N-linked Glycosylation Sites Research Articles

Rhesus Macaque Killer Cell Ig-like Receptor Domain 0 Glycans Impact Surface Expression and Ligand Specificity.

Defining the MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as a model for infectious diseases and comparative immunogenetics. Several rhesus macaque KIRs belong to a phylogenetically distinct group with a three-amino acid deletion in domain 0 (D0). This deletion results in polymorphic differences in potential N-linked glycosylation (PNG) sites adjacent to a predicted KIR-MHC class I contact site. Whereas most KIRs have two tandem PNG sites in D0 (N36FTN39FT), the KIRs containing the deletion only have a single site in this region (N36FT). To discern the contribution of glycosylation to KIR expression and ligand recognition, we constructed PNG mutants for six lineage II KIR genes that eliminate or create sites for N-glycan addition at these locations. The impact of these mutations on total and surface expression was determined by immunoblotting and flow cytometry. Ligand engagement was assessed by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. We found that N36FT is glycosylated in KIR with a single site, and at least one site is glycosylated in KIRs with two tandem sites. In general, for rhesus KIRs with a single D0 glycosylation site, that site contributes to surface expression. For KIRs with two tandem sites, the first site can contribute to ligand specificity. This study establishes that D0 glycosylation of rhesus macaque KIRs modulates surface expression and contributes to ligand specificity.

Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion.

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1-97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H6-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H6-trC1INH(MGS)-MSA), and H6-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H6-trC1INH(MGS)-MSA was significantly increased versus that of H6-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H6-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H6-trC1INH(MGS)-MSA by 33% relative to that of H6-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H6-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018–2021

ABSTRACT We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

CoNglyPred: Accurate Prediction of N-Linked Glycosylation Sites Using ESM-2 and Structural Features With Graph Network and Co-Attention.

N-Linked glycosylation is crucial for various biological processes such as protein folding, immune response, and cellular transport. Traditional experimental methods for determining N-linked glycosylation sites entail substantial time and labor investment, which has led to the development of computational approaches as a more efficient alternative. However, due to the limited availability of 3D structural data, existing prediction methods often struggle to fully utilize structural information and fall short in integrating sequence and structural information effectively. Motivated by the progress of protein pretrained language models (pLMs) and the breakthrough in protein structure prediction, we introduced a high-accuracy model called CoNglyPred. Having compared various pLMs, we opt for the large-scale pLM ESM-2 to extract sequence embeddings, thus mitigating certain limitations associated with manual feature extraction. Meanwhile, our approach employs a graph transformer network to process the 3D protein structures predicted by AlphaFold2. The final graph output and ESM-2 embedding are intricately integrated through a co-attention mechanism. Among a series of comprehensive experiments on the independent test dataset, CoNglyPred outperforms state-of-the-art models and demonstrates exceptional performance in case study. In addition, we are the first to report the uncertainty of N-linked glycosylation predictors using expected calibration error and expected uncertainty calibration error.

Genetic and antigenic characterization of influenza A(H3N2) virus after 13 consecutive years of influenza surveillance in Senegal, 2010-2022.

Despite decades of influenza surveillance in many African countries, little is known about the evolutionary dynamics of seasonal influenza viruses. This study aimed to characterize the epidemiological, genetic and antigenic profiles of A/H3N2 viruses in Senegal from 2010 to 2022. A/H3N2 infection was confirmed using reverse transcription-polymerase chain reaction. Subsequently, a representative of A/H3N2 isolates was selected for genome sequencing. Predicted vaccine efficacy was measured using the Pepitope model. During the study period, 22638 samples were tested and influenza was detected in 31.8%, among which type A was confirmed in 78.1%. Of the Influenza A cases, the H3N2 subtype was detected in 29.8%, peaking at expected times during the rainy season. Genome sequencing of 123A/H3N2 isolates yielded 24 complete and 99 partial genomic sequences. Phylogenetic analysis revealed the circulation of multiple clades of A/H3N2 in Senegal, including 2a.3, 3C.2 and 3C.3a. A/H3N2 isolates were mainly susceptible to the influenza antiviral drugs oseltamivir and zanamivir, but the primary adamantine-resistance marker, S31N was encountered in all isolates. At least nine potential N-linked glycosylation sites were predicted among A/H3N2 strains, six of which (at positions 24, 38, 79, 181, 262 and 301) remains conserved among all isolates. Antigenic distances between circulating strains and vaccine viruses indicated varying vaccine efficacies, from suboptimal to moderate protection. The findings emphasize the need to enhance local genomic and antigenic surveillance and further research on influenza epidemiology and genetic evolution in sub-Saharan Africa.

Immunogenicity Analysis of Chikungunya Virus DNA Vaccine Based on Mutated Putative N-Linked Glycosylation Sites of the Envelope Protein.

Chikungunya fever is a mosquito-borne infectious disease caused by the chikungunya virus (CHIKV). Recently, CHIKV has spread rapidly worldwide, raising global concerns. However, there is only one approved vaccine is available to prevent CHIKV infection; therefore, different platform vaccines development is a public health priority. The CHIKV genome encodes four non-structural polyproteins (nsP1-4) and one structural polyprotein (capsid, envelope 3, envelope 2, 6 K, and envelope 1). Previous studies have shown that N-linked glycans in viral proteins play important roles in regulating immune responses. Accordingly, in this study, we designed four CHIKV DNA vaccine candidates with mutated N-glycosylation sites in the full-length E and E I/II proteins. Our results indicated that immunization of mice with the vaccine elevated the cytokines levels, including IFN-γ, associated with T cell immune response. Furthermore, the truncated E protein with a deleted E III domain (E I/II) exhibited better immunogenicity than the full-length E protein, and N-linked glycosylation of E I/II protein induced a higher cell-mediated immune response. Overall, our study demonstrates that N-linked glycosylation of the E I/II proteins of CHIKV significantly enhances cell-mediated immune responses, laying the foundation for the development of potential vaccination strategies against CHIKV.

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry.

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C'D loop region of PD-1 upon complex formation with nivolumab.

Molecular Cloning of the scd1 Gene and Its Expression in Response to Feeding Artificial Diets to Mandarin Fish (Siniperca chuatsi).

Background/Objectives: Stearoyl-coenzyme A desaturase 1 (SCD1) plays a crucial role in fatty acid metabolism. However, its roles in the feeding habit transformation of mandarin fish (Siniperca chuatsi) remain largely unknown. Methods: Juvenile mandarin fish (10.37 ± 0.54)g were trained to feed on an artificial diet and then divided into artificial diet feeders and nonfeeders according to their feed preference. Afterwards, the scd1 gene of mandarin fish (Sc-scd1) was identified and characterized, and its transcription difference was determined between S. chuatsi fed live artificial diets and those fed prey fish. Results: Our results show that Sc-scd1 coding sequence is 1002 bp long, encoding 333 amino acids. The assumed Sc-SCD1 protein lacks a signal peptide, and it contains 1 N-linked glycosylation site, 24 phosphorylation sites, 4 transmembrane structures, and 3 conserved histidine elements. We found that Sc-SCD1 exhibits a high similarity with its counterparts in other fish by multiple alignments and phylogenetic analysis. The expression level of Sc-scd1 was detected with different expression levels in all tested tissues between male and female individuals fed either live prey fish or artificial diets. Conclusions: In particular, the Sc-scd1 expression level was the highest in the liver of both male and female mandarin fish fed artificial diets, indicating that scd1 genes may be associated with feed adaption of mandarin fish. Taken together, our findings offer novel perspectives on the potential roles of scd1 in specific domestication, and they provide valuable genetic information on feeding habits for the domestication of mandarin fish.

Systematic mutational analysis reveals an essential role of N275 in IgE stability.

Therapeutic antibodies have predominantly been IgG-based. However, the ongoing clinical trial of MOv18 IgE has highlighted the potential of using IgE antibodies in cancer therapy. While extensive studies targeting IgG glycosylation resulted in a rational basis for the development of enhanced biotherapeutics, IgE glycosylation remains an area with limited analyses. Previous studies on the role of IgE glycosylation present conflicting data with one study emphasizing the importance of N275 and T277 residues for FcεRI binding whereas another asserts the nonsignificance of IgE glycosylation in receptor interaction. While existing literature underscores the significance of glycans at the N275 position for binding to FcεR1 receptor and initiation of anaphylaxis, the role of other IgE glycosylation sites in folding or receptor binding remains elusive. This study systematically investigates the functional significance of N-linked glycosylation sites in the heavy chain of IgE which validates the pivotal role of N275 residue in IgE secretion and stability. Replacement of this asparagine to non-amine group moieties does not affect IgE function in vitro, yet substitution with aspartic acid compromises antibody yield. The deglycosylated IgE variant exhibits superior efficacy, challenging the conventional importance of glycosylation for effector function. In summary, our study unveils an intricate relationship between N-glycosylation sites and the structural-functional dynamics of IgE antibodies. Furthermore, it offers novel insights into the IgE scaffold, paving the way for the development of more effective and stable IgE-based therapeutics.

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants.

The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc). The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35mg/kg leaf fresh weight). Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.

First report of molecular epidemiology and phylogenetic characteristics of feline herpesvirus (FHV-1) from naturally infected cats in Kunshan, China

BackgroundFeline herpesvirus type 1 (FHV-1) is a life threatening highly contagious virus in cats and typically causes upper respiratory tract infections as well as conjunctival and corneal ulcers. Genetic variability could alter the severity of diseases and clinical signs. Despite regular vaccine practices against FHV-1 in China, new FHV-1 cases still commonly occur. The genetic and phylogenetic characteristics of FHV-1 in Kunshan city of China has not been studied yet. Therefore, this study was planned to investigate the prevalence, molecular characteristics of circulating strains, and phylogenetic analyses of FHV-1. This is the first report of molecular epidemiology and phylogenetic characteristics of FHV-1 from naturally infected cats in Kunshan, China.MethodsThe occulo-nasal swabs were collected from diseased cats showing respiratory distress, conjunctivitis, and corneal ulcers at different veterinary clinics in Kunshan from 2022 to 2023. Clinical data and general information were recorded. Swab samples were processed for preliminary detection of FHV-1. Thymidine kinase (TK), glycoprotein B (gB) and glycoprotein D (gD) genes were sequenced and analyzed to investigate genetic diversity and evolution of FHV-1.ResultsThe FHV-1 genome was detected in 43 (43/200, 21.5%) samples using RT-PCR targeting the TK gene. Statistical analysis showed a significant correlation between age, vaccination status and living environment (p < 0.05) with FHV-1 positivity, while a non-significant correlation was observed for FHV-1 positivity and sex of cats (p > 0.05). Additionally, eight FHV-1 positive cats were co-infected with feline calicivirus (8/43,18.6%). FHV-1 identified in the present study was confirmed as FHV-1 based on phylogenetic analyses. The sequence analyses revealed that 43 FHV-1 strains identified in the present study did not differ much with reference strains within China and worldwide. A nucleotide homology of 99-100% was determined among gB, TK and gD genes nucleotide sequences when compared with standard strain C-27 and vaccine strains. Amino acid analysis showed some amino acid substitutions in TK, gB and gD protein sequences. A potential N-linked glycosylation site was observed in all TK protein sequences. Phylogenetic analyses revealed minor variations and short evolutionary distance among FHV-1 strains detected in this study.ConclusionsOur findings indicate that genomes of 43 FHV-1 strains are highly homogenous and antigenically similar, and the degree of variation in major envelope proteins between strains is low. This study demonstrated some useful data about prevalence, genetic characteristics, and evolution of FHV-1 in Kunshan, which may aid in future vaccine development.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Increased α2,3-sialyl N-glycosylated prostate-specific membrane antigen (PSMA) in post-DRE urine is associated with high grade group prostate cancer.

Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate-specific membrane antigen (PSMA), a highly glycosylated protein with 10 N-linked glycosylation sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized. We investigated whether urinary PSMA sialylation correlate with high-grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin-antibody enzyme-linked immunoassay was used to quantify α2,3-sialyl PSMA in post-DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease. There are significant increases in α2,3-sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3-sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947). Our study shows that there are significant differences in the abundance of α2,3-sialylated PSMA in post-DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post-DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3-sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer.

Abstract 3136: Towards development of a novel biparatopic trop2-ADC

Abstract Background: Trophoblast cell surface antigen 2 (TROP-2) is a member of Tumor-associated Calcium Signal Transducer family (TACSTD2), which is a 35kDa transmembrane protein with 4 N-linked glycosylation sites. The overexpression of TROP2 in various cancers is associated with poor prognosis and increased risk of metastasis. Anti-TROP2 antibody drug conjugates (ADCs) are emerging as an innovative therapeutic intervention for cancer treatment. While these efforts were rewarded by the FDA approval of Trodelvy (Sacituzumab govitecan), a Trop2 targeted ADC, in 2020 for the TNBC patients, tumor heterogeneity could present one of the limitations for the Trop2-targeting therapy, leading to drug resistance. Here, we report our effort in developing a novel biparatopic TROP2 ADC which demonstrate superior cell killing activity compared with the monospecific TROP2 ADC, including certain resistant cell lines for the approved therapy. Methods: Various biparatopic Abs were engineered using two different mAbs targeting the two non-overlapping epitopes of Trop2. The physical stability and biological activities were characterized. Binding competition assay was utilized for epitope binning using SPR. Cell binding affinity was determined using flow cytometry. Cellular internalization activity was measured using Incucyte system, and tumor cell killing efficacy was evaluated in multiple cancer cell lines. CDX mouse models were utilized to investigate the TROP2-ADC in vivo efficacy. Results: We identified a biparatopic TROP2 mAb using two monospecific TROP2 mAbs with A recognizing the same epitope as Trodelvy, and B binding to a non-overlapping epitope vs Sacituzumab’s. mAb-B demonstrates superior ability to recognize a few resistance cell lines to mAb-A. The biparatopic TROP2 mAb-A/B displays a KD of 0.81 nM protein binding compared with 6.42 nM and 0.26 nM, respectively for mAb-A and mAb-B. Importantly, the biparatopic mAb-A/B demonstrates significantly faster cellular internalization activity compared to those of the individual mAbs, signifying the biparatopic scaffold could drive receptor clustering and subsequently enhance the internalization, trafficking to the lysosome and degradation of the target. As expected, the ADC of the biparatopic mAb-A/B conjugated with a TOPi payload shows cancer cell killing efficacy in both in vitro and in vivo experiments. Furthermore, we tested mAb-A, -B and biparatopic mAb-A/B for their binding affinity to 14 missense mutations in TROP2 ECD expressed in HEK293 cells. Interestingly, mAb-A showed decreased binding in 5 mutant cell lines, while mAb-B and biparatopic mAb-A/B maintains activity. Conclusion: We have engineered a novel TROP2 biparatopic antibody with unique biological features as well as superior in vitro and in vivo activities. Such a biparatopic ADC presents its potential to be a best-in-class ADC therapy by overcoming the limitation of the monospecific ADC therapy such as drug resistance. Citation Format: Yinan Wu, Brittany Jiang, Julliet Rivera, Fang Wang, Jing Zhou, Lin Liu, Silvia Hudson, Will Draper, Jennifer Li, Xiaoshan Min, Hang Chen, Zhulun Wang. Towards development of a novel biparatopic trop2-ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3136.

The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαμR, FcμR, and CDC activities.

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N-linked glycosylation site located in the Fc-domain is directly involved in high affinity binding to the respective receptors and initiation ofcorresponding immune response. IgM molecules have five N-glycosylation sites and one N-glycosylation site in the J-chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N-glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N-glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαμR, and the specific receptor for IgM, FcμR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcμR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcμR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcμR.

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1–8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57–60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Hydrophilic Peptide and Glycopeptide as Immobilized Sorbents for Glycosylation Analysis.

Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment in shot-gun glycoproteomics to enhance the glycopeptide signal and minimize the ionization competition of peptides. In this work, we have developed a novel hydrophilic material (glycoHILIC) based on glycopeptides and peptides to provide hydrophilic properties. GlycoHILIC was synthesized by oxidizing cotton and then reacting the resulting aldehyde with the N-terminus of the glycopeptide or peptide by reductive amination. Due to the large amount of hydrophilic carbohydrates and hydrophilic amino acids contained in glycopeptides, glycoHILIC showed significantly better enrichment of glycopeptides than cotton itself. Our results demonstrate that glycoHILIC has high selectivity, a low detection limit, and good stability. Over 257 unique N-linked glycosylation sites in 1477 intact N-glycopeptides from 146 glycoproteins were identified from 1 μL of human serum using glycoHILIC. Serum analysis of pancreatic cancer patients found that 38 N-glycopeptides among 21 glycoproteins changed significantly, of which 7 N-glycopeptides increased and 31 N-glycopeptides decreased. These results demonstrate that glycoHILIC can be used for glycopeptide enrichment and analysis.

Inter and intra-host diversity of RSV in hematopoietic stem cell transplant adults with normal and delayed viral clearance.

Respiratory syncytial virus (RSV) infection in immunocompromised individuals often leads to prolonged illness, progression to severe lower respiratory tract infection, and even death. How the host immune environment of the hematopoietic stem cell transplant (HCT) adults can affect viral genetic variation during an acute infection is not understood well. In the present study, we performed whole genome sequencing of RSV/A or RSV/B from samples collected longitudinally from HCT adults with normal (<14 days) and delayed (≥14 days) RSV clearance who were enrolled in a ribavirin trial. We determined the inter-host and intra-host genetic variation of RSV and the effect of mutations on putative glycosylation sites. The inter-host variation of RSV is centered in the attachment (G) and fusion (F) glycoprotein genes followed by polymerase (L) and matrix (M) genes. Interestingly, the overall genetic variation was constant between normal and delayed clearance groups for both RSV/A and RSV/B. Intra-host variation primarily occurred in the G gene followed by non-structural protein (NS1) and L genes; however, gain or loss of stop codons and frameshift mutations appeared only in the G gene and only in the delayed viral clearance group. Potential gain or loss of O-linked glycosylation sites in the G gene occurred both in RSV/A and RSV/B isolates. For RSV F gene, loss of N-linked glycosylation site occurred in three RSV/B isolates within an antigenic epitope. Both oral and aerosolized ribavirin did not cause any mutations in the L gene. In summary, prolonged viral shedding and immune deficiency resulted in RSV variation, especially in structural mutations in the G gene, possibly associated with immune evasion. Therefore, sequencing and monitoring of RSV isolates from immunocompromised patients are crucial as they can create escape mutants that can impact the effectiveness of upcoming vaccines and treatments.