Influenza Hemagglutinins H2, H5, H6, and H11 are not Targets of Pulmonary Surfactant Protein D: N-glycan subtypes in host-pathogen interactions

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low-pathogenicity avian influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortment and emergence in human populations. Since the presence of head region high-mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here, we investigate the activities of two recombinant human SP-D forms against representative LPAIV strains, including H2N1, H5N1, H6N1, H11N9, an avian H3N8, and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPAIV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between the protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high-mannose glycans in H3 HA but by complex glycans in all LPAIV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high-mannose glycan on the head region, our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three-dimensional structural analysis.IMPORTANCE Low-pathogenicity avian influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations, as was seen in the 1957 pandemic, in which an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first-line innate immunity defense, removes influenza type A virus (IAV) through interaction with hemagglutinin (HA) head region high-mannose glycan(s). While it is known that both H1 and H3 HAs have one or more key high-mannose glycosites in the head region, little is known about similar glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high-mannose glycans and do not interact with SP-D, and that sequence analysis can predict glycan subtype, thus predicting the presence or absence of this virulence marker.