asparagine deamidation
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2021 ◽  
Author(s):  
Ramiro Lorenzo ◽  
Lucas A. Defelipe ◽  
Lucio Aliperti ◽  
Stephan Niebling ◽  
Tânia F. Custódio ◽  
...  

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 °C, respectively. This process is significantly slowed down at 4 °C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.


2021 ◽  
Author(s):  
Li Cao ◽  
Moshe Beiser ◽  
Joseph D Koos ◽  
Margarita Orlova ◽  
Hader E Elashal ◽  
...  

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.


Author(s):  
Jared A. Delmar ◽  
Jihong Wang ◽  
Seo Woo Choi ◽  
Jason A. Martins ◽  
John P. Mikhail

2020 ◽  
Vol 21 (20) ◽  
pp. 7566
Author(s):  
Maria-Eleni Androutsou ◽  
Agathi Nteli ◽  
Areti Gkika ◽  
Maria Avloniti ◽  
Anastasia Dagkonaki ◽  
...  

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35–55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35–55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35–55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35–55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide.


2020 ◽  
Vol 19 (7) ◽  
pp. 1236-1247 ◽  
Author(s):  
Shutao Mei ◽  
Rochelle Ayala ◽  
Sri H. Ramarathinam ◽  
Patricia T. Illing ◽  
Pouya Faridi ◽  
...  

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.


2020 ◽  
Vol 109 (1) ◽  
pp. 656-669 ◽  
Author(s):  
Magfur E. Alam ◽  
Thomas R. Slaney ◽  
Lina Wu ◽  
Tapan K. Das ◽  
Sambit Kar ◽  
...  

2019 ◽  
Vol 15 ◽  
pp. 264-274 ◽  
Author(s):  
Jared A. Delmar ◽  
Jihong Wang ◽  
Seo Woo Choi ◽  
Jason A. Martins ◽  
John P. Mikhail

2019 ◽  
Author(s):  
X. Lu ◽  
S. Lin ◽  
N. De Mel ◽  
A. Parupudi ◽  
M. Pandey ◽  
...  

ABSTRACTAsparagine deamidation is a common posttranslational modification in which asparagine is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kilodalton (kDa) truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which suggests a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide a possible explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest an approach that can be used for process control to ensure product quality and process consistency.Statement of SignificanceAsparagine deamidation can have a potentially significant impact on protein therapeutics. Previous studies have revealed that deamidation at a single site significantly reduces the biological activity of moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin developed for the treatment of B-cell malignancies. Surprisingly, despite the fact that deamidation introduced a negative charge, the deamidated variant eluted earlier than its unmodified form on anion exchange chromatography. In order to understand these observations, we isolated the deamidated variant using an anion exchange column and characterized it by differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry. The results revealed the conformational change caused by the deamidation, which explains the diminished biological activity of the variant and its early elution time on anion exchange chromatography.


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