Glycan Profile Of Hemagglutinin Of High Pathogenic Influenza H5N1Virus

Background:Hemagglutinin(HA) is one of the most important viral surface glycoprotein, which plays an important role in receptor recognition, binding and virus infection. It is a glycan-binding protein and also glycoprotein, the oligosaccharides on which are closely related with the host specificity, host range and virulence of the virus. This study characterized the structures of the oligosaccharides on HA which bind to receptors of hosts by recognizing SAa2-3Gal or SAa2-6Gal.Method:The epoxy magnetic particles were modified to hydroxyl magnetic particles. And then two oligosaccharides (SAa2-3Gal or SAa2-6Gal) were immobilized to the hydroxyl magnetic particles, which could specifically binding with HA of influenza virus. Now that this glycan-magnetic particle conjugates were used for purifying HA from the virus solution having been pretreated with ethyl. Subsequently, the isolated proteins were identified with SDS-PAGE and the glycan profile of the HA were characterized using lectin microarray then the HA was treated by PNGase F to release the oligosaccharides, use sepharose CL-4B purified the oligosaccharides, and the last use MALDI-TOF/TOF-MS to analysis the glycan structure。Result:The molecular weight of the isolated hemagglutinin was consistent with the former reports. The results of lectin microarray showed that PHA-E, RCA120, DSA, ACA and SNA were positive signals with the HA purified by SAa2-3Gal glycan-magnetic particle conjugates whereas PHA-E, LTL, RCA120, ConA, DSA, ACA, PWM, GNA and SNA were positive signals with the HA purified by SAa2-6Gal glycan-magnetic particle conjugates. The results indicated that the hemagglutinin of H5N1(DK/GD/17/08) avian influenza virus contained two groups of glycans. One group, consisting of Bisecting GlcNAc and biantennary N-glycans, Fucose, p-gal, GlcNAc, Galp1-3GalNAca-Ser/Thr(Tn), Gal(31-3GalNAc, biantennary N-glycans, tetraantennary complex-type and Sia2-6Galβ1-4Glc(NAc),, were involved in recognition and binding with the receptor with an a2-3linkage. While the other group, consisting of bisecting GlcNAc and biantennary N-glycans, Fucosea-1,3GlcNAc(core fucose), Sia-Lex and Lex, P-gal, branched and terminal mannose, terminal GlcNAc, GlcNAc, Galβ1-3GalNAca-Ser/Thr(Tn), Terminala-1,3mannose and Sia2-6Galβ1-4Glc(NAc), were participate in the recognition and binding with receptor with an a2-6linkage.In addition, both HAs purified by glycan-magnetic particle conjugates were treated by PNGase F, then the glycan profile of the HA were analysis with MALDI-TOF/TOF-MS. And the obtained spectrums were analyzed with Bruker flexAnalysis3.3. The results shown that eleven identical major glycans were presented in both HAs, and glycan peaks intensities of SAa2-3Gal purified HA were much higher than that of SAa2-6Gal purified HA. Additional, Five specific low-abundance glycan were only presented in the HA purified by SAa2-3Gal while two glycans purified by SAa2-6Gal HA.Conclusions:In conclusion, using lectin microarray together with MS to analysis the glycan structure of HA, our results shows that the high pathogenic H5N1subtype avian influenza virus HA contains two glycan groups, which play a different role in recognization and binding with different receptors of the influenza virus. Our study provides a new theoretical basis for the prevention and treatment of influenza.