| Background:Avian influenza virus (AIV) can cause acute infectious diseases in poultry,as well as severe respiratory infections and even death after infecting humans. It was regarded as the beginning of infecting that the hemagglutinin(HA) of influenza virus bind to sialic acid (SA) alpha 2-3 galactosyl (Gal) or SA alpha 2-6 Gal receptor which is present on the host cell surface. HA plays a pivotal role in identification and bond with specific host receptors and in fusion of virus membrane with host cell membrane during viral infection. It is also the important target of neutralizing antibodies and vaccine development. HA is not only a glycoprotein but also a glycan binding protein. The glycosylation of HA can affect not only its binding activity,but also the virulence as well as evolution of virus.The glycans of HA has a significant role in life cycle of AIV, and it will contribute to a further understanding of the important biological functions of HA in influenza virus infection if known the glycan structure of HA as well as the effects of these glycan structural changes. However, little is known about the precise glycan structures of HA at the present time. Therefore,it is necessary to analyzes precise glycan structures of HA, hoping to provide a new targeted goals for the prevention and treatment of influenza and vaccine design on the level of glycans.Methods:HAs,from 3 strains of duck derived avian influenza virus, were separated and purified by Specific glycan-magnetic particles (SAa2-3Gal and SAa2-6Gal glycan-magnetic particle components) separately,and two sets of HA (defined as HA2,3and HA2,6) was obtained from each strains. Their glycan profiles were analyzed by lectin microarray subsequently. Glycans were then cut off from each HA2,3 and HA2,6 by PNGaseF aiding with filter membrane. The matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF/TOF-MS) was employed to determine the actual structure of these glycans. Two technologies complement each other, and analysis N-Glycan profiles of HA from duck derived avian influenza virus jointly.Results and Discussion:1.According to SDS-PAGE, HA2,3 and HA2,6 can be both separated and purified from 3 different strains of duck derived avian influenza virus (A/Duck/Guangdong/17/2008(H5N1), A/Mallard/Jiangxi/16/2005(H5N2), A/Duck/Guangdong/S-7-134/2004(H9N2))with specific glycan-magnetic particles (SAa2-3Gal or SAa2-6Gal glycan-magnetic particle components).2. Lectin microarray was used to analysis glycan profiles of HAs, and found that:(1) There exist 5 lectins, including DSA, RCA120, ACA, PHA-E and SNA,showed strongger positive signals both in HA2,3 and HA2,6 derived from A/Duck/Guangdong/17/2008 (H5N1),and the corresponding high expression of glycan structures are β-D-GlcNAc, (GlcNAcβ1-4)n, Galβ1-4GlcNAc;β-Gal,Galβ-1,4GlcNAc(type Ⅱ), Galβ1-3GlcNAc(typeI); Galβ1-3GalNAc α-Ser/Thr(T-antigen); Bisecting GlcNAc and biantennary N-glycans; Sia2-6Galβ1-4Glc(NAc). These glycans may associated with the basic structure and function of HA from this strain. In addition, BPL, which could identify the glycans like Galβ1-3GalNAc, showed obvious positive signals in H5N1HA2,3; while GNA which could identify the glycans like Terminalα-1,3 mannose, and ConA which could identify the glycans like High-Mannose, Manα1-6(Manal-3)Man, terminal GlcNAc showed obvious positive signals in H5N1HA2,6· These specific glycans may associated with the receptor activity of HA. (2)There exist 6 lectins, including RCA 120, DSA, PHA-E, ACA, SNA and ConA, showed strongger positive signals both in HA2,3 and HA2,6 derived from A/Mallard/Jiangxi/16/2005 (H5N2), and the high expression of glycan structures are Galβ1-3GalNAc a-Ser/Thr(T-antigen);P-D-GlcNAc, (GlcNAcβ1-4)n, Galβ1-4GlcNAc;β-Gal, Galβ-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); biantennary N-glycans and Bisecting GlcNAc;Sia2-6Galβ1-4Glc(NAc); High-Mannose, Manal-6(Manal-3)Man, terminal GlcNAc. These glycans may associated with the basic structure and function of HA from this strain. In addition, GNA which could identify the glycans like Terminala-1,3 mannose, also showed obvious positive signals in H5N2HA2,3; while STL which could identify the glycans like trimers and tetramers of GlcNAc, core (GlcNAc) of N-glycan, oligosaccharide containing GlcNAc and MurNAc showed obvious positive signals in H5N2HA2,6.These specific glycans may associated with the receptor activity of HA. (3)The majority of lectins showed positive signals both in HA2,3 and HA2,6 derived from A/Duck/Guangdong/S-7-134/2004(H9N2), and 5 lectins (AAL, RCA120, GNA, SNA, DBA) showed strongger positive signals, others such as BPL,ACA,PWM showed positive signals, too. The results suggesting that several glycans including β-Gal, Galβ-1,4GlcNAc (typeⅡ), Galβ1-3GlcNAc (typeⅠ); Fucal-6 GlcNAc(core fucose), Fucα1-3(Galβ1-4)GlcNAc; Terminalα-1,3 mannose; Sia2-6Galβ1-4Glc(NAc), and αGalNAc, GalNAcα1-3((Fucal-2))Gal (blood group A antigen) exist on HA from this H9N2. H9N2HA expressed more positive signals in lectin microarray when compared with H5N1 and H5N2, and there shown little differences between H9N2HA2,6 and H9N2HA2,3 except their expression level, it may because of the lower pathogenicity of this H9N2 strain. (4)RCA120,SNA and ACA showed positive signals in HA2,3 and HA2,6 of all 3 strains, and the high expression of glycan structures are P-Gal, Galβ-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ), Sia2-6Galβ1-4Glc(NAc), and Galβ1-3GalNAca-Ser/Thr(Tn), the result may indicate that these common glycans may associated with the basic structure and function of HA. Besides, HA of H5N1 and H5N2 have some highly expressed glycans in common,while H9N2 have much bigger differences with those two strains. It probably because the former two virus belongs to homologous H5 subtype.3. Analysis the glycan structure of HA using MALDI-TOF/TOF-MS, we found:(1)①H5N1 HA2,3 showed 18 glycan peaks while H5N1HA2,6 showed 16, they shared 15 peaks. Moreover, 3 glycan peaks only exsit on H5N1HA2,3 and 1 peak only exsit on H5N1HA2,6.②H5N2HA2,3 showed 17 glycan peaks while H5N2HA2,6 showed 20, they shared 15 peaks.2 glycan peaks only exsit on H5N2HA2.3 and 5 glycan peaks only exsit on H5N2 HA2,6.(3)H9N2HA2,3 showed 20 glycan peaks, while H9N2HA2;6 showed 21, they shared 18 peaks.2 glycan peaks only exsit on H9N2HA2,3 and 3 glycan peaks only exsit on H9N2HA2.6. These glycans peaks concern several monosaccharides including Mannose, Galactose, Fucose, GlcNAc, GalNAc, etc. Those common glycans may associated with the basic structure and function of HA while those specific glycans may associated with the binding activity between HA and it’s receptor. (2)A11 of these 6 HAs from 3 strains virus, have the same 12 glycan peaks, involved the glycan structures like Mannose, Galactose, Gal-GlcNAc, GalNAc-GlcNAc, fucose-GlcNAc, etc. it may suggest that these glycan structures may be required for the basic structure and function of HA. Moreover, the same glycan peak also have different expression intensity. HA2,3 showed highly expression than HA2.6 approximately, it maybe because the samples were originated with poultry. (3) SNA showed positive signal in these 3 different duck derived AIVs according to lectin microarray, meanwhile Sia2-6Galpl-4Glc(NAc) was discovered in the Mass analysis. It may suggest that this glycan structure can urge the virus combine and reunion with each other, then result in the subdued pathogenicity. It maybe the reason that many AIVs lurked in the host for a long period while showed low pathogenicity.4.In summary, there exist 2 sets of glycan systems on the influenza HA, one of which (named after HA23) could be used to control the virus to infect poultry, and another (named after ) could be used to control the virus to infect human. |
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