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.
