Francesco Giansanti

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86-258) and a small peptide (324-329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.

Physiological roles of ovotransferrin.

BACKGROUND Ovotransferrin is an iron-binding glycoprotein, found in avian egg white and in avian serum, belonging to the family of transferrin iron-binding glycoproteins. All transferrins show high sequence homology. In mammals are presents two different soluble glycoproteins with different functions: i) serum transferrin that is present in plasma and committed to iron transport and iron delivery to cells and ii) lactoferrin that is present in extracellular fluids and in specific granules of polymorphonuclear lymphocytes and committed to the so-called natural immunity. To the contrary, in birds, ovotransferrin remained the only soluble glycoprotein of the transferrin family present both in plasma and egg white. SCOPE OF REVIEW Substantial experimental evidences are summarized, illustrating the multiple physiological roles of ovotransferrin in an attempt to overcome the common belief that ovotransferrin is a protein dedicated only to iron transport and to iron withholding antibacterial activity. MAJOR CONCLUSIONS Similarly to the better known family member protein lactoferrin, ovotransferrin appears to be a multi-functional protein with a major role in avian natural immunity. GENERAL SIGNIFICANCE Biotechnological applications of ovotransferrin and ovotransferrin-related peptides could be considered in the near future, stimulating further research on this remarkable protein. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Avian Cytokines - An Overview

In recent years the knowledge of avian cytokines has advanced and new data are continuously added. Nevertheless, some discontinuities persist and the correlations between molecular and functional levels are not completely clear. Most of the studies are focused on chicken, and comparative aspects with other avian groups are limited. The existence of T1 and T3 avian cytokines was assessed long ago and the recent relevant demonstration of the existence of T2 cytokines in birds is a further step in depicting a more complete view on avian immunology. The progressive knowledge of avian cytokines can hopefully help in developing new strategies in prophylaxis and therapy of avian diseases, not always completely controlled due to the emergence of more pathogenic strains.

Lactoferrin from Milk: Nutraceutical and Pharmacological Properties

Lactoferrin is an iron-binding protein present in large quantities in colostrum and in breast milk, in external secretions and in polymorphonuclear leukocytes. Lactoferrin’s main function is non-immune protection. Among several protective activities shown by lactoferrin, those displayed by orally administered lactoferrin are: (i) antimicrobial activity, which has been presumed due to iron deprivation, but more recently attributed also to a specific interaction with the bacterial cell wall and extended to viruses and parasites; (ii) immunomodulatory activity, with a direct effect on the development of the immune system in the newborn, together with a specific antinflammatory effects; (iii) a more recently discovered anticancer activity. It is worth noting that most of the protective activities of lactoferrin have been found, sometimes to a greater extent, also in peptides derived from limited proteolysis of lactoferrin that could be generated after lactoferrin ingestion. Lactoferrin could therefore be considered an ideal nutraceutic product because of its relatively cheap production from bovine milk and of its widely recognized tolerance after ingestion, along with its well demonstrated protective activities. The most important protective activities shown by orally administered bovine lactoferrin are reviewed in this article.

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 μM and 0.03 s−1, respectively, the optimum pH value is 7.5 at 25 °C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys 416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.

Metal-induced self-assembly of peroxiredoxin as a tool for sorting ultrasmall gold nanoparticles into one-dimensional clusters†

Nanomanipulation of matter to create responsive, ordered materials still remains extremely challenging. Supramolecular chemistry has inspired new strategies by which such nanomaterials can be synthesized step by step by exploiting the self-recognition properties of molecules. In this work, the ring-shaped architecture of the 2-Cys peroxiredoxin I protein from Schistosoma mansoni, engineered to have metal ion-binding sites, is used as a template to build up 1D nanoscopic structures through metal-induced self-assembly. Chromatographic and microscopic analyses demonstrate the ability of the protein rings to stack directionally upon interaction with divalent metal ions and form well-defined nanotubes by exploiting the intrinsic recognition properties of the ring surfaces. Taking advantage of such behavior, the rings are then used to capture colloidal Ni(2+)-functionalized ultrasmall gold nanoparticles and arrange them into 1D arrays through stacking into peapod-like complexes. Finally, as the formation of such nano-peapods strictly depends on nanoparticle dimensions, the peroxiredoxin template is used as a colloidal cut-off device to sort by size the encapsulated nanoparticles. These results open up possibilities in developing Prx-based methods to synthesize new advanced functional materials.

Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ∼12–18 months post‐diagnosis. GBM usually recurs within 12 months post‐resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4‐Pseudomonas aeruginosa exotoxin A (IL4‐PE, NBI‐3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha‐TP‐38, IL13‐PE38, and a transferrin‐C diphtheriae toxin mutant (Tf‐CRM107). In this work, we studied the effects of the plant ribosome‐inactivating saporin and of its chimera transferrin–saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways. J. Cell. Physiol. 227: 939–951, 2012.

Antifungal and Antiparasitic Activities of Lactoferrin

The first function attributed to lactoferrin, an iron-binding protein belonging to the non-immune natural de- fences, was antimicrobial activity related to its capability of sequestering iron. Many other antimicrobial and antiviral functions have been ascribed to lactoferrin. In vitro activity towards human pathogenic fungi on the part of both human and bovine lactoferrin has been well documented as well. The antifungal activity appears to be related to lactoferrin inter- ference with the fungal cell surface rather than iron deprivation and some host-mediated mechanisms of action cannot be ruled out. Lactoferrin also displays anti-parasitic activity, although the molecular mechanisms of such activity are even more complex.

Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems

BackgroundAntibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.ResultsAn anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range.ConclusionsWe undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.

Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants

A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.