AN ACTIVE-SITE MUTATION (GLY(633)-] ARG) OF DIPEPTIDYL PEPTIDASE-IV CAUSES ITS RETENTION AND RAPID DEGRADATION IN THE ENDOPLASMIC-RETICULUM

被引:51
作者
TSUJI, E
MISUMI, Y
FUJIWARA, T
TAKAMI, N
OGATA, S
IKEHARA, Y
机构
[1] FUKUOKA UNIV,SCH MED,DEPT BIOCHEM,RADIOISOTOPE LAB,JONAN KU,FUKUOKA 81401,JAPAN
[2] FUKUOKA UNIV,SCH MED,JOINT LAB PATHOL BIOCHEM,JONAN KU,FUKUOKA 81401,JAPAN
关键词
D O I
10.1021/bi00162a035
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. Taken together, these results suggest that the substitution of Gly633 --> Arg at the active site causes a conformational change of DPPIV coupled with its rapid degradation in the ER, accounting for the enzyme defect in the substrain 344/CRJ.
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页码:11921 / 11927
页数:7
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