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 forrn of human brain L-glutamic decarboxylase 65 (tGAD(65)) in vivo as well as in vitro and found that tGAD(65) was more active than the full-length GAD(65) (Wei et al., J. Biomed. Sci., 10: 617-624, 2003). Here, we report the presence of two shorter forms of hGAD(67), namely, hGAD(67) (Delta 1-70) and hGAD(67) (Delta 1-90), referring to a deletion of 1-70 and 1-90 amino acids from the N-terminal, respectively. The molecular masses of hGAD(67) (Delta 1-70) and hGAD(67) (Delta 1-90) were found to be 59 kDa and 57 kDa, respectively. Both shorter forms were cloned, expressed, and characterized. In contrast to hGAD(65), the shorter forms of hGAD(67) 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 GAD(67) were detected in porcine brain extract. Furthermore, the full-length GAD(67) 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 GAD(67) seems to be Ca2+-dependent. The model for the conversion of GAD from full-length GAD to shorter forms of GAD and its physiological implications was proposed. (c) 2005 Elsevier B.V. All rights reserved.