摘要
哺乳动物的多种细胞在多种条件诱导凋亡过程中会表达乙酰胆碱酯酶
(acetylcholinesterase,AChE),用反义核酸抑制 AChE 的表达可以抑制一部分细
胞的凋亡,但 AChE 在凋亡中的功能仍不清楚。为研究其可能的功能,我们通过
转基因方法在 NRK(Normal Rat Kidney)细胞中过表达 AChE。正常 NRK 细胞
中检测不到 AChE 的酯酶活性,但用 Western blot 方法可检测到 AChE 蛋白的存
在。在多种细胞凋亡诱导因子刺激下,凋亡的 NRK 细胞中出现了 AChE 酯酶活
性,并且 AChE 的 mRNA 和蛋白也随着增加。在 NRK 细胞中过表达 AChE 蛋白
没有诱导细胞凋亡,并且我们得到了稳定表达的细胞株,但这种细胞株的生长明
显延缓了,而用稳定表达 AChE 反义核酸抑制 AChE 蛋白的表达却能促进细胞的
生长。在无血清诱导下,MTT 实验结果显示 AChE 表达株的残留细胞活性比对
照的低,而 AChE 反义核酸表达株的残留细胞活性比对照的高。这些结果说明在
凋亡过程中 AChE 有两个作用,先是增加的 AChE 抑制细胞的生长,然后是促进
细胞凋亡的进程。为检测 NRK 细胞中的无酯酶活性 AChE 在凋亡中可能的功能,
我们用一种蛋白合成的抑制剂 G418 来诱导细胞凋亡。G418 诱导的凋亡 NRK 细
胞中有非常明显的 AChE 酯酶活性,而这种 AChE 酯酶活性不依赖于新蛋白的合
成和 caspases 的活性。用共聚焦显微镜观察 AChE 的细胞定位,观察到正常 NRK
细胞中 AChE 定位于内质网上,而在凋亡的细胞中 AChE 分布于整个细胞。用透
射电镜观察 AChE 酯酶活性的产物分布,发现产物主要定位在细胞核中,并有一
部分位于胞质和一些囊泡的外膜上。缺失内质网定位信号肽的 AChE 表达质粒使
在胞质直接表达的 AChE 不能诱导细胞凋亡。但长时间培养后,在胞质中表达
AChE 细胞的凋亡比例明显比在内质网中表达 AChE 细胞的高。有趣的是在胞质
中表达 AChE 蛋白的 C 末端肽片段也有促进细胞凋亡的功能。根据上述结果我
们提出一个假设:在 NRK 细胞中无酯酶活性的 AChE 在内质网中比较稳定地存
在,当受到凋亡诱导因子刺激后以某种机制从内质网转移到胞质并部分转到细胞
核内,在胞质和核中其 C 末端肽段同其它的凋亡因子相互作用并促进细胞凋亡,
同时 AChE 本身的构象也发生改变,成为有酯酶活性的形式。为进一步分析无酯
酶活性 AChE 蛋白,我们制备了 AChE 抗体亲和层析柱,分离出总的 AChE 蛋白。
我们还制备了以 AChE 抑制剂(tacrine)为配体的亲和层析柱,分离有酯酶活性的
AChE。这两个方法为在蛋白水平上比较无酯酶活性和有酯酶活性 AChE 之间的
差异奠定了基础。
Acetylcholinesterase (AChE) is expressed in a number of cell lines upon
induction of apoptosis by various stimuli, and inhibiting expression of AChE by
AChE antisense prevents part of the cells from apoptosis. However, the function of
AChE in apoptosis is still elusive. We made AChE overexpresse in Normal Rat
Kidney (NRK) cells to investigate its possible function. AChE activity was not
presented in living NRK cells, but the AChE protein existed. Upon induction of
apoptosis, apoptotic NRK cells presented AChE activity, and AChE mRNA and
protein also increased. Overexpression of AChE in NRK cells did not induce
apoptosis. However, the proliferation of cell lines expressing AChE was retarded, and
the effect was reversed in cell lines overexpressing AChE antisene, in which AChE
expression decreased. Under induction by serum deprivation, the residual cell activity
of cells overexpressing AChE was lower than control, and the effect was reversed in
cell lines expressing AChE antisense. It suggested that AChE had two functions in the
process of apoptosis: increased AChE protein first inhibited cell proliferation, and
then promoted apoptosis. To detect the possible function of inactive AChE during
apoptosis, NRK cells were induced apoptosis by G418, which is an inhibitor of
protein synthesis. Apoptotic NRK cells induced by G418 exhibited strong AChE
activity, which was independent of new protein synthesis and capases activity. In
living NRK cells, AChE protein located in ER, and in apoptotic cells, AChE
distributed over the whole cell. The products of AChE enzymatic reaction observed
under electron microscope were located in nuclei, cytosole, and on the membrane of
some vacuoles. Expression of AChE without ER location signal (ERLS) did not
induce apoptosis. However, the percentage of apoptotic cells in NRK cells transfected
with AChE without ERLS was higher than that in NRK cells transfected with intact
AChE. Interestingly, cells expressed C-terminal of AChE had the same effect with
cells expressed AChE without ERLS, and moreover, AChE directly expressed in
cytosole had no activity. Base on these experiments, we brought forward a hypothesis
on AChE in apoptosis: in the process of apoptosis, inactive AChE, which stably stood
in ER in NRK cells, was translocated into cytosole and nuclei, where the C-terminal
of AChE interacted with some apoptotic factors and promoted apoptosis, and at the
same time, inactive AChE was transformed into active AChE. To further analyse the
inactive AChE protein, we prepared antibody affinity chromatography to isolate the
total AChE. This method with tacrine affinity chromatography which was to isolate
active AChE provided precondition for analysis of the difference between inactive and
active AChE at the protein level.
引文
1. Massoulie J. and Bon S. (1982) The molecular forms of cholinesterase and
acetylcholinesterase in vertebrates. Annu Rev Neurosci 5: 57-106
2. Massoulie J., Pezzementi L., Bon S., Krejci E. and Vallette F. M. (1993)
Molecular and cellular biology of cholinesterases. Prog Neurobiol 41: 31-91
3. Li Y., Camp S., Rachinsky T. L., Getman D. and Taylor P. (1991) Gene
structure of mammalian acetylcholinesterase. Alternative exons dictate
tissue-specific expression. J Biol Chem 266: 23083-23090
4. Li Y., Camp S. and Taylor P. (1993) Tissue-specific expression and alternative
mRNA processing of the mammalian acetylcholinesterase gene. J Biol Chem
268: 5790-5797
5. Rosenberry T. L., Toutant J. P., Haas R. and Roberts W. L. (1989)
Identification and analysis of glycoinositol phospholipid anchors in membrane
proteins. Methods Cell Biol 32: 231-255
6. Ferguson M. A. and Williams A. F. (1988) Cell-surface anchoring of proteins
via glycosyl-phosphatidylinositol structures. Annu Rev Biochem 57: 285-320
7. Krejci E., Thomine S., Boschetti N., Legay C., Sketelj J. and Massoulie J.
(1997) The mammalian gene of acetylcholinesterase-associated collagen. J
Biol Chem 272: 22840-22847
8. Perrier A. L., Massoulie J. and Krejci E. (2002) PRiMA: the membrane anchor
of acetylcholinesterase in the brain. Neuron 33: 275-285
9. Kronman C., Chitlaru T., Elhanany E., Velan B. and Shafferman A. (2000)
Hierarchy of post-translational modifications involved in the circulatory
longevity of glycoproteins. Demonstration of concerted contributions of
glycan sialylation and subunit assembly to the pharmacokinetic behavior of
bovine acetylcholinesterase. J Biol Chem 275: 29488-29502
10. Legay C., Huchet M., Massoulie J. and Changeux J. P. (1995) Developmental
regulation of acetylcholinesterase transcripts in the mouse diaphragm:
alternative splicing and focalization. Eur J Neurosci 7: 1803-1809
11. Kaufer D., Friedman A., Seidman S. and Soreq H. (1998) Acute stress
facilitates long-lasting changes in cholinergic gene expression. Nature 393:
373-377
12. Meshorer E., Erb C., Gazit R., Pavlovsky L., Kaufer D., Friedman A., et al.
(2002) Alternative splicing and neuritic mRNA translocation under long-term
neuronal hypersensitivity. Science 295: 508-512
13. Soreq H. and Seidman S. (2001) Acetylcholinesterase--new roles for an old
actor. Nat Rev Neurosci 2: 294-302
14. Layer P. G., Weikert T. and Alber R. (1993) Cholinesterases regulate neurite
growth of chick nerve cells in vitro by means of a non-enzymatic mechanism.
Cell Tissue Res 273: 219-226
15. Small D. H., Reed G., Whitefield B. and Nurcombe V. (1995) Cholinergic
regulation of neurite outgrowth from isolated chick sympathetic neurons in
culture. J Neurosci 15: 144-151
16. Koenigsberger C., Chiappa S. and Brimijoin S. (1997) Neurite differentiation
is modulated in neuroblastoma cells engineered for altered
acetylcholinesterase expression. J Neurochem 69: 1389-1397
17. Grifman M., Galyam N., Seidman S. and Soreq H. (1998) Functional
redundancy of acetylcholinesterase and neuroligin in mammalian
neuritogenesis. Proc Natl Acad Sci U S A 95: 13935-13940
18. Darboux I., Barthalay Y., Piovant M. and Hipeau-Jacquotte R. (1996) The
structure-function relationships in Drosophila neurotactin show that
cholinesterasic domains may have adhesive properties. Embo J 15: 4835-4843
19. Llinas R. R. and Greenfield S. A. (1987) On-line visualization of dendritic
release of acetylcholinesterase from mammalian substantia nigra neurons.
Proc Natl Acad Sci U S A 84: 3047-3050
20. Holmes C., Jones S. A., Budd T. C. and Greenfield S. A. (1997)
Non-cholinergic, trophic action of recombinant acetylcholinesterase on
mid-brain dopaminergic neurons. J Neurosci Res 49: 207-218
21. Inestrosa N. C., Alvarez A., Perez C. A., Moreno R. D., Vicente M., Linker C.,
et al. (1996) Acetylcholinesterase accelerates assembly of
amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral
site of the enzyme. Neuron 16: 881-891
22. Wright C. I., Geula C. and Mesulam M. M. (1993) Neurological
cholinesterases in the normal brain and in Alzheimer's disease: relationship to
plaques, tangles, and patterns of selective vulnerability. Ann Neurol 34:
373-384
23. Lev-Lehman E., Deutsch V., Eldor A. and Soreq H. (1997) Immature human
megakaryocytes produce nuclear-associated acetylcholinesterase. Blood 89:
3644-3653
24. Kawashima K. and Fujii T. (2000) Extraneuronal cholinergic system in
lymphocytes. Pharmacol Ther 86: 29-48
25. Soreq H., Patinkin D., Lev-Lehman E., Grifman M., Ginzberg D., Eckstein F.,
et al. (1994) Antisense oligonucleotide inhibition of acetylcholinesterase gene
expression induces progenitor cell expansion and suppresses hematopoietic
apoptosis ex vivo. Proc Natl Acad Sci U S A 91: 7907-7911
26. Robitzki A., Mack A., Hoppe U., Chatonnet A. and Layer P. G. (1998)
Butyrylcholinesterase antisense transfection increases apoptosis in
differentiating retinal reaggregates of the chick embryo. J Neurochem 71:
p413-420
27. Lev-Lehman E., Ginzberg D., Hornreich G., Ehrlich G., Meshorer A., Eckstein
F., et al. (1994) Antisense inhibition of acetylcholinesterase gene expression
causes transient hematopoietic alterations in vivo. Gene Ther 1: 127-135
28. Ehrlich G., Viegas-Pequignot E., Ginzberg D., Sindel L., Soreq H. and Zakut
H. (1992) Mapping the human acetylcholinesterase gene to chromosome 7q22
by fluorescent in situ hybridization coupled with selective PCR amplification
from a somatic hybrid cell panel and chromosome-sorted DNA libraries.
Genomics 13: 1192-1197
29. Stephenson J., Czepulkowski B., Hirst W. and Mufti G. J. (1996) Deletion of
the acetylcholinesterase locus at 7q22 associated with myelodysplastic
syndromes (MDS) and acute myeloid leukaemia (AML). Leuk Res 20:
235-241
30. Zhang X. J., Yang L., Zhao Q., Caen J. P., He H. Y., Jin Q. H., et al. (2002)
Induction of acetylcholinesterase expression during apoptosis in various cell
types. Cell Death Differ 9: 790-800
31. Li Y., Camp S., Rachinsky T. L., Bongiorno C. and Taylor P. (1993) Promoter
elements and transcriptional control of the mouse acetylcholinesterase gene. J
Biol Chem 268: 3563-3572
32. Atanasova E., Chiappa S., Wieben E. and Brimijoin S. (1999) Novel
messenger RNA and alternative promoter for murine acetylcholinesterase. J
Biol Chem 274: 21078-21084
33. Shapira M., Tur-Kaspa I., Bosgraaf L., Livni N., Grant A. D., Grisaru D., et al.
(2000) A transcription-activating polymorphism in the ACHE promoter
associated with acute sensitivity to anti-acetylcholinesterases. Hum Mol Genet
9: 1273-1281
34. Chan R. Y., Boudreau-Lariviere C., Angus L. M., Mankal F. A. and Jasmin B.
J. (1999) An intronic enhancer containing an N-box motif is required for
synapse- and tissue-specific expression of the acetylcholinesterase gene in
skeletal muscle fibers. Proc Natl Acad Sci U S A 96: 4627-4632
35. Plageman L. R., Pauletti G. M. and Skau K. A. (2002) Characterization of
acetylcholinesterase in Caco-2 cells. Exp Biol Med (Maywood) 227: 480-486
36. Coleman B. A. and Taylor P. (1996) Regulation of acetylcholinesterase
expression during neuronal differentiation. J Biol Chem 271: 4410-4416
37. Grisaru D., Lev-Lehman E., Shapira M., Chaikin E., Lessing J. B., Eldor A., et
al. (1999) Human osteogenesis involves differentiation-dependent increases in
the morphogenically active 3' alternative splicing variant of
acetylcholinesterase. Mol Cell Biol 19: 788-795
38. Lapidot-Lifson Y., Prody C. A., Ginzberg D., Meytes D., Zakut H. and Soreq
H. (1989) Coamplification of human acetylcholinesterase and
butyrylcholinesterase genes in blood cells: correlation with various leukemias
and abnormal megakaryocytopoiesis. Proc Natl Acad Sci U S A 86: 4715-4719
39. Galyam N., Grisaru D., Grifman M., Melamed-Book N., Eckstein F., Seidman
S., et al. (2001) Complex host cell responses to antisense suppression of
ACHE gene expression. Antisense Nucleic Acid Drug Dev 11: 51-57
40. Fuentes M. E. and Taylor P. (1993) Control of acetylcholinesterase gene
expression during myogenesis. Neuron 10: 679-687
41. Chan R. Y., Adatia F. A., Krupa A. M. and Jasmin B. J. (1998) Increased
expression of acetylcholinesterase T and R transcripts during hematopoietic
differentiation is accompanied by parallel elevations in the levels of their
respective molecular forms. J Biol Chem 273: 9727-9733
42. Rotundo R. L. (1988) Biogenesis of acetylcholinesterase molecular forms in
muscle. Evidence for a rapidly turning over, catalytically inactive precursor
pool. J Biol Chem 263: 19398-19406
43. Brockman S. K., Usiak M. F. and Younkin S. G. (1986) Assembly of
monomeric acetylcholinesterase into tetrameric and asymmetric forms. J Biol
Chem 261: 1201-1207
44. Chatel J. M., Grassi J., Frobert Y., Massoulie J. and Vallette F. M. (1993)
Existence of an inactive pool of acetylcholinesterase in chicken brain. Proc
Natl Acad Sci U S A 90: 2476-2480
45. Eichler J. and Silman I. (1995) The activity of an endoplasmic
reticulum-localized pool of acetylcholinesterase is modulated by heat shock. J
Biol Chem 270: 4466-4472
46. Torriglia A., Negri C., Chaudun E., Prosperi E., Courtois Y., Counis M. F., et
al. (1999) Differential involvement of DNases in HeLa cell apoptosis induced
by etoposide and long term-culture. Cell Death Differ 6: 234-244
47. Gong J., Traganos F. and Darzynkiewicz Z. (1994) A selective procedure for
DNA extraction from apoptotic cells applicable for gel electrophoresis and
flow cytometry. Anal Biochem 218: 314-319
48. Karnovsky M. J. and Roots L. (1964) A "Direct-Coloring" Thiocholine
Method for Cholinesterases. J Histochem Cytochem 12: 219-221
49. Legay C., Bon S. and Massoulie J. (1993) Expression of a cDNA encoding the
glycolipid-anchored form of rat acetylcholinesterase. FEBS Lett 315: 163-166
50. Bar-Nun S., Shneyour Y. and Beckmann J. S. (1983) G-418, an elongation
inhibitor of 80 S ribosomes. Biochim Biophys Acta 741: 123-127
51. Carson K. A., Geula C. and Mesulam M. M. (1991) Electron microscopic
localization of cholinesterase activity in Alzheimer brain tissue. Brain Res 540:
204-208
52. Chatel J. M., Eichler J., Vallette F. M., Bon S., Massoulie J. and Grassi J.
(1994) Two-site immunoradiometric assay of chicken acetylcholinesterase:
active and inactive molecular forms in brain and muscle. J Neurochem 63:
1111-1118
53. Choi R. C., Leung P. W., Dong T. T., Wan D. C. and Tsim K. W. (1996)
Calcitonin gene-related peptide increases the expression of
acetylcholinesterase in cultured chick myotubes. Neurosci Lett 217: 165-168
54. Belbeoc'h S., Massoulie J. and Bon S. (2003) The C-terminal T peptide of
acetylcholinesterase enhances degradation of unassembled active subunits
through the ERAD pathway. Embo J 22: 3536-3545
55. Hu W., Gray N. W. and Brimijoin S. (2003) Amyloid-beta increases
acetylcholinesterase expression in neuroblastoma cells by reducing enzyme
degradation. J Neurochem 86: 470-478
56. Melo J. B., Agostinho P. and Oliveira C. R. (2002) Amyloid beta-peptide
25-35 reduces [3H]acetylcholine release in retinal neurons. Involvement of
metabolic dysfunction. Amyloid 9: 221-228
57. Alvarez A., Alarcon R., Opazo C., Campos E. O., Munoz F. J., Calderon F. H.,
et al. (1998) Stable complexes involving acetylcholinesterase and
amyloid-beta peptide change the biochemical properties of the enzyme and
increase the neurotoxicity of Alzheimer's fibrils. J Neurosci 18: 3213-3223
58. Bartos E. M. and Glinos A. D. (1976) Properties of growth-related
acetylcholinesterase in a cell line of fibroblastic origin. J Cell Biol 69:
638-646
59. Park S. E., Kim N. D. and Yoo Y. H. (2004) Acetylcholinesterase plays a
pivotal role in apoptosome formation. Cancer Res 64: 2652-2655
60. Giles K. (1997) Interactions underlying subunit association in cholinesterases.
Protein Eng 10: 677-685
61. Cottingham M. G., Hollinshead M. S. and Vaux D. J. (2002) Amyloid fibril
formation by a synthetic peptide from a region of human acetylcholinesterase
that is homologous to the Alzheimer's amyloid-beta peptide. Biochemistry 41:
13539-13547
62. Bon S., Dufourcq J., Leroy J., Cornut I. and Massoulie J. (2004) The
C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports
homomeric and heteromeric interactions. Eur J Biochem 271: 33-47
63. Perry C., Sklan E. H., Birikh K., Shapira M., Trejo L., Eldor A., et al. (2002)
Complex regulation of acetylcholinesterase gene expression in human brain
tumors. Oncogene 21: 8428-8441
64. Breckenridge D. G., Germain M., Mathai J. P., Nguyen M. and Shore G. C.
(2003) Regulation of apoptosis by endoplasmic reticulum pathways.
Oncogene 22: 8608-8618
65. Nutt L. K., Pataer A., Pahler J., Fang B., Roth J., McConkey D. J., et al. (2002)
Bax and Bak promote apoptosis by modulating endoplasmic reticular and
mitochondrial Ca2+ stores. J Biol Chem 277: 9219-9225
66. Grisaru D., Sternfeld M., Eldor A., Glick D. and Soreq H. (1999) Structural
roles of acetylcholinesterase variants in biology and pathology. Eur J Biochem
264: 672-686
67. Day T. and Greenfield S. A. (2003) A peptide derived from
acetylcholinesterase induces neuronal cell death: characterisation of possible
mechanisms. Exp Brain Res 153: 334-342
68. Gomez-Ramos P., Mufson E. J. and Moran M. A. (1992) Ultrastructural
localization of acetylcholinesterase in neurofibrillary tangles, neuropil threads
and senile plaques in aged and Alzheimer's brain. Brain Res 569: 229-237