Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line

Abstract

Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM.

Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

“…DMEM containing 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin at 37° was used in a 5% CO 2 atmosphere to maintain 2D and 3D NB cell cultures. The NB_1(- Mgat1 ) cell line was rescued by transient transfection with pCDNA3.1 recombinant vector coding the mouse Mgat1 cDNA using the Lipofectamine ® 2000 (Thermofisher Scientific, MA, USA) protocol [ 18 ]. The mouse Mgat1 coding sequence was obtained from Dr. Pamela Stanly, College of Albert Einstein [ 21 ] and was modified to have BamHI at both 5’ and 3’ ends using PCR and then cloned into pCDNA3.1 vector at the BamHI restriction site for expression in mammalian cell lines.…”

“…RIPA buffer (PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibitor cocktail set III (EMD Biosciences, San Diega, CA, USA) was used to make whole cell lysates from 2D and 3D cell cultures, as we previously described [ 18 ]. Total membranes of 2D cell cultures, along with 3D cell cultures were purified as described [ 19 ].…”

“…Total membranes of 2D cell cultures, along with 3D cell cultures were purified as described [ 19 ]. Conditioned serum-free media was prepared as described [ 18 ], except cells were cultured in serum-free medium for 72–80 h, instead of 48 h. Further both 2D and 3D cell cultures were used to generate conditioned medium. In all cases, protein levels were determined by a modified Lowry assay.…”

“…Previously, our lab demonstrated that knockdown of MGAT2/Mgat2 gene suppresses tumorgenicity in 2D cultured human [ 18 ] and rat NB cell lines [ 19 , 20 ]. The knockdown of GnT-II prevents the conversion of hybrid to complex type N-glycans, thus yielding a glycosylation mutant cell line.…”

“…The knockdown of GnT-II prevents the conversion of hybrid to complex type N-glycans, thus yielding a glycosylation mutant cell line. Further, substitution of complex with hybrid types of N-glycans diminished aberrant NB cell properties [ 18 , 20 ]. Given the implications of alterations in N-glycan processing on suppression/promotion of cancerous cell properties, herein, we further manipulate the N-glycan pathway via knockdown of Mgat1 , thereby creating a mutant cell line with even less processed N-glycans.…”