Resistance to antibiotic treatments continues to be a significant health challenge and necessitates investigating novel antibacterial targets including noncoding regulatory RNA. The antiterminator RNA element of the T-box riboswitch is one such novel target. In this report, we investigated a spatially dispersed polycationic pharmacophore model for targeting the T-box antiterminator RNA. Decapeptides were designed based on a known alkyl polyamine agonist of the T-box riboswitch. Antiterminator model RNA binding was assessed using native mass spectrometry and modulation of T-box riboswitch function determined using a previously validated fluorescence-based assay. One peptide was identified that formed a complex with antiterminator model RNA and selectively inhibited T-box riboswitch transcription readthrough (both with and without tRNA induction). The inhibitory activity of this polycationic peptide, which was designed to mimic biogenic polyamines, has implications for future drug discovery efforts targeting the T-box riboswitch and other medicinally important noncoding regulatory RNA.

Early diagnosis of cancer offers the best chance for effective treatment. Serological tests for the detection of cancer testis antigens (CTA) could aid in cancer diagnosis, prognosis, and treatment evaluation. Since NY-ESO-1 induces a strong immune response in several cancer types, it is considered an attractive CTA for antibody assay development. The full-length NY-ESO-1 protein was recombinantly produced in Escherichia coli, purified, and used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of NY-ESO-1 specific antibodies. This ELISA was optimized and validated using serum samples collected from 78 confirmed cancer patients and control sera from healthy donors (n = 20) and rheumatoid arthritis patients (n = 43). The optimized NY-ESO-1 ELISA provided a sensitivity of 96.2 % in cancer patients and a specificity of 100 % for non-cancer donors. A follow-up study of patients undergoing cancer treatment indicated that the antibody levels were responsive to therapy, becoming very low or negative after successful treatment and rising again before tumor metastasis recurrence was confirmed. Comparative peptide ELISA using overlapping peptides of the full NY-ESO-1 sequence did not improve the detection rate, but did reveal common epitopes. The NY-ESO-1 protein-based ELISA could be used to detect potential cancer patients and is useful for monitoring cancer therapy and tumor recurrence after treatment.

Resistance to fluoroquinolone antibiotics has serious implications for healthcare; here, we conjugate the widely used fluoroquinolone ciprofloxacin to a proline-rich antimicrobial peptide (PrAMP) oncocin to improve oncocin's potency in ciprofloxacin-sensitive and ciprofloxacin-resistant strains of Escherichia coli. The conjugate molecule (oncocin-cipro-c) is ∼3× more potent than the parent oncocin, as determined by MIC, while retaining Gram-negative selectivity. We have characterized oncocin-cipro-c's interactions with three intracellular targets, two from oncocin (DnaK and 70S ribosome) and a third from ciprofloxacin (gyrase). Oncocin-cipro-c is also able to facilitate mast cell degranulation at a lower concentration than the parent peptide. The development of multimode antibiotics like oncocin-cipro-c is essential in the coming decades of antibiotic resistance.

SUMMARY The unicellular soil alga Chlamydomonas reinhardtii forms diploid zygotes during its sexual cycle. The process of a zygote maturing into a highly resistant zygospore remains poorly understood despite its importance for survival under adverse environmental conditions. Here we describe the detailed timeline of morphological and physiological changes during zygote maturation in darkness on ammonium‐free Tris‐acetate‐phosphate agar plates. The formation of a multilayered cell wall is primarily responsible for the increase in cell size in the first few days after zygote formation. Desiccation and freezing tolerance also develop in the period 3–7 days. Photosynthetic and respiratory activity decrease to reach minimal levels after 7–10 days, accompanied by a partial dedifferentiation of the chloroplast that includes chlorophyll degradation followed by the possible disappearance of the pyrenoid. In contrast to the decreasing concentrations of most carotenoids in the first few days after zygote formation, ketocarotenoids can first be detected after 3 days and their accumulation is completed after 10 days. Furthermore, the zygote degrades a large proportion of its starch and enriches oligosaccharides that may serve as osmoprotectants. The storage lipid triacylglycerol is accumulated at the expense of thylakoid membrane lipids, which mirrors the conversion of a metabolically active cell into a dormant spore on the metabolic level. Taken together, zygote maturation is a multifaceted process that yields mature zygospores after ~ 3 weeks. This work sheds light on the complete time course of the remodeling of a photosynthetically active eukaryotic cell into a dormant, highly resistant spore.

IntroductionKlebsiella pneumoniae (K. pneumoniae) is a major human nosocomial infectious agent and an important veterinary pathogen, frequently resistant to various antibiotics. It causes diseases such as pneumonia, urinary tract infections, surgical wound infections and septicemia. Biofilm formation of K. pneumoniae promotes persistent infection and contributes to resistance against antimicrobial agents. The objective of this study was to comparatively evaluate the effect of selected AMPs on the formation, metabolic activity and viability of Klebsiella pneumoniae biofilms of veterinary and human origin.MethodsBiofilm formation of three K. pneumoniae strains was quantified using the crystal violet assay and visualized by scanning electron microscopy (SEM). The inhibitory effects of eight different AMPs on the formation and metabolic activity of K. pneumoniae biofilms, as well as on planktonic growth, were examined using crystal violet, resazurin and broth microdilution assays, respectively. The effect on living and dead bacteria in mature biofilms was investigated using the fluorescent dyes SYTO™ 9 and propidium iodide. In addition, the distribution of rhodamine B-labeled peptide DJK-5 in mature biofilms of strain 17349 was visualized by confocal laser scanning microscopy (CLSM).ResultsBiofilm formation was confirmed for all three K. pneumoniae strains. Depending on the strain, we found that planktonic growth was affected by the AMPs DJK-5, DJK-6, Onc72, and Onc112. Biofilm formation of all three strains was inhibited by hbD3, LL-37, DJK-5, and DJK-6, with biofilm mass reduced to less than 40% of the untreated control. In addition to the inhibition of biofilm formation, a reduction in the metabolic activity of the biofilm-associated bacteria was also observed. These four AMPs also showed an effect on mature biofilms by reducing the number of both viable and dead bacteria in 22 h-old biofilms. Rhodamine B-labeled DJK-5 took 7 h to visibly accumulate in the planktonic bacteria. Multi-layered biofilm aggregations were mainly negative for rhodamine B-labeled DJK-5, even 44 h after AMP treatment, indicating that certain parts of mature K. pneumoniae biofilms are not accessible for this AMP.ConclusionIn conclusion, we found differences in the effect of AMPs on biofilms including both increases and decreases in biofilm mass and viability.

Background: The well-studied 18-residue-long proline-rich antimicrobial designer peptide Api137 utilizes at least two lethal intracellular mechanisms that target the bacterial 70S ribosome. First, Api137 stalls the ribosome by binding to the peptidyl-transferase center, trapping the release factor, and inhibiting protein expression. Second, Api137 disrupts the assembly of the large 50S subunit of the ribosome, resulting in partially assembled pre-50S dead-end particles that are unable to form the functional 70S ribosome. Methods: All six proline residues in Api137 were substituted with 4S- and 4R-fluoro-l-proline (Fpr), which promote the cis- and trans-conformer ratio of the preceding Xaa-Pro-bond, respectively. The effect on the antibacterial activity was studied using Escherichia coli. The underlying mechanisms were investigated by studying 70S ribosome binding, inhibition of in vitro translation, and ribosome profile analysis. Results: Interestingly, the analogs were equipotent to Api137, except for the 4S-Fpr11 and 4S-Fpr16 analogs, which were four times more or less active, respectively. The most active 4S-Fpr11 analog competed the least with Api137 for its ribosome binding site, suggesting a shifted binding site. Both Fpr14 and the 4S-Fpr16 analogs disturbed 50S subunit assembly less than Api137 or not at all. The strongest effect was observed with the 4R-Fpr16 analog resulting in the lowest 70S ribosome content and the highest pre-50S particle content. This peptide also showed the strongest competition with Api137 for its binding site. However, its antibacterial activity was similar to that of Api137, possibly due to its slower cellular uptake. Conclusions: Api137 inhibits protein translation and disrupts 50S assembly, which can be adjusted by substituting specific proline residues with fluoroproline. 4R-Fpr16 potently inhibits ribosome assembly and offers a novel, unexploited clinical mechanism for future antibiotic development.

The proline-rich antimicrobial designer peptide Api137 inhibits protein expression in bacteria by binding simultaneously to the ribosomal polypeptide exit tunnel and the release factor (RF), depleting the cellular RF pool and leading to ribosomal arrest at stop codons. This study investigates the additional effect of Api137 on the assembly of ribosomes using an Escherichia coli reporter strain expressing one ribosomal protein per 30S and 50S subunit tagged with mCherry and EGFP, respectively. Separation of cellular extracts derived from cells exposed to Api137 in a sucrose gradient reveals elevated levels of partially assembled and not fully matured precursors of the 50S subunit (pre-50S). High-resolution structures obtained by cryogenic electron microscopy demonstrate that a large proportion of pre-50S states are missing up to five proteins (uL22, bL32, uL29, bL23, and uL16) and have misfolded helices in 23S rRNA domain IV. These data suggest a second mechanism for Api137, wherein it disrupts 50S subunit assembly by inducing the formation of misfolded precursor particles potentially incapable of evolving into active ribosomes, suggesting a bactericidal mechanism.

New strategies are needed to prevent and control upcoming outbreaks of SARS‐CoV‐2 infections, independent of vaccination. SARS‐CoV‐2 binds to the human ACE‐2 receptor through the receptor binding domain (RBD) of the spike (S) protein, allowing the virus to enter human cells and begin replication. When peptides corresponding to four regions of RBD containing previously reported ACE‐2 interaction sites were explored, the sequence 392 to 421, peptide p392wt, bound strongly to ACE‐2 and inhibited wild‐type RBD binding to ACE‐2. Interestingly, p392 peptides corresponding to mutated sequences from different SARS‐CoV‐2 VOCs, including the current VOC BA.5 and KP.3, bound less strongly to ACE‐2, but showed partially better inhibition of the ACE‐2 interaction of all tested RBDs. When studied in a SARS‐CoV‐2 pseudovirus assay, the p392 peptides showed a good inhibition rate of 98.8±8.1 % at a peptide concentration of ~244 μmol/L, while none of the p392 peptides inhibited antibody binding to the RBD, suggesting that peptide treatment is sufficient in the presence of anti‐RBD antibodies. Interestingly these peptides were active in the presence of diluted human serum and non‐toxic to human cell lines.

Excessive oxidative stress, associated with various diseases, can induce protein carbonylation-nonenzymatic modifications involving aldehyde or keto group formation. These modifications are structurally diverse and low in abundance, which complicates their detection and quantitation. Here, we developed a strategy to identify and quantify protein carbonylation in human serum proteins from 39 rheumatoid arthritis patients and 29 healthy donors. Reactive carbonyl groups were derivatized with an aldehyde reactive probe (ARP), digested with trypsin, enriched via avidin affinity chromatography, and analyzed using RP-HPLC-ESI-IMS-MS/MS. Ion mobility spectrometry (IMS) was applied in both data-dependent (DDA) and data-independent acquisition (DIA) modes. DDA generated spectral libraries of ARP-derivatized peptides (ARP-peptides), which enabled peptide-centric detection in DIA data. We manually confirmed 86 ARP-peptides, with 93.8% of peak areas showing signal-to-background ratios >3. Among the 32 unique carbonylation sites, 28 were on human serum albumin, with hotspots at Cys58, Lys214, Lys219, Lys223, Lys456, Lys543, Lys549, and Lys565. Six previously unreported species were identified using IMS, DIA, ARP-reporter ions, and de novo sequencing. The ARP-peptides were quantified with ≥ 75% intrabatch reproducibility (coefficient of variation <20%). Similar modification levels were observed in both groups, suggesting basal, disease-independent carbonylation in abundant serum proteins.

Protein glycation, or non-enzymatic glycosylation, refers to the reaction of reducing sugars with amino groups in proteins to form Amadori and Heyns products for aldoses (e.g., glucose) and ketoses (e.g., fructose), respectively. While Amadori peptides have been well studied after enrichment by boronate affinity chromatography (BAC), it is often assumed that BAC also enriches the isomeric Heyns peptides, although the binding of Heyns rearrangement products seems unlikely due to the very low content of 1,2- and 1,3-cis-diols in their dominant tautomeric forms. For seven different tryptic peptide sequences derived from human plasma digests, we showed that the synthetic glucose-modified Amadori peptides can be enriched by BAC with high recovery rates, while the corresponding fructose-modified Heyns peptides did not bind, independent of the buffers and pH used. Reduction of the carbonyl groups with borohydride, yielding the corresponding hexitol-modified peptides, allowed enrichment of both the former Amadori and, more importantly, Heyns peptides.

The rise in antibiotic resistance has meant that there is a need for new strategies and one avenue is the use of proline-rich antimicrobial peptides (PrAMPs). Here we investigate how different metal ion environments (Na+, Mg2+, Ca2+) affect antimicrobial activity of PrAMPs derived from apidaecin 1b (Api88, Api137) and Oncopeltus antibacterial peptide 4 (Onc72, Onc112) against Pseudomonas aeruginosa and Escherichia coli . Initial antimicrobial testing in an ion-rich media (ion levels similar to mammalian body fluids) found that the PrAMPs were effective against E. coli but not P. aeruginosa . Both Api88 and Api137 were bactericidal, while Onc72 and Onc112 were bacteriostatic against E. coli . In a lower ion-media the activity of the PrAMPs significantly improved against both bacteria and Onc72 and Onc112 altered the mode of action to bactericidal. In low Na+, Ca2+ and Mg2+ ion conditions all of the peptides were able to penetrate the outer membrane of P. aeruginosa , however at higher ion concentrations none of the peptides were able to penetrate the outer membrane. PrAMPs were found to cause E. coli cells to swell and have a hyperpolarised membrane indicating a new mechanism of action for PrAMPs. Our data indicates that bacteria reduce susceptibility to AMPs by stabilising their LPS layer with metal ions and that the PrAMPs have secondary modes of action affecting the functionality of the bacterial membrane. Combining an ion chelator with PrAMPs may be a novel solution to combat weak antimicrobial activity in ion-rich environments such as host tissues.

AbstractAlthough the intrinsic electronic properties of polyoxometalates (POMs) can be greatly influenced by modifying them with organic substituents, their resistive switching behavior on surfaces dependent on the organic substituents remains largely unexplored. In this work, we assessed the importance of electron‐withdrawing and electron‐donating ligand substituents on the material properties of a series of hybrid Lindqvist‐type hexavanadates TBA2[V6O13((OCH2)3CCH2OH)2] (TBA2V6‐OH), TBA2[V6O13((OCH2)3CMe)2] (TBA2V6‐Me), TBA2[V6O13((OCH2)3CNHCOCH2Cl)2] (TBA2V6‐Cl), and TBA2[V6O13((OCH2)3CNHCOCH2‐OOCC10H15)2] (TBA2V6‐Ad) as potential resistive random‐access memory (ReRAM) components. Compared to their redox behavior in solution, changing the ligand substituents on surfaces results in no significant effect on the potential and, thus, no effect on the resistance steps in the current‐voltage profiles. However, while the current‐voltage characteristics do not change, the peripheral metal‐free substituents in the trisalkoxide framework of Lindqvist‐type hexavanadate molecules influence the adsorption and switching stability of these POMs on gold. This work highlights the noticeable differences between hexavanadate's redox properties in solution (which follow the trend observed in the gas phase) and hexavanadate's resistive switching properties on conducting surfaces. Importantly, their multi‐state switching behavior is not significantly altered by the different type of substituent at the periphery of the trisalkoxo ligands.

Muscle wasting diseases, such as cancer cachexia and age-associated sarcopenia, have a profound and detrimental impact on functional independence, quality of life, and survival. Our understanding of the underlying mechanisms is currently limited, which has significantly hindered the development of targeted therapies. In this study, we explored the possibility that the streptococcal quorum sensing peptide Competence Stimulating Peptide 7 (CSP-7) might be a previously unidentified contributor to clinical muscle wasting. We found that CSP-7 selectively triggers muscle cell inflammation in vitro, specifically the release of IL-6. Furthermore, we demonstrated that CSP-7 can traverse the gastrointestinal barrier in vitro and is present in the systemic circulation in humans in vivo. Importantly, CSP-7 was associated with a muscle wasting phenotype in mice in vivo. Overall, our findings provide new mechanistic insights into the pathophysiology of muscle inflammation and wasting.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts. Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis. Here, the authors show that the honey-bee derived PrAMPs Api137 and Api88 inhibit bacterial ribosomes through multiple mechanisms, promising for drug development.

This study proposes an innovative strategy to enhance the pharmacophore model of antimicrobial bismuth thiolato complex drugs by substituting hydrocarbon ligand structures with boron clusters, particularly icosahedral closo -dicarbadodecaborane (C 2 B 10 H 12 , carboranes). The hetero- and homoleptic mercaptocarborane complexes BiPh 2 L ( 1 ) and BiL 3 ( 2 ) (L=9-S-1,2-C 2 B 10 H 11 ) were prepared from 9-mercaptocarborane (HL) and triphenylbismuth. Comprehensive characterization using NMR, IR, MS, and XRD techniques confirmed their successful synthesis. Evaluation of antimicrobial activity in a liquid broth microdilution assay demonstrated micromolar to submicromolar minimum inhibitory concentrations (MIC) suggesting high effectiveness against S. aureus and limited efficacy against E. coli . This study highlights the potential of boron-containing bismuth complexes as promising antimicrobial agents, especially targeting Gram-positive bacteria, thus contributing to the advancement of novel therapeutic approaches.

Serological assays for SARS-CoV-2 play a pivotal role in the definition of whether patients are infected, the understanding of viral epidemiology, the screening of convalescent sera for therapeutic and prophylactic purposes, and in obtaining a better understanding of the immune response towards the virus. The aim of this study was to investigate the performance of a bead-based multiplex assay. This assay allowed for the simultaneous testing of IgG antibodies against SARS-CoV-2 spike, S1, S2, RBD, and nucleocapsid moieties and S1 of seasonal coronaviruses hCoV-22E, hCoV-HKU1, hCoV-NL63, and hCoV-OC43, as well as MERS and SARS-CoV. We compared the bead-based multiplex assay with commercial ELISA tests. We tested the sera of 27 SARS-CoV-2 PCR-positive individuals who were previously tested with different ELISA assays. Additionally, we investigated the reproducibility of the results by means of multiple testing of the same sera. Finally, the results were correlated with neutralising assays. In summary, the concordance of the qualitative results ranged between 78% and 96% depending on the ELISA assay and the specific antigen. Repeated freezing-thawing cycles resulted in reduced mean fluorescence intensity, while the storage period had no influence in this respect. In our test cohort, we detected up to 36% of sera positive for the development of neutralising antibodies, which is in concordance with the bead-based multiplex and IgG ELISA.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance. Glycation sites on human serum albumin (HSA) appear to be potential biomarkers for diabetes mellitus. In contrast to previous mass spectrometry techniques, immunoassays including ELISA using specific and sensitive antibodies directed against the glycation site (e.g. Lys414) are suitable for high-throughput screening and thus for routine laboratory analysis.image

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

BackgroundCryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.ResultsA preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI− cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI− cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.