Protein and Peptide Letters - Volume 24, Issue 8, 2017

Over the last years, a novel class of anti-cancer drugs named antibody-drug conjugates (ADCs) has been developed. Due to their limited off-target toxicity but highly potent cytotoxicity at tumor sites, ADCs have proven to be a good alternative to ordinary cancer treatment, such as chemotherapy or combination therapy. Numerous enhancements in antibody-drug engineering led to highly potent tumor targeting drugs with a wide therapeutic window. Two ADCs (Brentuximab vedotin and Trastuzumab emtansine) are already on the market and many others are in clinical trials. However, unstable linkers, low drug potency and unwanted bystander-effects are only some of the drawbacks of ADCs. Enzymes used in combination with prodrugs happen to be a promising alternative. The glyco-enzyme horseradish peroxidase (HRP) has proven to activate the hormone indole-3-acetic acid (IAA) to a highly potent cytotoxic drug. This combination of IAA and HRP has been investigated for the use in strategies such as gene-directed enzyme prodrug therapy (GDEPT) and antibody-directed enzyme prodrug therapy (ADEPT). This article reviews the current state of research in ADC engineering and describes the potential major enhancements through use of glycoenzymes in combination with a prodrug.

Glycoside phosphorylases catalyse the reversible synthesis of glycosidic bonds by glycosylation with concomitant release of inorganic phosphate. The equilibrium position of such reactions can render them of limited synthetic utility, unless coupled with a secondary enzymatic step where the reaction lies heavily in favour of product. This article surveys recent works on the combined use of glycan phosphorylases with other enzymes to achieve synthetically useful processes.

Background: Peptide: N-glycanase is a deglycosylation enzyme releasing N-glycan from glycoproteins. Although glycan specificity analysis of this enzyme has been reported, recognition requirements for the peptide sequence have not been precisely elucidated. Objective: In this study, we carried out peptide specificity analysis of several peptide:N-glycanases. Methods: Using synthetic chitobiose-pentapeptide substrates having a systematic series of amino acid sequences composed of hydrophobic leucine and hydrophilic serine, we examined the peptide specificities of peptide: N-glycanases comprising yeast cytoplasmic PNGase, bacterial PNGase F, and plant PNGase A by ultra-performance liquid chromatography combined with electrospray ionization mass spectrometry. Results: We found that each of the PNGases had higher activity for the more hydrophobic (leucinerich) chitobiose-pentapeptides, although the sensitivities of the PNGases for hydrophobicity varied. Cytoplasmic PNGase showed broad specificity. In contrast, PNGase A showed moderate specificity. PNGase F showed the highest specificity. Conclusion: PNGases from different origins had similar but significantly independent peptide specificities.

Background: The biocatalytic oxidation of UDP-glucose in the presence of NAD+ is catalyzed by UDP-glucose dehydrogenases. Objectives: The main objective of this study was the characterization of a UDP-glucose dehydrogenase (AmUGD) from Akkermansia muciniphila, a bacterium originally isolated from human faeces in an anaerobic medium containing gastric mucin as the sole carbon source. Methods: The biochemical analysis of AmUGD was performed using a plate reader-based assay measuring the reaction by-product NADH. Furthermore, HPLC- and MALDI-ToF-MS- based methods were used for the enzyme characterization. Results: The recombinant form of the protein was expressed in E. coli and the purified enzyme exhibited optimum levels of activity at 37°C and pH 9.0. While the enzyme is active in the absence of metal ions, the presence of Zn2+ ions results in markedly enhanced levels of catalysis. Conclusion: This study describes the first characterization of a nucleotide-processing enzyme from A. muciniphila. The ease of expression and purification of this enzyme make it ideal for biotechnological applications such as the enzymatic synthesis of nucleotide sugars, which may in turn be used for the synthesis of complex carbohydrates or glycoconjugates.

Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology - particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. Objectives: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. Conclusion: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label-free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.

Background: Candida species infections are an important worldwide health issue since they do not only affect immunocompromised patients but also healthy individuals. The host developed different mechanisms of protection against Candida infections; specifically the immune system and the innate immune response are the first line of defence. Defensis are a group of antimicrobial peptides, components of the innate immunity, produced at mucosal level and known to be active against bacteria, virus but also fungi. Objectives: The aim of the current work was to review all previous studies in literature that analysed defensins in the context of Candida spp. infections, in order to investigate and clarify the exact mechanisms of defensins anti-fungal action. Methods: Several studies were identified from 1985 to 2017 (9 works form years 1985 to 1999, 44 works ranging from 2000 to 2009 and 35 from 2010 to 2017) searched in two electronic databases (PubMed and Google Scholar). The main key words used for the research were “Candida”, “Defensins”,“ Innate immune system”,“fungi”. Results and conclusion: The findings of the reviewed studies highlight the pivotal role of defensins antimicrobial peptides in the immune response against Candida infections, since they are able to discriminate host cell from fungi: defensins are able to recognize the pathogens cell wall (different in composition from the human ones), and to disrupt it through membrane permeabilization. However, further research is needed to explain completely defensins' mechanisms of action to fight C. albicans (and other Candida spp.) infections, being the information fragmentary and only in part elucidated.

Objective: In our previous study, we showed that Bacillus Calmette-Guerin (BCG)- activated macrophages have the ability to directly kill tumor cells. One of the main properties of these macrophages is the high expression of tripartite motif family protein 59 (TRIM59). This study was conducted to investigate the mechanism of BCG-induced TRIM59 expression on macrophages and to identify the subcellular localization of TRIM59. Methods: TRIM59 expression and TNF-α secretion were compared in RAW264.7 macrophage cells that were stimulated using BCG with or without Toll-like receptor 2/4 (TLR2/4)-neutralizing antibodies. Next, small interfering RNA (siRNA) was used to down-regulated interferon regulatory factor 5 (IRF5) gene expression in RAW264.7 cells. Transfected cells were stimulated with BCG, after which TRIM59 expression and TNF-α secretion were evaluated in cells pre-treated with siRNA or scramble control. After treatments, supernatants were co-cultured with MCA207, and cell viabilities were determined. Moreover, BCG-stimulated RAW264.7 cells were stained for TRIM59 and F4/80 expression. Results: In this study, we showed that TRIM59 was expressed on the membrane of RAW264.7 cells. After blocking TLR2/4, treatment with BCG failed to induce the expression of TRIM59, IRF5, and TNF-α on RAW264.7 cells. In addition, down-regulation of IRF5 inhibited TRIM59 and TNF-α expression. Conclusion: Our study showed that TRIM59 is a membrane protein, and that BCG treatment upregulated TRIM59 expression on macrophages via TLR2/4 and IRF5 pathways.