2026· Methods in molecular biology· Vol 3018, pp.
85-98
· 0 citations
Medicine
TL;DR
Hydrophilic interaction chromatography is utilized to enrich glycopeptides from both peptides bound to HLA and digest of HLA proteins that obtained from immunoprecipitation of MHC complex from cell lysate that enables the characterization of glycosylation of MHC by database search.
Abstract The Human Cytomegalovirus (HCMV) gH/gL/UL128/UL130/UL131A complex (Pentamer) is among the candidates for the development of a vaccine against HCMV; indeed, it brought to more immunogenic formulations which were able to protect the fetus against vertical transmission in pregnant women. Pentamer-specific antibodies have shown to be hundredfold more potent than gB or gH/gL antibodies, endowed with strong neutralizing activity. The often inadequate antibody immune response elicited by glycoprotein antigen has been, in part, attributed to the abundance of glycosylation on the protein. Here, we determine the composite glycan population of each of the N-linked and O-linked glycosylation sites present on the Pentamer by multienzymatic proteolysis and mass spectrometry; furthermore, total N-glycans profiles of Pentamer expressed in different cell systems have been compared exploiting the glycan fluorescent labeling and the immunogenicity of Pentamer with different glycosylation patterns has been investigated. Our analysis reveals the presence of underprocessed oligomannose-type glycans on some glycosylation sequons, which are probably derived as a result of sterically reduced accessibility to glycan processing enzymes. Furthermore, the antigen glycosylation pattern affects the elicitation of neutralizing antibodies in mice.
Antonio Lembo, Gian Luca Sardone, Michela Aurilia et al.· Glycobiology· 0 citations
Peptides presented by class-I Human Leukocyte Antigen (HLA-I) proteins provide the basis of immune surveillance. Conversely, reduced surface HLA-I expression is a hallmark of immune evasion in cancers, which confounds the identification of peptide antigens and neoantigens. Here, we outline a system (HLA-Shuttle) for in vitro manipulation of cells with engineered components of the HLA-I processing pathway that improves recovery of the immunopeptidome of immunologically “cold” tumors.
HLA-Shuttle is comprised of an engineered variant of the HLA class I chaperone tapasin that bypasses its native degradation and ER retention signals, enabling improved expression and escape from the ER. HLA-Shuttle provides a continuum of chaperoning activity for HLA-I complexes from their point of assembly in the ER to the cell surface, improving antigen presentation in those cells.
Our data suggest that HLA-Shuttle functions in a multimodal fashion, both enhancing HLA-I complex production in the ER while stabilizing the folded conformation of HLA-I molecules globally. This is evidenced by increased surface expression of HLA-I, while cellular trafficking assays and single particle tracking reveal an extension of their cell-surface lifetimes and microdomain formation, implying an enhancement in their stability. Leveraging this technology, we captured the immunopeptidomes of neuroblastoma cell lines. We observed improved immunoprecipitation of HLA-I complexes, which correlated with a significant expansion of the observable immunopeptidome in immunologically cold neuroblastoma cells. Following bioinformatics analysis to search for therapeutically relevant peptides, we identified multiple novel tumor associated antigens (TAAs) from both known and novel cancer immunotherapy targets.
In conclusion, HLA-Shuttle restores antigen presentation in immunologically cold tumor cells, facilitating identification of TAAs with favorable therapeutic potential.
T32 Fellowship
Classical and Non-Classical Antigen Presenting Cells (APC)
Daniel Hwang, Molly C. Erdman, Santosh Adhikari et al.· Journal of Immunology· 0 citations
Abstract Class I MHC peptide (MHC-Ip) multimers are well-established reagents that detect antigen-specific T cells. The classical method for production of MHC-Ip multimers begins with the expression of MHC heavy chains (HCs) and β2-microglobulin (β2m) subunits as inclusion bodies in Escherichia coli and is followed by denaturant solubilization, in vitro folding in the presence of a defined peptide ligand, and purification by size exclusion chromatography. This protocol is labor intensive, difficult to scale, and represents a significant bottleneck in application of the technology. Herein, we present a novel method for the expression in eukaryotic cells of secreted peptide exchange–competent class I MHC proteins in their native conformation. In this method, expression constructs are engineered as bimolecular complexes composed of an MHC HC and a β2m molecule covalently linked at its amino terminus to an MHC-binding peptide through a flexible peptide linker containing a defined protease site. Upon proteolysis, the original peptide occupant of the MHC binding site dissociates and is easily replaced with a synthetic peptide. When leucine zippers are added to the carboxyl terminus of each subunit, protease cleavage of the linker results in a stable HC/β2m complex that can be isolated and stored for subsequent peptide loading. Using this method, we have produced homogeneous MHC-Ip complexes for 25 class I MHC alleles and demonstrated that tetramers produced in this way are equivalent to conventionally produced tetramers for T-cell staining.
V. Ramachandiran, J. Shires, Richard Willis et al.· Journal of Immunology· 0 citations
The main mechanisms of antigen acquisition, endocytic/lysosomal factors controlling MHCII-restricted processing and presentation, and evidence linking lysosomal dysfunction to autoimmunity are explored.
Gabriele Sergio Colangelo, Kyra J Cowan, Federico Riccardi Sirtori et al.· Journal of Immunology· 0 citations
Background
. The active ingredient of a new-generation antiviral drug for the treatment of herpes and human papillomavirus infection is alloferon, an oligopeptide with the amino acid sequence HGVSGHGQHGVHG. By inducing interferon biosynthesis, alloferon improves the immune status of men and women with various viral and bacterial-viral infections. The precise mechanism of alloferon's molecular pharmacological action is unknown.
Objective
: To establish possible molecular mechanisms of the anti-infective, immunostimulatory, and other effects of alloferon.
Material and methods.
Biophysical modeling of the structure and properties of the alloferon oligopeptide and bioinformatic analysis of its amino acid sequence in proteomic databases.
Results
. Expert analysis of bioinformatics and biophysical modeling results revealed that the primary hypothesis for alloferon's action is its role as an antigenic epitope, similar in structure to fragments of viral capsid proteins (including hemagglutinin). By interacting with T-cell receptors (TCRs) via major histocompatibility complex (MHC) proteins, alloferon and/or its fragments activate NK lymphocytes, which facilitate the destruction of viral and bacterial pathogens. This mechanism is supported not only by alloferon's similarity to known TCR antigenic epitopes but also by the results of biophysical prediction of epitopes, processing, and binding of alloferon to MHC proteins. The second most significant molecular mechanism involves alloferon's properties as an antimicrobial peptide (AMP) with antiviral activity. This hypothesis is supported by (1) the similarity of the amino acid sequence and amino acid composition of alloferon with known AMPs (piscidins, CA-1, bacteriocin plantaricin, etc.); (2) the potential alpha-helical structure of alloferon; (3) the results of bioinformatics and biophysical prediction of AMP activity against bacterial (E. coli, P. aeruginosa, K. pneumoniae, S. aureus, etc.) and viral (DENV-1, JEV, MERS-CoV, SARS-CoV, SARS-CoV-2, hepatitis C virus, herpes simplex virus) pathogens. Other potentially important molecular mechanisms of action of alloferon include (1) activation of formyl peptide receptors FPR1/2 on the surface of neutrophils (causes chemotaxis of lymphocytes to the site of infection); (2) inhibition of the interleukin-17 receptor (anti-inflammatory effect); (3) blocking the interaction of viruses with sialic acids; (4) cytoprotective properties of peptide fragments within the alloferon molecule; (5) inhibition of proteins containing the potassium channels tetramerization domain (KCTD) (important for modulating neurotransmission and for antitumor activity).
Conclusion
. The results of this study indicate verifiable mechanisms of molecular action of alloferon.
I. Torshin, A. N. Gromov, O. A. Gromova· FARMAKOEKONOMIKA. Modern Pha...· 0 citations
Human leukocyte antigen (HLA) class I presents intracellular peptides to the immune system on the cell surface. Since this process is crucial for the recognition of cancer cells and the initiation of anti-tumor immunity, peptides presented by HLA are valuable immunotherapy targets. More stable peptide HLA (pHLA) complexes provoke superior immune responses. However, how peptide sequence motifs contribute to pHLA stability is not well understood.
We developed a high-throughput assay to quantify stability of thousands of user-defined pHLA produced in E. coli. Peptide libraries and the desired HLA are produced and form pHLA complexes in E. coli. pHLA are purified and stability is evaluated by treating pHLA with a thermal gradient and recovering only the peptides which remain HLA-bound after heat treatment. Peptide depletion over the temperature range is monitored by quantitative tandem mass tag (TMT) enabled mass spectrometry.
Our new E. coli-based method is reliable for assessing pHLA stability. Detected HLA-binding peptides have the expected binding motifs, and stability data strongly correlates with current gold-standard data. We are able to generate large peptide stability datasets (1,800+ peptides) in one scaled experiment — five times larger than currently available datasets. We show that peptide motifs and anchor residue combinations potentially drive pHLA stability. Additionally, peptides were included in user-defined libraries with public immunogenicity annotations. We observed that immunogenic peptides were significantly more stable than non-immunogenic peptides.
We generated customizable pHLA stability datasets which show how peptide sequence motifs affect pHLA stability, and may be helpful for improving our mechanistic understanding of pHLA stability. Further, since peptide stability is related to immunogenicity, these large-scale pHLA stability datasets will be useful for improving peptide immunogenicity predictions for the development of immunotherapeutics.
NIH R01CA155010, Mark Foundation for Cancer Research, Moderna
Classical and Non-Classical Antigen Presenting Cells (APC)
M. Wilbrink, Luis O Correa-Medero, Emma C Duggan et al.· Journal of Immunology· 0 citations