Biology subjects

Bouwman, K. M.

Publications and source records attributed to Bouwman, K. M..

3 recordsLinked to original sources

bioRxiv2021

3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRRS2) to facilitate viral-host membrane fusion. ACE2 and TMPRRS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq level, however, biologically relevant protein receptor organization in whole tissues is still poorly understood. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRRS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals were stained using antibodies against ACE2 and TMPRRS2, combined with fluorescent spike protein and SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize expression patterns. The data demonstrates that infection is restricted to sites with both ACE2 and TMPRRS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the terminal bronchioles and alveoli. Conversely, infection completely overlaps at these sites where ACE2 and TMPRSS2 co-localize. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/435771v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@150fc65org.highwire.dtl.DTLVardef@1ea6a56org.highwire.dtl.DTLVardef@eb3c43org.highwire.dtl.DTLVardef@1c1877e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

N-glycolylneuraminic acid binding of avian H7 influenza A viruses

Influenza A viruses initiate infection by binding to glycans with terminal sialic acids present on the cell surface. Hosts of influenza A viruses variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in the majority of mammals including horses, pigs, and mice, but is absent in humans, ferrets, and birds. Intriguingly, the only known naturally occurring influenza A viruses that exclusively bind NeuGc are the extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with its NeuGc ligand and observed a high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs. We found that mutation A135E is key for binding 2,3-linked NeuGc but does not abolish NeuAc binding. Interestingly, additional mutations S128T, I130V, or a combination of T189A and K193R, converted from NeuAc to NeuGc specificity as determined by glycan microarrays. However, specific binding to NeuGc-terminal glycans on our glycan array did not always correspond with full NeuGc specificity on chicken and equine erythrocytes and tracheal epithelium sections. Phylogenetic analysis of avian and equine H7 HAs that investigated the amino acids at positions 128, 130, 135, 189, and 193 reveals a clear distinction between equine and avian residues. The highest variability in amino acids (four different residues) is observed at key position 135, of which only the equine glutamic acid leads to binding of NeuGc. The results demonstrate that avian H7 viruses, although genetically distinct from equine H7 viruses, can bind NeuGc after the introduction of two to three mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. Author summaryInfluenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A virus, these keys (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority binds NeuGc. NeuGc is present in species like horses, pigs, and mice, but not in humans, ferrets, and birds. Therefore, NeuGc binding could be a determinant of an Influenza A virus species barrier. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

molecular biology↗

bioRxiv2020

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.

molecular biology↗