Glycosylation and secretion of human α-amylases
Affiliation(s)
Laboratory of Glycobiology, The Noguchi Institute, Tokyo, Japan. New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Laboratory of Glycobiology, The Noguchi Institute, Tokyo, Japan. New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
ABSTRACT
Three human α-amylases exist: Amy1 (salivary amylase), Amy2A (pancreatic amylase), and Amy2B (expressed in various tissues). These amylases share a 97% - 99% amino acid sequence identity, and two potential N-glycosylation sites (N427 and N476) are commonly found in the C-terminal region. In general, salivary amylase is more frequently glycosylated than pancreatic amylase, and it is still uncertain why differences in the glycosylation pattern among human amylase iso-zymes occur. In this study, we found that there was no significant change of ratio of glycosylated molecules among isozymes produced by the same cultured cells, indicating that glycosylation of amylase is influenced by the type of cell producing the enzyme rather than being an inherent property of the amylase isozymes. We analyzed the glycosylation efficiency of N-glycosylation sites in recombinant Amy2A mutants produced by HEK293 cells and found that glycosylation efficiencies of N427 and N476 were 3% - 18% and 40% - 52%, respectively, indicating that the major N-glycosylation site of glycosylated Amy2A produced by HEK293 cells is N476. The difference in the glycosylation efficiency of each N-glycosylation site also seemed to contribute in part to generate different glycosylation patterns of human amylases. We also confirmed that the C-terminal region of human amylase plays a critical role in secretion, although glycosylation does not play a part in this effect.
Three human α-amylases exist: Amy1 (salivary amylase), Amy2A (pancreatic amylase), and Amy2B (expressed in various tissues). These amylases share a 97% - 99% amino acid sequence identity, and two potential N-glycosylation sites (N427 and N476) are commonly found in the C-terminal region. In general, salivary amylase is more frequently glycosylated than pancreatic amylase, and it is still uncertain why differences in the glycosylation pattern among human amylase iso-zymes occur. In this study, we found that there was no significant change of ratio of glycosylated molecules among isozymes produced by the same cultured cells, indicating that glycosylation of amylase is influenced by the type of cell producing the enzyme rather than being an inherent property of the amylase isozymes. We analyzed the glycosylation efficiency of N-glycosylation sites in recombinant Amy2A mutants produced by HEK293 cells and found that glycosylation efficiencies of N427 and N476 were 3% - 18% and 40% - 52%, respectively, indicating that the major N-glycosylation site of glycosylated Amy2A produced by HEK293 cells is N476. The difference in the glycosylation efficiency of each N-glycosylation site also seemed to contribute in part to generate different glycosylation patterns of human amylases. We also confirmed that the C-terminal region of human amylase plays a critical role in secretion, although glycosylation does not play a part in this effect.
Cite this paper
Takashima, S. and Amano, J. (2012) Glycosylation and secretion of human α-amylases. Advances in Biological Chemistry, 2, 10-19. doi: 10.4236/abc.2012.21002.
Takashima, S. and Amano, J. (2012) Glycosylation and secretion of human α-amylases. Advances in Biological Chemistry, 2, 10-19. doi: 10.4236/abc.2012.21002.
References
[1] [1] Doyon, Y., Home, W., Daull, P. and LeBel, D. (2002) Effect of C-domain N-glycosylation and deletion on rat pancreatic α-amylase secretion and activity. Biochemical Journal, 362, 259-264. [2] Yamashita, K., Tachibana, Y., Nakayama, T., Kitamura, M., Endo, Y. and Kobata, A. (1980) Structural studies of the sugar chains of human parotid α-amylase. The Journal of Biological Chemistry, 255, 5635-5642. [3] Yamashita, K., Tachibana, Y., Takeuchi, T. and Kobata, A. (1981) Structural study of the sugar chains of α-amylases produced ectopically in tumors. The Journal of Biochemistry, 90, 1281-1289. [4] Moriyama, T. (2008) Sialyl salivary-type amylase associated with ovarian cancer. Clinica Chimica Acta, 391, 106- 111. doi:10.1016/j.cca.2008.01.025 [5] Stiefel, D.J. and Keller, P.J. (1973) Preparation and some properties of human pancreatic amylase including a com- parison with human parotid amylase. Biochimica et Biophysica Acta, 302, 345-361. [6] Ito, K. (2006) Formation of glycoisoforms of human sa- livary α-amylase and endo-β-N-acetylglucosaminidase HS. Trends in Glyososcience and Glycotechnology, 18, 73-84. [7] Ito, K., Okada, Y., Ishida, K. and Minamiura, N. (1993) Human salivary endo-β-N-acetylglucosaminidase HS spe- cific for complex type sugar chains of glycoproteins. The Journal of Biological Chemistry, 268, 16074-16081. [8] Misaghi, S., Pacold, M.E., Blom, D., Ploegh, H.L. and Korbel, G.A. (2004) Using a small molecule inhibitor of peptide:N-glycanase to probe its role in glycoprotein turn- over. Chemistry & Biology, 11, 1677-1687. doi:10.1016/j.chembiol.2004.11.010 [9] Migashi, S., Korbel, G.A., Kessler, B., Spooner, E. and Ploegh, H.L. (2006) z-VAD-fmk inhibits peptide: N-gly- canase and may result in ER stress. Cell Death & Differentiation, 13, 163-165. doi:10.1038/sj.cdd.4401716 [10] Kim, Y.K., Kim, K.R., Kang, D.G., Jang, S.Y., Kim, Y.H. and Cha, H.J. (2009) Suppression of β-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells. Glycobiology, 19, 301-308. doi:10.1093/glycob/cwn138 [11] Aebi, M., Bernasconi, R., Clerc, S. and Molinari, M. (2010) N-glycan structures: recognition and processing in the ER. Trends in Biochemical Sciences, 35, 74-82. doi:10.1016/j.tibs.2009.10.001 [12] Panteghini, M. and Pagani, F. (1989) Time course of changes in serum activity of the P3 isoform of pancreatic amylase isoenzyme in patients with acute pancreatitis. Clinical Biochemistry, 22, 479-482.
[1] [1] Doyon, Y., Home, W., Daull, P. and LeBel, D. (2002) Effect of C-domain N-glycosylation and deletion on rat pancreatic α-amylase secretion and activity. Biochemical Journal, 362, 259-264. [2] Yamashita, K., Tachibana, Y., Nakayama, T., Kitamura, M., Endo, Y. and Kobata, A. (1980) Structural studies of the sugar chains of human parotid α-amylase. The Journal of Biological Chemistry, 255, 5635-5642. [3] Yamashita, K., Tachibana, Y., Takeuchi, T. and Kobata, A. (1981) Structural study of the sugar chains of α-amylases produced ectopically in tumors. The Journal of Biochemistry, 90, 1281-1289. [4] Moriyama, T. (2008) Sialyl salivary-type amylase associated with ovarian cancer. Clinica Chimica Acta, 391, 106- 111. doi:10.1016/j.cca.2008.01.025 [5] Stiefel, D.J. and Keller, P.J. (1973) Preparation and some properties of human pancreatic amylase including a com- parison with human parotid amylase. Biochimica et Biophysica Acta, 302, 345-361. [6] Ito, K. (2006) Formation of glycoisoforms of human sa- livary α-amylase and endo-β-N-acetylglucosaminidase HS. Trends in Glyososcience and Glycotechnology, 18, 73-84. [7] Ito, K., Okada, Y., Ishida, K. and Minamiura, N. (1993) Human salivary endo-β-N-acetylglucosaminidase HS spe- cific for complex type sugar chains of glycoproteins. The Journal of Biological Chemistry, 268, 16074-16081. [8] Misaghi, S., Pacold, M.E., Blom, D., Ploegh, H.L. and Korbel, G.A. (2004) Using a small molecule inhibitor of peptide:N-glycanase to probe its role in glycoprotein turn- over. Chemistry & Biology, 11, 1677-1687. doi:10.1016/j.chembiol.2004.11.010 [9] Migashi, S., Korbel, G.A., Kessler, B., Spooner, E. and Ploegh, H.L. (2006) z-VAD-fmk inhibits peptide: N-gly- canase and may result in ER stress. Cell Death & Differentiation, 13, 163-165. doi:10.1038/sj.cdd.4401716 [10] Kim, Y.K., Kim, K.R., Kang, D.G., Jang, S.Y., Kim, Y.H. and Cha, H.J. (2009) Suppression of β-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells. Glycobiology, 19, 301-308. doi:10.1093/glycob/cwn138 [11] Aebi, M., Bernasconi, R., Clerc, S. and Molinari, M. (2010) N-glycan structures: recognition and processing in the ER. Trends in Biochemical Sciences, 35, 74-82. doi:10.1016/j.tibs.2009.10.001 [12] Panteghini, M. and Pagani, F. (1989) Time course of changes in serum activity of the P3 isoform of pancreatic amylase isoenzyme in patients with acute pancreatitis. Clinical Biochemistry, 22, 479-482.