Glycopeptides have been engineered to structurally mimic both the glycan and peptide portions of the native glycoprotein to retain similar biological activity. Structural similarity to a native glycoprotein is important in order to maintain epitopes which bind to antibodies or receptors in a biologically relevant manner. Glycopeptides mimicking the structure of important disease biomarkers can therefore be used as antigens to develop highly sensitive and specific diagnostic assays for these biomarkers. For example, glycopeptides have been used to mimic tumor-associated antigens (TAAs), overexpressed in a number of cancers including Tn, STn, and sLeX. Glycopeptides containing these structures were developed as diagnostic antigens for immunoassays to detect biomarkers in cancer. Diagnostic assays deploy virally mimicking glycopeptides of influenza hemagglutinin and HIV gp120 to identify antibodies against these viruses. Since viral glycoproteins function as surface molecules that bind to host cell receptors for viral entry, antibodies that attach to these glycoproteins serve as definitive signs of previous viral exposure. The spike protein mimicking glycopeptides of SARS-CoV-2 which binds to the human ACE2 receptor to enter cells have proven effective in creating diagnostic tests that specifically detect SARS-CoV-2 antibodies from previous infections.
Glycopeptides have the benefit over linear peptides or whole proteins of conferring an additional level of specificity. Glycans confer an additional level of structural complexity to glycopeptides which can be interrogated by antibodies and which often results in highly specific binding. This is particularly attractive in diagnostics where the detection of antigens is often performed with assays such as ELISA or CLIA. These assays employ antibodies as capture and detection reagents which is inherently the possibility of high specificity. Antibodies may also be able to differentiate glycopeptides containing distinct glycans, which can significantly improve signal-to-noise ratios and decrease the occurrence of false positive or false negative results. Glycopeptide antigens can also be used to detect low abundance disease biomarkers which are difficult to target by traditional antibody assays. Glycopeptides may also be used to specifically target receptors or glycan-binding proteins (GBPs).
One of the important design features of diagnostic glycopeptides is the choice of glycan moiety. The Tn and STn antigens, for instance, are widely expressed in cancer cells and have been demonstrated to be immunogenic. Tn antigen and STn are both well-known tumor-associated carbohydrate antigens that are recognized by antibodies and immune cells. The sialyl Lewis X glycan structure, which is known to be involved in cell adhesion and immune cell recognition, is also relevant in the disease state of cancer and inflammation. This glycan moiety can also be conjugated with peptide structures that are diagnostic in nature, based on the specific epitopes that it presents. For example, Core1 is a starting point for most biosynthesis of more complex O-glycans and can be elongated to produce a number of specific terminal structures. An example of a Core1 elongation is sialyl Lewis X, a structure important in interactions with selectins of the immune system, which is known to be critical in cancer metastasis and immune cell recruitment. These and other glycans can be extended and modified to be specific for the natural antigens that appear in cancer and other disease states.
Fig.1 Classes of vertebrate glycan structures. 1,2
Peptide length together with scaffold selection stand as critical factors during the development of diagnostic glycopeptides. Length of the peptide moiety influences the stability, solubility, and overall properties of the glycopeptide. The extended peptide sequence provides essential stabilization and better antigen mimicry but faces synthesis challenges along with increased vulnerability to proteolytic breakdown. Optimization of the peptide length is of great importance. The scaffold decision affects the glycan's final structure and its presentation. Scaffolds can range from linear peptides to more complex protein-based scaffolds. Linear peptides have straightforward synthesis processes and simple glycan modification capabilities which makes them popular diagnostic tools. Cyclic peptides or protein scaffolds, on the other hand, can offer improved stability and specificity, especially when targeting complex receptors or GBPs.
Site-specific glycan (SSG) positioning can be used to build glycoconjugates. Efficient antibody or receptor recognition requires precise glycan positioning during glycopeptide synthesis for correct epitope display. The placement of glycans onto particular amino acids in peptides determines their binding affinity and specificity to glycopeptides. N-linked glycans attach to asparagine residues in the consensus sequence NXT/S while O-linked glycans frequently bind to serine or threonine residues. Natural glycoprotein antigenic determinants can be replicated by accurate positioning of glycans at binding sites. The spatial arrangement of glycans relative to the peptide backbone alters glycopeptide conformation which is essential for epitope exposure. SSG positioning can also be used to direct the accessibility of a glycan to the target antibody or receptor. Accessible positioning of a glycan allows the glycopeptide to reach maximal biological activity and binding specificity. Glycosylation site incorporation within peptide sequences is possible using solid-phase peptide synthesis followed by enzymatic glycosylation to achieve SSG positioning.
We offer expert design of high-performance glycopeptide antigens including one-on-one consultations to help you design the best glycopeptide antigen for your research needs. Our scientific team will work with you from the ground up through the design process from target selection to final sequence design to best meet the requirements of your diagnostic application. The design of your glycopeptide antigen is personalized to the needs of your assay, whether it is for the use of a diagnostic reagent to target specific cancer biomarkers or for the development of diagnostic reagents for infectious disease. Consultations cover selection of glycan moieties, peptide sequences, and scaffold structure to best meet the needs of your research project. Our expert advice is based on the most current scientific literature and our vast experience in the synthesis and characterization of glycopeptides.
Our services include rapid prototyping which accelerates your glycopeptide antigen development process together with personalized support. Our rapid prototyping facility enables you to manufacture small batches of glycopeptides for initial feasibility studies, so you can test and validate the performance of diagnostic assays in a timely manner. The approach allows for postponing large-scale production costs while providing necessary early-stage data to guide critical development decisions. The rapid prototyping of glycopeptides in our service includes creating diverse glycan structures and peptide sequences so you can select multiple options to assess during your feasibility studies. We provide analytical services to help you identify the glycopeptides you have synthesized. Our reports cover their purity, structure and function. The data enables evaluation of glycopeptide antigen efficiency in diagnostic assays for performance refinement.
Peptide Synthesis Services at Creative Peptides
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