The role of Thr5 in human neuron growth inhibitory factor.
GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer's disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.
Cai
Bin
B
Chemical Biology Laboratory, Department of Chemistry, Fudan University, 200433 Shanghai, China.
Zheng
Qi
Q
Teng
Xin-Chen
XC
Chen
Dong
D
Wang
Yang
Y
Wang
Ke-Qiang
KQ
Zhou
Guo-Ming
GM
Xie
Yi
Y
Zhang
Ming-Jie
MJ
Sun
Hong-Zhe
HZ
Huang
Zhong-Xian
ZX
D000818:Animals; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism; D000090023:Metallothionein 3; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000502:physiology
D000090023:Metallothionein 3; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D013912:Threonine; D017665:Hydroxyl Radical; D008668:Metallothionein