Elucidation of GlcNAc‐binding properties of type III intermediate filament proteins, using GlcNAc‐bearing polymers

Abstract

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin belong to type III intermediate filament family and are expressed in mesenchymal cells, skeletal muscle cells, astrocytes and peripheral neurons, respectively. Vimentin and desmin possess N-acetyl-d-glucosamine (GlcNAc)-binding properties on cell surfaces. The rod II domain of these proteins is a GlcNAc-binding site, which also exists in GFAP and peripherin. However, the GlcNAc-binding activities and behaviors of these proteins remain unclear. Here, we characterized the interaction and binding behaviors of these proteins, using various well-defined GlcNAc-bearing polymers synthesized by radical polymerization with a reversible addition-fragmentation chain transfer reagent. The small GlcNAc-bearing polymers strongly interacted with HeLa cells through vimentin expressed on the cell surface and interacted with vimentin-, desmin-, GFAP- and peripherin-transfected vimentin-deficient HeLa cells. These proteins present high affinity to GlcNAc-bearing polymers, as shown by surface plasmon resonance. These results show that type III intermediate filament proteins possess GlcNAc-binding activities on cell surfaces. These findings provide important insights into novel cellular functions and physiological significance of type III intermediate filaments.

Multimeric conformation of type III intermediate filaments but not the filamentous conformation exhibits high affinity to lipid bilayers

“…Cell surface‐expressed type III intermediate filaments can interact with a GlcNAc‐bearing polymer (AC‐GlcNAc; Ise et al, 2017). AC‐GlcNAc is a multivalent GlcNAc probe and is composed of acrylate as the main chain and GlcNAc as side chains.…”

“…Pierce High Sensitivity NeutrAvidin‐horse radish peroxidase (NeutrAvidin‐HRP) was purchased from Thermo Fisher Scientific Inc. Finally, AC‐GlcNAc was prepared as previously described (Ise et al, 2017).…”

“…Vimentin, desmin, GFAP, peripherin, the Cys‐2 domain (C328–I411) of vimentin and the rod II domain of these proteins were prepared as HQ‐tagged recombinant proteins using a KRX E. coli protein expression system (Promega Corporation) as described previously (Ise et al, 2017). The cDNA protein clones were purchased from the Kazusa DNA Research Institute.…”

“…These proteins are generally implicated in the preservation of cell integrity and architecture of various tissues (Herrmann, Bär, Kreplak, Strelkov, & Aebi, 2007; Ivaska, Pallari, Nevo, & Eriksson, 2007). However, we have previously reported that type III intermediate filament proteins are expressed on the cell surface of various cell types such as cancer cells, skeletal muscle cells, fibroblasts and mesenchymal stem cells; furthermore, they possess N‐acetyl‐D‐glucosamine (GlcNAc)‐binding activities through the interaction with multivalent GlcNAc‐bearing polymers (Ise, Yamasaki, Sueyoshi, & Miura, 2017; Kim, Ise, Goto, & Akaike, 2012; Ise et al, 2019). Moreover, vimentin is expressed on the cell surface of various cell types such as malignant tumor cells (Satelli & Li, 2011; Steinmetz, Cho, Ablack, Lewis, & Manchester, 2011), and cell surface‐expressed vimentin is a candidate biomarker for tumor cells including circulating tumor cells (Lindsay et al, 2016; Satelli, Brownlee, Mitra, Meng, & Li, 2015).…”

Development of a Gene Delivery System of Oligonucleotides for Fibroses by Targeting Cell-Surface Vimentin-Expressing Cells with N-Acetylglucosamine-Bearing Polymer-Conjugated Polyethyleneimine

“…To this end, the cell surface cytoskeletal proteins vimentin and desmin were used as markers to target myofibroblasts and activated hepatic stellate cells. Namely, the type III intermediate filament proteins vimentin, desmin, glial fibrillary acidic protein, and peripherin are expressed on the surface of various cells and possess N-acetylglucosamine (GlcNAc)-binding activity via GlcNAc-bearing polymers [ 14 , 15 , 16 , 17 , 18 , 19 ]. Moreover, vimentin and desmin are highly expressed on myofibroblasts and activated stellate cells in various lesion sites [ 20 , 21 ], and it is possible to target activated stellate cells in liver fibrosis with bio-imaging systems that utilize GlcNAc-conjugated polyethyleneimine (GlcNAc-PEI) and small interfering (siRNA) delivery systems [ 22 , 23 ].…”