Protein and Peptide Letters - Volume 22, Issue 11, 2015

Lectins are the (glyco)proteins that recognize and bind to specific sugar moieties without altering their structure. Galectins are mammalian lectins characterized by the presence of conserved 134 amino acids carbohydrate recognition domain and specificity for β-galactosides. The involvement of lectins in diverse biological spectrum, especially some deadly human diseases like cancer, neurological disorders and cardiovascular disorders has proclaimed them as one of the important components of glycobiology, thereby seeking the methods of their detection and identification heavily desirable. In the present manuscript, we have provided a comprehensive outline of various methods of detection and identification of lectins employed till date, with their needs and usage varying according to the level of infrastructure of laboratories and around the world. In addition, a vision for some quick, highly sensitive and advanced methods for lectin detection and identification for diagnostic and therapeutic of various diseases is also provided.

The aim of this study was to prepare PCADK/PLGA-blend microspheres for improving the stability of human immunoglobulin G (IgG). The short half-life of antibodies limit their development as therapeutic agents, thus PLGA microspheres were prepared to sustained release antibodies and prolong their half-life. However, the acidic intra-microsphere environment causes the loss of antibody stability and activity. In this study, the effect of PCADK or PLGA degradation products on IgG was investigated by size exclusion chromatography (SEC-HPLC), circular dichroism (CD), fluorescence spectroscopy and antigenicity detection. The degradation products of PCADK exerted a larger influence on IgG than that of PLGA. Then PCADK/PLGA microspheres were prepared by the emulsionsolvent evaporation method and systematically characterized and 20% PCADK were selected as the optimal proportion. In addition, the release profile of microspheres and the stability of the released IgG were investigated. The stability of the IgG released from the PCADK/PLGA microspheres was better than that of IgG released from the PLGA microspheres. Confocal laser scanning microscopy (CLSM) was used to determine the pH inside the microspheres. The IgG-loaded PCADK/PLGA microspheres have important advantages over the PLGA microspheres in terms of IgG stability and could be a good carrier to deliver antibodies for the treatment of disease.

Proteases hydrolyze polypeptides to release peptides and/or amino acids. This subclass of enzymes is among those with the most sales worldwide, particularly those produced by microorganisms. Proteases may be applied in the several industries, including the food industry, leather, detergents, and bioremediation. Myceliophthora thermophila protease was produced by a submerged bioprocess and then purified 185-fold by anion exchange and hydrophobic chromatography with a 37% yield. The molecular mass was estimated at 36.2 kDa, and mass spectrometry identified two sequences: GVVANMSLGGSYSASINNAAAALVR and STGNAAITGVPSGTTNR. The isolated protein was characterized biochemically, showed an optimum pH of 6.5 and optimum temperature of 45 °C, and stability at wide range of pH and temperatures and in the presence of reducing agents and some surfactants. Kinetic assays for this enzyme showed a greater catalytic efficiency when the substrate had alanine at position P'2. The protease presented characteristics that may be of interest to many industrial areas.

Tandem mass spectrometry (MS/MS) has emerged as a major technology for peptide sequencing. Typically, there are three kinds of methods for the peptide sequencing: database searching, peptide tagging, and de novo sequencing. De novo sequencing has drawn increasing attention because of its independence from existing protein databases and potential for identifying new proteins, proteins resulting from mutations, proteins with unexpected modifications and so on. Recently, with the improvements in the accuracy of MS/MS and development of alternative fragmentation modes of MS/MS, many new de novo sequencing methods have been formulated. This paper reviews these recently developed sequencing methods including those for alternative MS/MS spectra. The paper first introduces background knowledge on peptide sequencing and mass spectrometry, and then reviews de novo peptide sequencing methods for traditional CID spectra. After that, it focuses on the recent development of de novo methods for alternative MS/MS spectra. In addition, methods using multiple spectra from the same peptide are surveyed. Finally, conclusions and some directions of future work are discussed.

B-cell activating factor (BAFF) belonging to the TNF family, plays an important role in the proliferation and differentiation of B cells which renders it an attractive target for autoimmune diseases. Some peptides have been designed to target BAFF for the treatment of autoimmune diseases. Our previous studies suggested that peptides TA and DX-814 had competitive bioactivities compared to the natural peptide trans-membrane activator and calcium modulator and cyclophilin ligand interactor (TACI) in different binding orientations. In this study, we carried out molecular modeling and dynamics and molecular docking calculations to explore the structural and chemical features responsible for the binding affinities of these peptides. Binding free energy calculations, mutational analyses were also conducted to validate our findings. The result showed that hydrophobic and electrostatic interactions are the dominant forces for binding. DX-814 had a similar binding orientation with BCMA in a conserved hydrophobic pocket and formed electrostatic interaction with conserved arginine residues on the BAFF surface, compared with TA which might interact with a sub-pocket of BAFF in a different orientation. These results provide a thorough understanding of the binding mode between BAFF and its peptide inhibitors at the molecular level and further guide inhibitor design.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with tumor-selective apoptotic activity. Formation of aggregates as trimer is the prerequisite for TRAIL’s function as an apoptosis inducer. However the polymerization property of TRAIL has also brought difficulties for its production. RGD-TRAIL is an integrin-targeting TRAIL mutant with enhanced apoptosisinducing activity towards tumor cells both in vitro and in vivo. When expressed in E. coli, TRAIL or its mutant RGDTRAIL usually formed inclusion bodies. Their extreme aggregation propensity for aggregation destabilizes the protein, leading to poor recovery and therefore low yield from the purification process. The low purification efficiency of TRAIL retards its industrial application and large-scale production. To avoid the above problems during RGD-TRAIL production, we employed elastin-like polypeptides (ELPs) for the fusion-expression of recombinant RGD-TRAIL. Recombinant RGD-TRAIL-ELP was expressed in a soluble form and efficiently purified from the clarified cell extracts by three rounds of inverse transition cycling (ITC). SDS–PAGE and Western blotting analyses of purified RGD-TRAIL-ELP showed that RGD-TRAIL-ELP was successfully purified and the yield was up to 10 mg/L of bacterial culture. Apoptosis assay was performed in human colorectal carcinoma cells (COLO-205) and human breast cancer cell line (MDA-MB-231) to assess the potency of the fusion protein. Fusion with hydrophobic ELP effectively enhanced RGD-TRAIL’s biological activity. The higher activity and appropriate particle size of RGD-TRAIL-ELP could be used for RGD-TRAIL delivery in tumor therapy.

Pediocin PA-1 is a well-known Class IIa bacteriocin which shows strong inhibitory effect against Listeria monocytogenes. In this work, in order to improve the antimicrobial activity, eight single- site mutants and six combination mutants on nine interesting sites in the C-terminal region of pediocin PA-1 were constructed and expressed in Escherichia coli heterologously. Ten mutants demonstrated enhancement activity when performed the agar diffusion test to the indicator strain L. monocytogenes. The substitution of glycine in position 29 to alanine showed the most distinct increase of antimicrobial activity which clarified that 29G acted as a significant role as to guide the pediocin PA-1 molecule to dip into receptor membrane. Combination mutants of sites 29G to 32A illustrated that a hydrophobic tip of the hairpin-like structure and a smaller bundle of the α-helix domain facilitate the penetrating of pediocin PA-1 into a hydrophobic domain of the membrane-embedded subunits of the mannose-phosphotransferase system (MPTs).

A de novo protein named as EAF (Escherichia antifungal protein) from the cytoplasmic pool of an Escherichia coli strain (MTCC 1652), has been purified to homogeneity using anion exchange (Q-XL Sepharose) and cation exchange (SP-Sepharose) chromatography. The MIC (minimum inhibitory concentration) values of purified protein against A. fumigatus (the major pathogenic species) were found to be comparable with standard drugs i.e. 3.90 μg/ml, 3.90 μg/ml and 1.25 μg/disc via microbroth dilution assay (MDA), percentage spore germination inhibition (PSGI) and disc diffusion assay (DDA) respectively. Toxicity results confirmed that it causes no haemolysis against human RBCs upto a concentration of 1000.0 μg/ml as compared to Amphotericin B (conventional antifungal drug) that causes hundred percent haemolysis at a concentration of 37.50 μg/ml only.The purified protein demonstrated a molecular mass of 28 kDa on SDS-PAGE which was further authenticated by MALDI-TOF. Proteomic and bioinformatics studies deciphered its significant homology (72 %) with chain A-D-ribose binding protein (cluster 2 sugar binding periplasmic proteins; sequence homologues of transcription regulatory proteins) from E. coli. Single dimensional page analysis of A. fumigatusproteins with due effect of EAF (at MIC50) revealed the inhibition of two major proteins; a heat shock protein 70-Hsp70 (68 kDa); having role in protein folding and functioning andphenylanalyl-t RNA synthetase PodG subunit protein (74 kDa); involved in growth polarity in fungi. Scanning electron microscopic studies depicted homologous results. We suggest that EAF most likely belongs to a new group of proteins with potent antifungal characteristics, negligible toxicity and targeting vital proteins of fungal metabolism.

We previously reported that neostatin, a proteolytic fragment of collagen XVIII that includes endostatin, inhibits basic fibroblast growth factor-induced corneal angiogenesis and lymphangiogenesis. In experiments to determine which fragments in neostatin are responsible for binding to VEGF receptors (VEGFRs), we previously showed that a 28- mer sequence at the C-terminal of endostatin, known as endostatin peptide 9, preferentially binds VEGFR3-Fc over VEGFR1-Fc and VEGFR2-Fc. In the present study, we show that a different endostatin fragment, endostatin peptide 4 (26 mers long), also selectively binds VEGFR3-Fc and not VEGFR1-Fc or VEGFR2-Fc. From surface plasmon resonance data, the KD and Chi2 (RU2) values for endostatin peptide 4 binding to VEGFR3-Fc are 5.72x10-8 M and 0.354, respectively. In conclusion, endostatin peptides 4 and 9 may be responsible for endostatin binding to VEGFR3-Fc, and this improved understanding of endostatin peptide binding to VEGFR3-Fc may support the development of therapeutics targeting lymphangiogenic processes.

In this work, results of studies on the interactions of CopC and its mutants with the cationic surfactant Cetyltrimethyl Ammonium Bromide (CTAB) monitored by fluorescence spectroscopy, far- UV circular dichroism (CD) and fluorescence lifetime have been presented and discussed. The interaction of CTAB with CopC leads to stabilize a second intermediate, which is considered that the structure of C-terminal was unfolded completely and the structure of the N-terminal was partially unfolded. Notably, GdnHCl leads to refolding of the second intermediate and form the first intermediate, which has also been found on unfolding of CopC by sodium dodecyl sulfate (SDS). The refolding of a protein by GdnHCl in the presence of CTAB was reported. The results suggested that the formation of micelle-like aggregates interaction with the protein results in formation of intermediates of apoCopC and the effect of GdnHCl on micelle-like aggregates result in the folding of the second intermediate.

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.