The Synthesis of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase (GNE), α-dystroglycan, and β-galactoside α-2,3-sialyltransferase 6 (ST3Gal6) By Skeletal Muscle Cell As a Response To Infection with Trichinella Spiralis

Abstract

The Nurse cell of the parasitic nematode Trichinella spiralis is a unique structure established after genetic, morphological and functional modification of a small portion of invaded skeletal muscle fiber. Even if the newly developed cytoplasm of the Nurse cell is no longer contractile, this structure remains well integrated within the surrounding healthy tissue. Our previous reports suggested that this process is accompanied by an increased local biosynthesis of sialylated glycoproteins. In this work we examined the expressions of three proteins, functionally associated with the process of sialylation. The enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key initiator of the sialic acid biosynthetic pathway. The α-dystroglycan was the only identified sialylated glycoprotein in skeletal muscles by now, bearing sialyl-α-2,3-Gal-β-1,4-Gl-cNAc-β-1,2-Man-α-1-O-Ser/Thr glycan. The third protein of interest for this study was the enzyme β-galactoside α-2,3-sialyltransferase 6 (ST3Gal6), which transfers sialic acid preferably onto Gal-β-1,4-GlcNAc as an acceptor, and thus it was considered as a suitable candidate for the sialylation of the α-dystroglycan. The expressions of the three proteins were analyzed by real time-PCR and immunohistochemistry on modified methacarn fixed paraffin tissue sections of mouse skeletal muscle samples collected at days 0, 14 and 35 post infection. According to our findings, the up-regulation of GNE was a characteristic of the early and the late stage of the Nurse cell development. Additional features of this process were the elevated expressions of α-dystroglycan and the enzyme ST3Gal6. We provided strong evidence that an increased local synthesis of sialic acids is a trait of the Nurse cell of T. spiralis, and at least in part due to an overexpression of α-dystroglycan. In addition, circumstantially we suggest that the enzyme ST3Gal6 is engaged in the process of sialylation of the major oligosaccharide component of α-dystroglycan.

Keywords: GNE; Nurse cell; Sialic acid; Skeletal muscle; Trichinella spiralis; α-dystroglycan.

Fig. 1

Immunohistochemistry. Modified methacarn fixed sections from mouse skeletal muscles with Trichinella spiralis at days 14 and 35 post invasion (d.p.i.) were stained with rabbit polyclonal antibodies against glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), α-dystroglycan and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3Gal6). Paralel sections were subjected to H&E staining to facilitate the histological orientation. Strong expressions of GNE and α-dystroglycan, and moderate expression of ST3Gal6 were observed on days 14 and 35 after invasion, suggesting these proteins as permanent characteristics of the Nurse cell of T. spiralis. The brown colour indicates positive immunohistochemical reaction, hashtag indicates the occupied sarcoplasm, star – non-occupied skeletal muscle cell, arrow – enlarged nucleus, L– larva. H&E, HRP anti-rabbit IgG, DAB. Scale bar 20 μm.

Fig. 2

Agarose gel analysis of Trichinella spiralis ESV fragment PCR. Polymerase chain reaction was performed on modified methacarn fixed mouse skeletal muscle tissue sections, selected on days 0, 14 and 35 after T. spiralis invasion. Genomic DNA from T. spiralis infectious larvae was used as a positive control sample. Presence of 173 bp fragment of expansion segment V of the T. spiralis genome was detected only in the mouse samples collected on days 14 and 35 after invasion. The photograph is a representative of three randomly selected samples from each experimental group.

Fig. 3

Expressions of mouse glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (Gne), dystroglycan 1 (Dag1) and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (St3gal6) analysed by real time RT-PCR in modified methacarn fixed mouse skeletal muscle tissue sections, selected on days 0, 14 and 35 after T. spiralis invasion. The graphs show the relative quantification of the gene expressions calculated by the ΔΔCt method versus glyceraldehyde phosphate dehydrogenase (Gapdh) as a reference gene from five individual samples in triplicate. The bars show the standard error of mean. The products of amplification were loaded on 2.5% agarose gel versus Perfect 100-1000 bp DNA Ladder.