Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells

Abstract

Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS3 and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER− breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome–lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation.

Metabolic Shift Induced by ω -3 PUFAs and Rapamycin Lead to Cancer Cell Death

“…Autophagy is a highly dynamic process in mammalian cells. Therefore, monitoring autophagic flux is necessary for determining the rate of autophagy [32,33]. Autophagic flux was measured by comparing LC3II/β-actin levels in the cases of combination treatment with or without CQ (by increasing lysosomal pH) and long-lived protein p62 degradation.…”

The E3 Ubiquitin Ligase Siah-1 Suppresses Avian Reovirus Infection by Targeting p10 for Degradation

“…LAMP-1 is a multifunctional protein. It was reported previously that LAMP-1 was involved in apoptosis (31)(32)(33), autophagy (34,35), bacterial binding (36), and ARV p10 degradation (13). In this study, we show a new function of LAMP-1 in which LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection.…”

Podocyte Lysosome Dysfunction in Chronic Glomerular Diseases

“…In HeLa cells, HIV-1 counteracts metabolic and environmental stress-induced intracellular repositioning of lysosomes through enhancement of mTORC1 activity [51]. In another study, knockdown of FUT1 has been found to elevate the peripheral distribution of lysosomes by inhibition of mTORC1 activity, leading to the enhancement of autophagic flux [52]. All these previous findings demonstrate that the mTOR signaling pathway plays an important role in the regulation of lysosome positioning and trafficking.…”