Multiple Omicron subvariants of SARS-CoV-2 have emerged as dominant global concerns. Although the spike (S) proteins of Omicron subvariants harbor more than 30 mutations compared to the original wild type (WT), N-glycosylation sites within these S proteins are highly conserved. Site-specific glycosylation of S proteins from Omicron subvariants, particularly in the receptor binding domain (RBD) involved in binding to neutralizing antibodies, remain largely unexplored. Here, we purified recombinant S proteins and their corresponding RBDs from two Omicron subvariants (BA.5 and XBB.1) as well as the WT, and characterized site specific glycosylation of these proteins. Our glycoproteomic analysis revealed smaller glycans with mono-fucosylation at the site N331 in the RBD region of trimeric S proteins of Omicron subvariants relative to WT, which might reduce steric constraint for antibody binding to this region. Besides, higher levels of multi-fucosylation and sialylation at the site N331 were detected in monomeric RBDs compared to corresponding trimeric S proteins, suggesting more susceptible of RBDs to modification mediated by the glycan processing enzymes. We believe that the glycosylation profiles of Omicron subvariants will facilitate our understanding of the increased infectivity and transmissibility of Omicron subvariants, and thus assist the diagnosis, prevention, and treatment of COVID-19 infection.

Amino acid transporters are essential for maintaining intracellular and extracellular amino acid homeostasis. The sodium-dependent neutral amino acid transporter B⁰AT1 (SLC6A19) requires the accessory protein ACE2 or Collectrin to form a functional heteromeric complex for transmembrane transport. B⁰AT1 is primarily expressed at the brush border of epithelial cells in the small intestine and kidney, playing a vital role in the absorption and reabsorption of neutral amino acids, including Leucine (Leu), Methionine (Met), Glutamine (Gln), Tryptophan (Trp) and Phenylalanine (Phe). Mutations or functional impairments in B⁰AT1 lead to Hartnup disorder, characterized by aminoaciduria and neurological symptoms. Although its physiological roles are increasingly understood, the molecular mechanisms underlying selective substrate recognition, particularly for aromatic amino acids, remain poorly understood. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the human ACE2-B⁰AT1 complex bound to Phe and Trp at the overall resolution of 2.87 Å and 3.24 Å, respectively. Structural comparisons reveal conserved substrate backbone anchoring and distinct side-chain recognition mechanisms, identifying key residues that modulate substrate specificity. Our findings elucidate the substrate recognition landscape of B⁰AT1 and provide a mechanistic framework for understanding its function and disease-associated mutations.