Mapping the T cell epitopes of the M-type transmembrane phospholipase A2 receptor in primary membranous nephropathy.

The M-type phospholipase A2 receptor (PLA2R) is the major autoantigen of primary membranous nephropathy (MN). Despite many studies on B-cell epitopes recognized by antibodies, little is known about T-cell epitopes. Herein, we synthesized 123 linear peptides, each consisting of 15-22 amino acids with 8-12 amino acid overlaps, across ten domains of PLA2R. Their binding capacity to risk (DRB1∗1501, DRB1∗0301) and protective (DRB1∗0901, DRB1∗0701) HLA molecules was then assessed by flow cytometry. Proliferation of CD4+ T cells from patients with anti-PLA2R positive MN was analyzed after peptide stimulation. Cytokines produced by activated peripheral blood mononuclear cells were measured by cytometric bead arrays. We identified 17 PLA2R peptides that bound to both DRB1∗1501 and DRB1∗0301 molecules with high capacity. Some of these peptides showed decreased binding to heterozygous DRB1∗1501/0901 and DRB1∗0301/0701. Ten of the 17 peptides (CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1 and CTLD7-2) induced significant proliferation of CD4+ T cells from patients with MN than cells from healthy individuals. Upon activation by these peptides, peripheral blood mononuclear cells from patients with MN produced higher levels of pro-inflammatory cytokines, predominantly IL-6, TNF-α, IL-10, IL-9 and IL-17. Thus, we mapped and identified ten peptides in the CysR, FnII, CTLD3, CTLD5, and CTLD7 domains of PLA2R as potential T-cell epitopes of MN. These findings are a first step towards developing peptide-specific immunotherapies.

A silyl ether-protected building block for O-GlcNAcylated peptide synthesis to enable one-pot acidic deprotection.

In this report, we introduce a novel building block for Fmoc/tBu solid phase peptide synthesis (SPPS) of ß-linked O-GlcNAcylated peptides. This building block carries acid labile silyl ether protecting groups, which are fully removed under TFA-mediated peptide cleavage conditions from the resin, thus requiring fewer synthetic steps and no intermediate purification as compared to other acid or base labile protecting group strategies.

Biomimetic enantioselective synthesis of ß,ß-difluoro-α-amino acid derivatives.

Although utilization of fluorine compounds has a long history, synthesis of chiral fluorinated amino acid derivatives with structural diversity and high stereoselectivity is still very appealing and challenging. Here, we report a biomimetic study of enantioselective [1,3]-proton shift of ß,ß-difluoro-α-imine amides catalyzed by chiral quinine derivatives. A wide range of corresponding ß,ß-difluoro-α-amino amides were achieved in good yields with high enantioselectivities. The optically pure ß,ß-difluoro-α-amino acid derivatives were further obtained, which have high application values in the synthesis of fluoro peptides, fluoro amino alcohols and other valuable fluorine-containing molecules.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase.

During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

Robust synthesis of C-terminal cysteine-containing peptide acids through a peptide hydrazide-based strategy.

A new robust strategy was reported for the epimerization-free synthesis of C-terminal Cys-containing peptide acids through mercaptoethanol-mediated hydrolysis of peptide thioesters prepared in situ from peptide hydrazides. This simple-to-operate and highly efficient method avoids the use of derivatization reagents for resin modification, thus providing a practical avenue for the preparation of C-terminal Cys-containing peptide acids.

One-pot multi-segment condensation strategies for chemical protein synthesis.

With the growing requirement for otherwise-difficult-to-obtain proteins, it is necessary to develop more efficient chemical protein synthesis methods for rapid access to designed protein samples. In particular, a one-pot multi-segment condensation method, with only one purification step to obtain the final product, is expected to demonstrate unique benefits in chemical protein synthesis, such as the requirement of fewer handling procedures and the higher efficiency in obtaining aimed protein samples. The utilization of the one-pot multi-segment condensation strategy is demonstrated via the synthesis of a series of post-translational modification (PTM) or disease-associated peptides or proteins for basic and advanced scientific research. This review summarizes the recent one-pot multi-segment condensation methods utilized in chemical protein synthesis, in which two aspects of drive-strategies will be mainly included: a kinetically controlled strategy and a protecting group-removal strategy, respectively. On one hand, the activities of peptides in N-terminal thiol amino acids or C-terminal acyl donors can be largely different based on the differences in properties, such as steric hindrance, migration rates, electrophilicity, and introduction of active elements such as selenium, etc. Using the different activities, regio-selective peptide ligation can be performed in a kinetically controlled manner. On the other hand, the protecting group-removal strategy involves various moieties, which can block the activity of functional groups arising from N-terminal thiol amino acids or C-terminal acyl donors, and they can be removed by using additives, and pH- or photo-stimulation conditions with further achievement of chemical protein synthesis by the one-pot strategy.

Acyl donors for native chemical ligation.

Native chemical ligation (NCL) has become one of the most important methods in chemical syntheses of proteins. Recently, in order to expand its scope, considerable effort has been devoted to tuning the C-terminal acyl donor thioesters used in NCL. This article reviews the recent advances in the design of C-terminal acyl donors, their precursors and surrogates, and highlights some noteworthy progress that may lead the future direction of protein chemical synthesis.

Recent advances in racemic protein crystallography.

Solution of the three-dimensional structures of proteins is a critical step in deciphering the molecular mechanisms of their bioactivities. Among the many approaches for obtaining protein crystals, racemic protein crystallography has been developed as a unique method to solve the structures of an increasing number of proteins. Exploiting unnatural protein enantiomers in crystallization and resolution, racemic protein crystallography manifests two major advantages that are 1) to increase the success rate of protein crystallization, and 2) to obviate the phase problem in X-ray diffraction. The requirement of unnatural protein enantiomers in racemic protein crystallography necessitates chemical protein synthesis, which is hitherto accomplished through solid phase peptide synthesis and chemical ligation reactions. This review highlights the fundamental ideas of racemic protein crystallography and surveys the harvests in the field of racemic protein crystallography over the last five years from early 2012 to late 2016.