Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Preservation of Cichoric Acid Antioxidant Properties Loaded in Heat Treated Lactoferrin Nanoparticles.

In the current research, a new cichoric acid (CA) encapsulation system was investigated. The optimal condition for the formation of lactoferrin-cichoric acid nanoparticles (LF-CA NPs) was determined by controlling the solution pH, the thermal treatment conditions, and the concentration of CA. Fluorescence indicated that the electrostatic force and the hydrophobic force were the main forces in the formation of LF-CA NPs. LF-CA NPs prepared under different conditions were spherical in shape with smaller particle sizes and good zeta potential demonstrating good colloidal stability. Especially, the prepared particle size of the LF-CA NPs at pH 7 and 95 °C was about 67.20 ± 1.86 nm. The circular dichroism (CD) and the Fourier transform infrared spectroscopy (FTIR) results showed that the combination of LF (lactoferrin) and CA affected the secondary structure of the LF. The differential scanning calorimetry (DSC) results indicated that the addition of CA increased the thermal stability of LF. In vitro antioxidant experiments confirmed the antioxidant capacity of LF-CA NPs was better than CA. CA was successfully encapsulated into LF NPs with high encapsulated efficiency (97.87⁻99.87%) by high performance liquid chromatography (HPLC). These results showed that LF could be used as the wall material of CA with excellent nature.

High-resolution atomic force microscopy visualization of metalloproteins and their complexes.

BACKGROUND: Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level. METHODS: In this study, the pairwise interactions of MPO, CP and LF molecules were investigated at a single-molecule level using high-resolution atomic force microscopy (AFM). Highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM) was used as a substrate for protein deposition. RESULTS: The procedure for reliable AFM investigation of metalloproteins and their complexes has been developed. Using this procedure, we have visualized, for the first time, single MPO, CP and LF molecules, characterized the morphology of MPO-CP and LF-CP complexes and confirmed the absence of direct contacts between MPO and LF molecules. Moreover, we have revealed the novel chainlike shape of MPO-CP conjugates. CONCLUSIONS: GM-HOPG was shown to be a convenient substrate for AFM investigation of metalloproteins and their complexes. Direct AFM visualization of MPO-CP and LF-CP complexes, on the one hand, complements previous data obtained from the "bulk techniques" and, on the other hand, provides new insight into the ultrastructure of MPO-CP complexes. GENERAL SIGNIFICANCE: The obtained results contribute to the better understanding of regulation of inflammation and oxidation stress mediated by collaborative action of the metalloproteins such as MPO, CP and LF.

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.

Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin.

The ability of laboratory and clinical strains of Porphyromonas gingivalis to bind lactoferrin has been assessed (FEMS Immunology and Medical Microbiology, 1996, 14, 135-143). Relative binding for P. gingivalis to lactoferrin varies among strains from 3.78 to 26.62%. We also observed that fimbriated strains of P. gingivalis bind more strongly to lactoferrin as compared to nonfimbriated strains of P. gingivalis. This observation led us to study fimbrial interaction with human lactoferrin and the fine structure of these interactions. Binding of iodinated purified fimbriae was studied using an overlay assay. Iodinated fimbriae bind specifically and strongly to human lactoferrin. When various sugars were used to inhibit binding, only N-acetylgalactosamine and fucose were inhibitory. To confirm further that oligosaccharide of lactoferrin is involved in the interaction, lactoferrin was chemically deglycosylated, and fimbriae failed to bind deglycosylated lactoferrin. Antifimbriae, as well as four antipeptide antibodies against different regions of the P. gingivalis fimbrillin, were used to inhibit the interaction. Antipeptide E, directed against amino acids 81-98 (AAGLIMTAEPKTIVLKAG-C), was found to be the most effective inhibitor for the lactoferrin-fimbriae interaction. These results suggest that the binding of P. gingivalis cells to lactoferrin is lectin like, directed to a oligosaccharide of lactoferrin. Furthermore, these studies suggest that the region of fimbriae that binds to lactoferrin is the N-terminus of the molecule. It is likely that binding of lactoferrin to P. gingivalis cells results in antimicrobial activity directed against these cells by virtue of its ability to deprive the bacterial cell of needed iron.

Combined immunocytochemistry and enzyme cytochemistry on ultra-thin cryosections: a new method.

We combined immunocytochemistry and enzyme cytochemistry to localize two different proteins on the same ultrathin cryosection. In this method the immunocytochemical localization is visualized with colloidal gold probes and the enzyme cytochemical detection is achieved with cerium as the capture agent. The immunocytochemistry is conducted first so that any potential adverse effects of the enzyme cytochemical procedure will not alter the antibody binding properties of the cryosections.

Comparison of anticancer activity between lactoferrin nanoliposome and lactoferrin in Caco-2 cells in vitro.

The anticancer activities of Lactoferrin (Lf) and Lf nanoliposomes in Caco-2 cells were observed in this study, and mitochondrial function (MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygen species (ROS) and apoptosis induction (AO/EB staining) assays were used to evaluate the anticancer activity. MTT results demonstrated that Lf nanoliposomes and Lf reduced the mitochondrial activity of cells in a manner of dose and time effect, and the viabilities of Caco-2 cell were significantly decreased in vitro following exposure to Lf nanoliposomes at the concentrations of 5 and 10 mg/mL. LDH leakage and ROS significantly increased in cells exposed to Lf nanoliposomes (≥5 mg/mL), while Lf induced ROS only at higher doses (10mg/mL). CCK-8 evaluation of cell proliferation and AO/EB double staining supported the anti-proliferative effects of Lf liposomes. Our findings demonstrated that the presence of Lf nanoliposome is more significant than Lf in inhibiting human tumor cells proliferation. Therefore, it can be concluded that Lf nanoliposomes are a potential therapeutic modality in the management of tumors.

Size evolution of luminescent lactoferrin protected gold clusters.

Earlier, we have reported the synthesis and thorough physicochemical characterization of Au(QC)@NLf, where NLf refers to native lactoferrin. In this paper, we show how the clusters are formed in the protein cavity as a function of time. MALDI MS of the protein samples measured as a function of time of incubation show the evolution of the clusters. The mechanism of cluster growth suggests metal ion transfer across molecules which require further study.

Non-destructive visualisation of protective proteins in the in situ pellicle.

Several salivary anti-microbial and buffering components are part of the acquired in vivo pellicle. The purpose of the present in situ study was to visualise these proteins within the in situ formed pellicle and to investigate their distribution with respect to pellicle formation time and intra-oral localisation. Bovine enamel slabs were fixed on individual splints. They were carried by 6 subjects buccally and palatally in the region of the upper first molar teeth over 30 and 120 min, respectively, for in situ pellicle formation. After intra-oral exposure, enamel specimens were processed for transmission electron microscopy. Secretory immunoglobulin A (sIgA), lactoferrin, lysozyme, carbonic anhydrase (CA) I and II were visualised successfully in the in situ pellicle layer by gold immuno-labelling. All components were found to be distributed randomly within all layers of the pellicle. Significantly higher amounts of the proteins were detected after 120 min of formation time. Furthermore, significantly more labelled lactoferrin and lysozyme were found on buccal surfaces compared with palatal sites. For CA I, CA II and sIgA, no significant influence of the localisation was detected. All investigated anti-bacterial and buffering proteins are distributed randomly in the in situ formed pellicle layer and thus could contribute to its protective properties as an early defence barrier.

Properties of the iron-binding site of the N-terminal lobe of human and bovine lactotransferrins. Importance of the glycan moiety and of the non-covalent interactions between the N- and C-terminal lobes in the stability of the iron-binding site.

The recent determination by X-ray diffraction of the tridimensional structure of human lactotransferrin has underlined the presence of two lobes, each composed of two domains, I and II, as well as the involvement of five ligands in the binding of iron. Only one of the ligands (Asp-61) is located in domain I (residues 1-90 and 252-320), while the others [two tyrosine, one histidine and one (bi)carbonate ion linked to an arginine residue] belong to domain II (residues 91-251). On the basis of these data and of our previous results concerning the isolation of the 30 kDa N-tryptic fragment (residues 4-281) and the 20 kDa N2-glycopeptide (N-terminal domain II; residues 91-253) from human and bovine lactotransferrins, we have compared the iron-binding properties of these two fragments. The results demonstrate that Asp-61, which is missing from domain II, does not take part in the stability upon protonation of the iron complex of both human and bovine lactotransferrins. Furthermore, by comparing the iron-binding properties of human and bovine lactotransferrins to those of isolated 30 kDa N-tryptic and 50 kDa C-tryptic fragments and of the reassociated N,C-tryptic complex of both proteins, it has been shown that the non-covalent interactions which occurred between the two lobes of lactotransferrins and in the reassociated N,C-tryptic complex can explain in part the high affinity of lactotransferrins for iron. Finally, deglycosylation experiments on the 30 kDa N-tryptic fragment and N-terminal domain II from human and bovine lactotransferrins demonstrate that full removal of the glycan moiety leads to the loss of iron-binding capacity and so underlines the importance of the glycan moiety in the stability upon protonation of the N-terminal iron-binding site of both lactotransferrins.

In vitro characterization of lactoferrin aggregation and amyloid formation.

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.