS)
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キーワード
- Dipeptidyl Peptidase 4
- Molecular Sequence Data
- Endoplasmic Reticulum
- Transfection
- Animals
- Amino Acid Sequence
- RNA, Messenger
- Cloning, Molecular
- Rats, Wistar
- Dipeptidyl-Peptidases and Tripeptidyl-Peptidases
- Microscopy, Immunoelectron
- Cells, Cultured
- Immunosorbent Techniques
- Binding Sites
- Base Sequence
- DNA
- Blotting, Northern
- Rats, Inbred F344
- Rats
- Liver
- Mutagenesis, Site-Directed