Chronic obstructive pulmonary diseases and lung cancer are two major medical problems for many veterans. The main causes of fatalities of these patients are mucus hypersecretion and metastasis, respectively. These diseases can result from alteration of mucin glycan synthesis and structure. Mucin-type glycans are elaborated in a stepwise manner from ser/thr as catalyzed by glycosyltransferases. Among them, branching enzymes are unique because they synthesize 26 N-acetylglucosaminide branch structures. These enzymes include core 2 N-acetylglucosaminyltransferase (C2GnT)-1/L, C2GnT-2/M, and C2GnT-3/T. C2GnT-L synthesizes core 2 in membrane glycoproteins and C2GnT-M synthesizes all three branch structures, core 2, core 4, and I antigen, in secreted mucins while the function of C2GnT-3 is unknown. Down regulation of C2GnT-L leads to defective immune function while overexpression causes cancer. Loss of function of C2GnT-M causes colon cancer. C2GnT-L and C2GnT-M will be the focus of this application. Glycan synthesis is determined by not only the amounts of glycosyltransferases but also their Golgi localization. The sub-Golgi localization of a glycosyltransferase closely matches the steps it participates in glycan synthesis. It is known that glycosyltransferases in colon cancer are mis-targeted, which results in the formation of short mucin glycans. The Golgi localization signal of glycosyltransferases resides mainly in the N-terminal cytoplasmic tail and transmembrane domain. However, the mechanism is not known. Our preliminary studies show that C2GnT-L and C2GnT-M are segregated intracellularly even though both synthesize core 2 and are expected to co- localize. In addition, C2GnT-M forms complexes with non-muscle myosin IIA &IIB and heat shock protein 70, while C2GnT-L co-localizes with Golgi Phosphoprotein 3. The nature of these interactions is not clear. We propose to test the hypothesis that Golgi localization of C2GnT-L and C2GnT-M is determined by their cytoplasmic tails via interaction with specific cytoplasmic proteins.
The specific aims of this application are to (1) Identify the cytoplasmic proteins that form complexes with C2GnT-M and characterize the structure of the complexes;(2) Identify the cytoplasmic proteins that form complexes with C2GnT-L and characterize the structure of the complexes;and (3) Characterize the functions of the proteins that form complexes with C2GnT-M and C2GnT-L in controlling intracellular trafficking and Golgi localization of these two enzymes, and mucin glycosylation. A549 and H292 cells with and without heat shock will be the test models because they express C2GnT-L, C2GnT-M, MUC1 and MUC5AC, which allows assessment of changes in mucin glycan structures in these two mucins following disruption of Golgi localization of these enzymes. Primary cultures of human bronchial epithelial cells will be used to confirm the findings made with these cells. The results will expand our fundamental understanding of the mechanism of intracellular trafficking and Golgi retention of glycosyltransferases and could help develop therapeutic interventions for these two lung diseases in veterans.

Public Health Relevance

Project Narrative Chronic obstructive pulmonary diseases and lung cancer are two major medical problems for many veterans. The main causes of the fatalities of these patients are mucus hypersecretion and metastasis, respectively. These diseases can result from alteration of mucin glycan structures in which branching structures play a key role. C2GnT-M and C2GnT-L control the branch structures of glycans in secreted and membrane-bound mucins and thus their respective functions. The proposed work aims at understanding how these two enzymes are localized to the Golgi and how disruption of their intracellular trafficking and Golgi localization could affect the structures of mucin glycans. The results will help understand the roles of these two enzymes in lung diseases and cancer, and may help develop therapeutic interventions for treating these two lung diseases.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX000985-01A1
Application #
8141882
Study Section
Respiration (PULM)
Project Start
2011-10-01
Project End
2015-09-30
Budget Start
2011-10-01
Budget End
2012-09-30
Support Year
1
Fiscal Year
2011
Total Cost
Indirect Cost
Name
Omaha VA Medical Center
Department
Type
DUNS #
844360367
City
Omaha
State
NE
Country
United States
Zip Code
68105
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Petrosyan, Armen; Cheng, Pi-Wan (2014) Golgi fragmentation induced by heat shock or inhibition of heat shock proteins is mediated by non-muscle myosin IIA via its interaction with glycosyltransferases. Cell Stress Chaperones 19:241-54
Petrosyan, Armen; Holzapfel, Melissa S; Muirhead, David E et al. (2014) Restoration of compact Golgi morphology in advanced prostate cancer enhances susceptibility to galectin-1-induced apoptosis by modifying mucin O-glycan synthesis. Mol Cancer Res 12:1704-16
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Petrosyan, Armen; Ali, Mohamed F; Verma, Shailendra Kumar et al. (2012) Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail. Int J Biochem Cell Biol 44:1153-65
Petrosyan, Armen; Ali, Mohamed F; Cheng, Pi-Wan (2012) Glycosyltransferase-specific Golgi-targeting mechanisms. J Biol Chem 287:37621-7