The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants. IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-Oacetylated sialic acid reported previously was from the C7-to C9hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-Oacetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutininesterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host−microbe interactions and potential application of the more stable synthetic N-acetyl mimics. Articles pubs.acs.org/acschemicalbiology

Viral Fusion Protein Structures and N-glycosylation Sites (NGS). Viral fusion proteins are typically trimers of heterodimers with a globular head domain and a stalk/transmembrane domain. A GlucNAc 1 or GlucNac 2 are modeled at each NGS (Blue). (a) Lassa virus (LASV) glycoprotein (GP) complex contains 11 (NGS) with 7 in GP1 and 4 in GP2 (PDB: 5VK2) [27]. (b) Human immunodeficiency virus (HIV-1) Envelope (Env) GP contains around 29 NGS with 25 in gp120 the outer Env domain and 4 in gp41 the transmembrane domain (PDB: 5FYK) [49 ]. (c) Ebola virus (EBOV) GP has 17 NGS with 15 on GP1 and 2 on GP2 (PDB: 6G9B) [101]. (d) Coronavirus (CoV) spike GP has 26 NGS with 15 in subunit 1 and 11 in subunit 2 (PDB: 6BFU) [89]. (e) Influenza virus hemagglutinin is a homotrimer with 11 NGS (PDB: 4FNK) [92].

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, YCS, in the complementarity-determining region 3 of the heavy chain variable region compared with a YCA sequence in similar isotype-matched IgG from healthy controls. We previously found that the S residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the YC(A/V) amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent β-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

Biological activity of human neutrophil peptide (HNP)‐1 in hemostasis under physiological conditions is not fully understood. HNP‐1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti‐adhesion activity appears to depend on the terminal free thiols of HNP‐1, which may inhibit VWF‐VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP‐1 for arterial thrombosis. Summary BackgroundHuman neutrophil peptides (HNPs), also known as α‐defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. ObjectiveThis study aims to determine the role of HNP‐1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. MethodsAnticoagulated whole blood from wild‐type or Adamts13−/− mice was incubated with a fluorescein‐conjugated anti‐human CD41 in the presence of increasing concentrations of a synthetic HNP‐1 and perfused over a collagen surface or a tumor necrosis factor (TNF)‐α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet‐decorated ULVWF strings were determined using the BioFlux microfluidic system. ResultsHNP‐1 inhibited the rate and final coverage of fluorescein‐labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2) in a concentration‐dependent manner and the anti‐adhesive activity of HNP‐1 depended on its terminal free cysteine thiols. HNP‐1 (20 μM) also dramatically inhibited the formation of platelets‐decorated ULVWF strings on TNF‐α activated murine endothelial surface under arterial flow. ConclusionsOur results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP‐1; this activity depends on its terminal free thiols, likely affecting VWF‐VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.