| Background:The past influenza pandemics in history have been shown to be associated with avian influenza virus(AIV),and the infection of pregnant women with highly pathogenic AIVs will be a serious threat to both the maternal lives and the safety of their fetuses.Statistical data suggest that during past influenza pandemics,pregnant women had higher rates of morbidity and mortality than non-pregnant women or general population at the same age,especially when they are in the second and third trimesters,but the underlying molecular mechanisms remain unclear.It has been well estabished that influenza viruses usually initiate the host invasion by specifically recognizing and binding of their hemagglutinin(HA)to the terminal sialic acids(Sia)of glycoproteins on the surface of host cells.Saliva,as the first immune line of defense,is known that has strong anti-influenza activity It is reported that saliva contains a large number of soluble salivary glycoproteins the Sia-containing glyans on which can resist the infection of viruses by binding to the HA of influenza virus efficiently.A lot of previous studies have domenstrated that the glycosylation of saliva glycoprotein can be changed in different physiology and pathology conditions,and was associated with the gender,age and chronic disease,etc.In this study,we systematically investigated the variation of the Sia-containing glycan expression level on salivary glycoproteins of women during pregnancy and postpartum at different stagesand its impact on the saliva binding ability to AIV.The results will help to further understand the high susceptibility of pregnant women to AIV,and provide the experimental basis for the protection of this population from AIV infection.Method:In this study,a total of 1200 saliva samples were collected from the healthy non-pregnant women and the women at different stages of pregnancy and postpartum.The saliva samples were divided into seven groups : the group of healthy non-pregnant women(HN),the groups of pregnant women in the first trimester(FT),second trimester(ST),and third trimester(TT),and the groups of postpartum women in the first-period(FP,within six months after delivery),second-period(SP,during 7th to 12 th month after delivery)and third-period(TP,during 13 th to 24 th month after delivery).Firstly,the expression levels of Siaα2-6Gal glycan recognized by SNA and Siaα2-3Gal glycochain recognized by MAL-II on the salivary proteins of each group were compared and analyzed by lectin microarray technology,and the amount of sialic acid content in each saliva group was also quantitatively detected by the periodate-resorcinol assay.Then,the Sia expression levels in individual samples from each group further validated using saliva microarray technology.After that,the proteins profiles and the variation of Siaα2-3/6Gal on salivary glycoproteins of different groups were further analyzed by SDS-PAGE and lectin blotting assay.Finally,the binding affinity of salivary samples were detected against three avian influenza viruses(H5NI-CK,H5N1-DK,H9N2)and the H1N1 human influenza vaccine.In addition,given that the MUC5 B and IgA are the primary glycoproteins,the protein and glycosylation level of Siaα2-3/6Gal on which were investigated in different groups using Western blotting and lectin-based ELISA methods.Result: The normalized fluorescent intensities(NFIs)of salivary glycoproteins binding to SNA on the lectin microarray showed that the expression level of Sia2-6Gal was not altered obviously in the groups of FT,ST,TT,FP,SP and TP compare to HN group.While the NFIs of MAL-II showed that the Siaα2-3Gal expression level was significantly changed a lot in different groups,which was significantly down-regulated in the saliva of women during pregancy,especially for the second and third trimester(fold change = 0.53 and 0.37,p < 0.001).The results of the saliva microarray analysis for the individual samples were consistent with those of lectin microarray.Quantitative determination of Sia content showed that the Sia in α2-3-linkage was decreased from 2.08% in the saliva groups of HN to 0.78% in the saliva of pregnant group of TT,while the sialic acids withα2-6-or other linkages were not varied too much.Lectin blotting results further showed that there were three bands of proteins highly glycosylated with Sia2-3Gal,and the level of Sia2-3Gal decreased significantly in the saliva group of pregnant women and recovered gradually after delivery according to the binding of relative fluorescence intensities(RFIs),which was in accordance with the results of lectin microarray.The anti-Virus blotting analysis showed that the binding ability of salivary glycoproteins to H5N1-CK,H5N1-DK,H9N2 AIV and H1N1 vaccine strains were significantly reduced in the second and third trimesters during pregnancy,but the saliva binding affinity to influenza A H1N1 vaccine were not changed too much in three avian influenza viruses.From the result,it can be seen that the anti-virus binding ability was significantly correlated with the alternation of Siaα2-3/6Gal expression level in different saliva goups.Western blotting and ELISA analysis showed that the viration of Siaα2-3/6Gals on IgA and MUC5 B could mainly occur on the glycosylation level,as the protein expression level of these two proteins were not changed too much during pregancy and postpartum.Conclusion: The expression level of Siaα2-3Gal on soluble glycoproteins of saliva was decreased obviously in the second and third trimester of women during pregnancy,which can significantly affect their slaiva binding ability to AIV.After delivary,the salivary expression level of Siaα2-3Gal and the glycoprotein binding ability to AIV recovered gradually.The results of this study indicate that the decreased expression level of Siaα2-3Gal on salivary glycoproteins of women could be one of the key factors that cause them to be more susceptible to AIV during pregnnacy.These results may provide new ideas for the prevention and control of AIV infection in pregnant women. |
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