Molecular cloning, expression, purification, and characterization of shorter forms of human glutamic decarboxylase 67 in an E. coli expression system.
Previously, we reported the presence of truncated form of human brain l-glutamic decarboxylase 65 (tGAD65) in vivo as well as in vitro and found that tGAD65 was more active than the full-length GAD65 (Wei et al., J. Biomed. Sci., 10: 617-624, 2003). Here, we report the presence of two shorter forms of hGAD67, namely, hGAD67 (Delta1-70) and hGAD(67) (Delta1-90), referring to a deletion of 1-70 and 1-90 amino acids from the N-terminal, respectively. The molecular masses of hGAD67 (Delta1-70) and hGAD67 (Delta1-90) were found to be 59 kDa and 57 kDa, respectively. Both shorter forms were cloned, expressed, and characterized. In contrast to hGAD65, the shorter forms of hGAD67 were much less active than the full-length due to decrease in affinity of PLP towards the shorter enzymes. Both the full-length and one of the shorter forms of GAD67 were detected in porcine brain extract. Furthermore, the full-length GAD67 could be converted to both shorter forms by crude brain extract, suggesting that an endogenous protease may be present in the brain, which is responsible for the conversion. The cleavage of GAD67 seems to be Ca+(2)-dependent. The model for the conversion of GAD from full-length GAD to shorter forms of GAD and its physiological implications was proposed.
Sha
Di
D
Department of Biomedical Sciences, Florida Atlantic University, Biomedical Science Program, 777 Glades Road, Boca Raton, FL 33431-0991, USA.
Wei
Jianning
J
Wu
Heng
H
Jin
Ying
Y
Wu
Jang-Yen
JY
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000201:enzymology*; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology*; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1