Analysis of sialylated N-linked glycans on fish cell lines permissive to nervous necrosis virus for predicting cellular receptors of the virus

Abstract

Nervous necrosis virus (NNV), one of the simplest spherical RNA-viruses, is the causative agent of severe diseases affecting many fish species. It appears that NNV can bind to sialic acid moieties of N-glycans on host cell surface. To predict cellular receptors for NNV, we analyzed sialylated N-glycans on six fish cell lines with differential permissiveness to NNV. SSN-1, SeGF and BF-2(H) cells were permissive to both red-spotted grouper NNV (RGNNV) and striped jack NNV (SJNNV), while BF-2(A) and RoBE-4 cells were permissive only to RGNNV. FHM cells were not permissive to RGNNV or SJNNV. Matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) analysis revealed that 12–35% of total N-glycans were complex type sialylated N-glycans and that almost all sialic acids at the non-reducing end were N-acetylneuraminic acid (Neu5Ac). Major sialylated N-glycans were mono-, bi- or tri-antennary glycans. A common motif containing Neu5Ac-N-acetyllactosamine (LacNAc) was always present on at least one of the antennae. Both RGNNV and SJNNV could bind to Neu5Ac-LacNAc regardless whether Neu5Ac was α2,3- or α2,6-linked, although RGNNV preferred the former whereas SJNNV preferred the latter. Therefore, N-glycan antennae with terminal Neu5Ac-LacNAc moieties are likely to function as cellular receptors for NNVs.