鳥糞嘌呤核苷酸交換蛋白(Brefeldin A inhibited guanine nucleotide-exchange protein factor) BIG1 和 BIG2 以GTP 取代GDP活化二磷酸腺苷核糖基化因子 (Arf1)。Arf1 調節高基氏體和質膜之間的囊泡運輸。多亞基束縛因子可調控高基氏體的內膜運輸和醣蛋白修飾。使用酵母雙雜交測定和免疫共沉澱分析高基氏體複合物上的多亞基束縛因子的其中一個亞基與BIG1和BIG2間的交互作用。BIG1和BIG2位於細胞核周圍的高基氏體結構。我們分析BIG1、BIG2、integrin β1和LAMP-1的蛋白質含量是否會因LoVo細胞處理布雷菲爾德菌素 (Brefeldin A, BFA)抑制Arf1的活化而改變。以shRNA 敲低(knockdown)BIG1或BIG2會使得 LoVo細胞的長度變長而改變細胞型態，細胞內O-GlcNAcylation 的蛋白修飾有增加的趨勢。我們也在 BIG1-、BIG2-或高基氏體束縛因子-敲低的細胞中分析integrin β1、CD44、LAMP-1 和組織蛋白酶 D (cathepsin D)的蛋白質量和凝膠電泳遷移率情形。我們也使用 Endo H、PNGaseF、neuraminidase和O-glycosidase分析敲低BIG1、BIG2或高基氏體束縛因子對於細胞的蛋白質醣基化修飾影響，並發現O-糖基化在控制組的細胞和敲低BIG1、BIG2或高基氏體束縛因子的細胞之間有明顯的區別。敲低BIG1、BIG2和高基氏體束縛因子不只在體外抑制細胞增殖並在體內抑制腫瘤生長，另外在 transwell遷移試驗(transwell migration assay)中，敲低BIG1或BIG2增加了 LoVo 細胞的粘附和遷移。綜上所述，我們的數據顯示 BIG1和 BIG2 與 高基氏體束縛因子之間的相互作用可能有助於調節參與細胞增殖、細胞粘附和細胞遷移的幾種膜蛋白表現和修飾。

Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and BIG2 act as guanine nucleotide exchange factors (GEFs) to catalyze the replacement of bound GDP with GTP to activate of ADP-ribosylation factor (Arf)1, which regulate vesicular transport between the Golgi and plasma membrane. The multi-subunit tethering factor at Golgi control intra-Golgi membrane trafficking and glycoprotein modification. Interaction of one subunit of the Golgi multi-subunit tethering factor with BIG1 and BIG2 was analyzed by yeast two-hybrid assays and co-immunoprecipitation. BIG1 and BIG2 were localized in the perinuclear Golgi structures. Treatment with BFA to inhibit Arf1 activation in LoVo cells, the protein abundance of BIG1, BIG2, integrin β1 and LAMP-1 was analyzed. Depletion of BIG1 or BIG2 by shRNA altered the LoVo cells morphology to become longer in cell length and increased the whole cell protein abundances of O-GlcNAcylation at several bands in gels. The protein abundance and gel electrophoretic mobility of integrin β1, CD44, LAMP-1 and cathepsin D were examined in BIG1-, BIG2- or Golgi tethering factor-knockdown cells. We also used Endo H, PNGaseF, neuraminidase and O-glycosidase to analyze the effects of BIG1, BIG2 or Golgi tethering factor-knockdown on protein glycosylation and found that protein O-glycosylation is the major different between control cells and cells that depletion of BIG1, BIG2 or Golgi tethering factor. BIG1, BIG2 and the Golgi tethering factor knockdown suppressed the cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, BIG1 or BIG2 knockdown increased the LoVo cell adhesion and migration in transwell migration assay. Hence, our data shown that BIG1 and BIG2 interact with tethering factor and regulate the several membrane protein abundance and modification that could contribute the cell proliferation, cell adhesion and cell migration.

1. Q. Yu, M. A. Ciemerych, P. Sicinski, Ras and Myc can drive oncogenic cell proliferation through individual D-cyclins. Oncogene 24, 7114-7119 (2005).
2. K. Kaibuchi, S. Kuroda, M. Amano, Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annual Review of Biochemistry 68, 459-486 (1999).
3. I. Kjos, K. Vestre, N. A. Guadagno, M. B. Distefano, C. Progida, Rab and Arf proteins at the crossroad between membrane transport and cytoskeleton dynamics. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1865, 1397-1409 (2018).
4. J. G. Donaldson, C. L. Jackson, ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nature Reviews Molecular Cell Biology 12, 362-375 (2011).
5. T. Taura, H. Krebber, P. A. Silver, A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export. Proceedings of the National Academy of Sciences of the United States of America 95, 7427-7432 (1998).
6. M. Nakamura et al., When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to γ-tubulin. Journal of Cell Biology 143, 1041-1052 (1998).
7. J. L. Bos, H. Rehmann, A. Wittinghofer, GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865-877 (2007).
8. R. Ishizaki, H.-W. Shin, H. Mitsuhashi, K. Nakayama, Redundant roles of BIG2 and BIG1, guanine-nucleotide exchange factors for ADP-ribosylation factors in membrane traffic between the trans-Golgi network and endosomes. Molecular Biology of the Cell 19, 2650-2660 (2008).
9. F. Boal, D. J. Stephens, Specific functions of BIG1 and BIG2 in endomembrane organization. PLoS One 5, e9898 (2010).
10. H. D. Jones, J. Moss, M. Vaughan, BIG1 and BIG2, Brefeldin A‐Inhibited Guanine Nucleotide‐Exchange Factors for ADP‐Ribosylation Factors. Methods in Enzymology 404, 174-184 (2005).
11. N. Morinaga, S.-C. Tsai, J. Moss, M. Vaughan, Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain. Proceedings of the National Academy of Sciences of the United States of America 93, 12856-12860 (1996).
12. A. Togawa, N. Morinaga, M. Ogasawara, J. Moss, M. Vaughan, Purification and cloning of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors. Journal of Biological Chemistry 274, 12308-12315 (1999).
13. R. Yamaji et al., Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex. Proceedings of the National Academy of Sciences of the United States of America 97, 2567-2572 (2000).
14. X. Shen, M.-S. Hong, J. Moss, M. Vaughan, BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1. Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 104, 1230-1235 (2007).
15. X. Shen et al., Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin β1 cycling and actin remodeling. Proceedings of the National Academy of Sciences of the United States of America 109, 14464-14469 (2012).
16. P. Stanley, Golgi glycosylation. Cold Spring Harbor Perspectives in Biology 3, a005199 (2011).
17. J. Shorter, G. Warren, Golgi architecture and inheritance. Annual Review of Cell and Developmental Biology 18, 379-420 (2002).
18. D. Ungar et al., Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. Journal of Cell Biology 157, 405-415 (2002).
19. M. Sohda et al., Interaction of Golgin‐84 with the COG complex mediates the intra‐Golgi retrograde transport. Traffic 11, 1552-1566 (2010).
20. F. Foulquier et al., Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II. Proceedings of the National Academy of Sciences of the United States of America 103, 3764-3769 (2006).
21. S.-H. Yu et al., Defective mucin-type glycosylation on α-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases. Journal of Biological Chemistry 293, 14534-14544 (2018).
22. A. Shestakova, S. Zolov, V. Lupashin, COG Complex-Mediated Recycling of Golgi Glycosyltransferases is Essential for Normal Protein Glycosylation. Traffic 7, 191-204 (2006).
23. S. N. Zolov, V. V. Lupashin, Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells. Journal of Cell Biology 168, 747-759 (2005).
24. I. D. Pokrovskaya et al., Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery. Glycobiology 21, 1554-1569 (2011).
25. Z. D’Souza, J. B. Blackburn, T. Kudlyk, I. D. Pokrovskaya, V. V. Lupashin, Defects in COG-mediated Golgi trafficking alter endo-lysosomal system in human cells. Frontiers in Cell and Developmental Biology 7, 118 (2019).
26. J. Bailey Blackburn, I. Pokrovskaya, P. Fisher, D. Ungar, V. V. Lupashin, COG complex complexities: detailed characterization of a complete set of HEK293T cells lacking individual COG subunits. Frontiers in Cell and Developmental Biology 4, 23 (2016).
27. C. Reily, T. J. Stewart, M. B. Renfrow, J. Novak, Glycosylation in health and disease. Nature Reviews Nephrology 15, 346-366 (2019).
28. J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman, R. S. Kerbel, Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236, 582-585 (1987).
29. M. Yoshimura, A. Nishikawa, Y. Ihara, S. i. Taniguchi, N. Taniguchi, Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proceedings of the National Academy of Sciences of the United States of America 92, 8754-8758 (1995).
30. M. R. Kudelka, T. Ju, J. Heimburg-Molinaro, R. D. Cummings, Simple sugars to complex disease—mucin-type O-glycans in cancer. Advances in Cancer Research 126, 53-135 (2015).
31. F. Dall’Olio, N. Malagolini, M. Trinchera, M. Chiricolo, Mechanisms of cancer-associated glycosylation changes. Frontiers in Bioscience 17, 670 (2012).
32. S. L. Bellis, Variant glycosylation: an underappreciated regulatory mechanism for β1 integrins. Biochimica et Biophysica Acta (BBA)-Biomembranes 1663, 52-60 (2004).
33. B. Von Lampe, A. Stallmach, E. Riecken, Altered glycosylation of integrin adhesion molecules in colorectal cancer cells and decreased adhesion to the extracellular matrix. Gut 34, 829-836 (1993).
34. O. Saitoh, W.-C. Wang, R. Lotan, M. Fukuda, Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. Journal of Biological Chemistry 267, 5700-5711 (1992).
35. A. K. Agarwal, R. P. Gude, R. D. Kalraiya, Regulation of melanoma metastasis to lungs by cell surface Lysosome Associated Membrane Protein-1 (LAMP1) via galectin-3. Biochemical and Biophysical Research Communications 449, 332-337 (2014).
36. T. Du et al., Cosmc disruption-mediated aberrant O-glycosylation suppresses breast cancer cell growth via impairment of CD44. Cancer Management and Research 12, 511 (2020).
37. S. Goodison, V. Urquidi, D. Tarin, CD44 cell adhesion molecules. Molecular Pathology 52, 189 (1999).
38. L. Du et al., CD44 is of functional importance for colorectal cancer stem cells. Clinical Cancer Research 14, 6751-6760 (2008).
39. P. Benes, V. Vetvicka, M. Fusek, Cathepsin D—many functions of one aspartic protease. Critical Reviews in Oncology/Hematology 68, 12-28 (2008).
40. G. Berchem et al., Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene 21, 5951-5955 (2002).
41. N. Zaidi, A. Maurer, S. Nieke, H. Kalbacher, Cathepsin D: a cellular roadmap. Biochemical and Biophysical Research Communications 376, 5-9 (2008).
42. L. K. Climer, M. Dobretsov, V. Lupashin, Defects in the COG complex and COG-related trafficking regulators affect neuronal Golgi function. Frontiers in Neuroscience 9, 405 (2015).
43. O. Laufman, H. H. Freeze, W. Hong, S. Lev, Deficiency of the Cog8 subunit in normal and CDG‐derived cells impairs the assembly of the COG and Golgi SNARE complexes. Traffic 14, 1065-1077 (2013).
44. C. Zhou et al., BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K–AKT and ERK signaling pathways. Neuroscience 254, 361-368 (2013).
45. T. Oka, D. Ungar, F. M. Hughson, M. Krieger, The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins. Molecular Biology of the Cell 15, 2423-2435 (2004).
46. W. Nickel, B. Brügger, F. T. Wieland, Vesicular transport: the core machinery of COPI recruitment and budding. Journal of Cell Science 115, 3235-3240 (2002).
47. X. Ren, G. G. Farías, B. J. Canagarajah, J. S. Bonifacino, J. H. Hurley, Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1. Cell 152, 755-767 (2013).
48. B. Xia et al., Modulation of cell adhesion and migration by the histone methyltransferase subunit mDpy-30 and its interacting proteins. PLoS One 5, e11771 (2010).
49. C.-C. Li et al., Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing. Proceedings of the National Academy of Sciences of the United States of America 108, 19228-19233 (2011).
50. B. T. Hennessy, D. L. Smith, P. T. Ram, Y. Lu, G. B. Mills, Exploiting the PI3K/AKT pathway for cancer drug discovery. Nature Reviews Drug Discovery 4, 988-1004 (2005).
51. J. A. McCubrey et al., Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1773, 1263-1284 (2007).