Genes Cells 22, 900-917 (2017)

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

 

Hirohiko Ise*,1, Sadanori Yamasaki2, Kazuaki Sueyoshi2, Yoshiko Miura3

1Institute for Materials Chemistry and Engineering, 2Department of Chemistry and Biochemistry, Graduate School of Engineering, 3Department of Chemical Engineering, Kyushu University, 744 Motooka Nishi-ku Fukuoka 819-0395, Japan

* Corresponding author: Hirohiko Ise, Kyushu University, 744 Motooka Nishi-ku Fukuoka 819-0395, Japan; E-mail: ise@ms.ifoc.kyushu-u.ac.jp

 

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-fragment 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 demonstrate 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.

 

Supplement:

Vimentin, desmin, GFAP, and peripherin are expressed in mesenchymal tissues, skeletal muscle tissues, astrocytes, and peripheral neurons, respectively. These proteins play important roles in the stabilization of cell integrity such as architecture and structure in each tissue [1, 2]. Recent studies indicated that these proteins are highly expressed in lesion sites of various chronic diseases such as fibrosis, cancer, and autoimmune diseases [3–7], and most studies focused on pathologic analyses and elucidation of a pathologic mechanism related to high expression of these proteins in chronic diseases [8–10]. Type III intermediate filament proteins may represent a new target molecule for multiple chronic diseases [1, 11–23]. We previously reported that the cytoskeletal protein, vimentin, is expressed on the cell surface and possesses N-acetyl-D-glucosamine (GlcNAc)-binding activity in various types of mesenchymal, skeletal muscle, and cancer cells [24, 25], as demonstrated by exploiting the interaction between multivalent GlcNAc-bearing polymers and vimentin. GlcNAc-binding sites exist in the rod II domain of vimentin and the rod II domain of tetrameric, but not filamentous vimentin is exposed on the cell surface [15, 24, 25]. Furthermore, the GlcNAc-binding property on the cell surface was discovered for the first time using multivalent GlcNAc-bearing polymers, but not GlcNAc monosaccharides [24]. Therefore, multivalent GlcNAc may be critical for the interaction of vimentin with GlcNAc. Desmin, GFAP, and peripherin also possess the rod II domain and these domains are very similar. Therefore, it is assumed that desmin, GFAP, and peripherin, like vimentin, possess GlcNAc-binding properties on the cell surface. Vimentin overexpression is involved in the progression of fibrosis and malignant tumors [3, 11, 26]. In addition, during the progression of these diseases, many mesenchymal cells, including myofibroblasts and cancer-associated fibroblasts (CAFs), accumulate at sites of fibrosis and carcinogenesis, where they may promote disease progression [27]. Therefore, the removal of these mesenchymal cells may be an effective strategy for the treatment of these diseases. Because vimentin is also overexpressed in these mesenchymal cells, fibroblasts and cancer cells overexpressing vimentin may be targeted in novel therapies [11]. Accordingly, multivalent GlcNAc-conjugated materials such as GlcNAc-conjugated polyethylenimine and GlcNAc-bearing polymer-coated liposomes are very useful for targeting vimentin-overexpressing cells in the treatment of cardiovascular diseases, liver fibrosis, and cancer [28–31]. However, it is unclear how many valences of GlcNAc-bearing polymers are needed to bind to vimentin although the valences are important to interact with vimentin. To design a molecular targeting system, the size of molecular targeting compounds should be smaller because high weight polymers increase the risk for vascular embolization. Therefore, the optimal GlcNAc valences to interact with vimentin should be determined for the design of effective vimentin-targeting GlcNAc molecules and for the elucidation of GlcNAc-binding behaviors of vimentin. Moreover, whether desmin, GFAP, and peripherin have GlcNAc-binding activities on the cell surface remains unknown. Targeting cells overexpressing these proteins with GlcNAc-bearing polymers can be applied for the treatment of Alzheimer’s disease and neurodegenerative diseases [4–6, 28, 31].

We aimed to design effective type III intermediate filament protein-targeting molecules by determining the optimal GlcNAc valences using various sized GlcNAc-bearing polymers produced by polymerizing GlcNAc with reversible addition-fragmentation chain transfer (RAFT) reagents. We then examined the interactions of various GlcNAc-bearing polymers with HeLa cells through binding to vimentin. Moreover, we investigated whether desmin, GFAP, and peripherin could interact with the GlcNAc-bearing polymers on the cell surface. Finally, the affinities and kinetics between these proteins and GlcNAc-bearing polymers were measured using surface plasmon resonance (SPR) analysis.

In this study, we demonstrated that GlcNAc-bearing polymers (AC-GlcNAc) with low valency (approximately 13 mers) could interact with not only vimentin, but also desmin, GFAP, and peripherin (Fig. 1) (Table 1). The small GlcNAc-bearing polymers can be easily conjugated to various gene products and drug delivery carriers. Therefore, we expect that the conjugation of small GlcNAc-bearing polymers can allow the targeting of diseases by various therapeutic agents. Additionally, these findings may facilitate the design of new GlcNAc-bearing polymers for the targeting of various chronic diseases and the unraveling of the mechanisms through which type III intermediate filament proteins recognize GlcNAc. In future studies, we will determine whether GlcNAc-bearing polymers such as AC-GlcNAc could be targeted to the surface of cells expressing type III intermediate filaments at disease sites in various mouse models of chronic disease.

 

Table 1 Binding kinetics of interaction of immobilized AC-GlcNAcs with type III intermediate filaments

ka (1/Ms) kd (1/s) KD (M)
Vimentin 1.42 × 104 4.07 × 10-4 2.87 × 10-8
Desmin 1.72 × 104 1.01 × 10-3 5.83 × 10-8
GFAP 5.22 × 104 2.97 × 10-3 5.69 × 10-8
Peripherin 1.34 × 104 8.07 × 10-4 6.18 × 10-8

 

 

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