Assessment of the Conformation Stability and Glycosylation Heterogeneity of Lactoferrin by Native Mass Spectrometry.

Lactoferrin (LTF) has diverse biological activities and is widely used in functional foods and active additives. Nevertheless, evaluating the proteoform heterogeneity, conformational stability, and activity of LTF remains challenging during its production and storage processes. In this study, we describe the implementation of native mass spectrometry (nMS), glycoproteomics, and an antimicrobial activity assay to assess the quality of LTF. We systematically characterize the purity, glycosylation heterogeneity, conformation, and thermal stability of LTF samples from different sources and transient high-temperature treatments by using nMS and glycoproteomics. Meanwhile, the nMS peak intensity and antimicrobial activity of LTF samples after heat treatment decreased significantly, and the two values were positively correlated. The nMS results provide essential molecular insights into the conformational stability and glycosylation heterogeneity of different LTF samples. Our results underscore the great potential of nMS for LTF quality control and activity evaluation in industrial production.

Assessment of Broad-Spectrum Antimicrobial, Antibiofilm, and Anticancer Potential of Lactoferrin Extracted from Camel Milk.

Lactoferrin is a multifunctional glycoprotein present in mammalian milk. It possesses antimicrobial, antioxidant, immunomodulatory, and several biological functions. Owing to the current trend of increasing antibiotic resistance, our study was designed to purify lactoferrin from camel milk colostrum using cation exchange chromatography on the SP-Sepharose high-performance column. The purity and molecular weight of lactoferrin were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The chromatogram of the purification procedure illustrated a single peak corresponding to lactoferrin, while the SDS-PAGE revealed 78 kDa molecular weight protein. Furthermore, lactoferrin protein and its hydrolysate form were assessed for its antimicrobial potential. The highest inhibitory effect of whole lactoferrin at the concentration (4 mg/ml) was observed against methicillin-resistant S. aureus (MRSA) and S. aureus, while 10 mg/ml concentration was effective against K. pneumonia, and 27 mg/ml was potent against multidrug-resistant (MDR) bacteria, P. aeruginosa. Likewise, MRSA was more sensitive toward iron-free lactoferrin (2 mg/ml) and hydrolyzed lactoferrin (6 mg/ml). The tested lactoferrin forms showed variability in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) among tested bacteria. The scanning electron microscopy (SEM) analysis images revealed distortions of the bacterial cells exposed to lactoferrin. The antibiofilm effect differed depending on the concentration and the type of the bacteria; biofilm inhibition ranged from 12.5 to 91.3% in the tested pathogenic bacteria. Moreover, the anticancer activity of lactoferrin forms exhibited a dose-dependent cytotoxicity against human lung cancer cell line (A549).

Secretory Expression of a Chimeric Peptide in Lactococcus lactis: Assessment of its Cytotoxic Activity and a Deep View on Its Interaction with Cell-Surface Glycosaminoglycans by Molecular Modeling.

Nowadays, cancer remains a major cause of death affecting millions of people. Currently, the antimicrobial peptides (AMPs) as potent anticancer therapeutic agents offer specificity and low levels of side effects in cancer therapy. In the present study, a cationic chimeric peptide (cLFchimera), derived from camel lactoferrin, was expressed as a secretory peptide using P170 expression system in L. lactis. Peptide purification was carried out using Ni-NTA agarose column from culture medium with 21 µ/mL concentration. The recombinant peptide was investigated for its activity against four tumor and one normal cell line. The cLFchimera was more active against two tumor cell lines (chondrosarcoma and colorectal cancer cells), but the activity against two other tumor cell lines (hepatoma and breast cancer cell line) and normal cells was low. Finally, to have better insight into the mode of action of the peptide on cytotoxic activity, we examined the interaction of cationic peptide with two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), as the two most anionic molecules on the cell surface by molecular dynamic simulation. The results of in silico analysis showed that the cLFchimera interacted with HS and CS with a totally different amino acid profile. Hydrogen bonding screening in GAGs-peptide complexes revealed K21, V23 and I3, R16 are the dominant amino acids involved in peptide-HS and CS interaction, respectively. Overall, the results of this investigation showed the P170 expression system successfully expressed a cationic peptide with potent anticancer activity. Moreover, molecular docking analysis revealed the pattern of peptide interaction with negatively charged membrane molecules.

Assessment of Binding Interaction between Bovine Lactoferrin and Tetracycline Hydrochloride: Multi-Spectroscopic Analyses and Molecular Modeling.

In this paper, the interaction between bovine lactoferrin (bLf) and tetracycline hydrochloride (TCH) was researched by microscale thermophoresis (MST), multi-spectroscopic methods, and molecular docking techniques. Normal fluorescence results showed that TCH effectively quenched the intrinsic fluorescence of bLf via static quenching. Moreover, MST confirmed that the combination force between bLf and TCH was very strong. Thermodynamic parameters and molecular docking further revealed that electrostatic forces, van der Waals, and hydrogen bonding forces played vital roles in the interaction between bLf and TCH. The binding distance and energy transfer efficiency between TCH and bLf were 2.81 nm and 0.053, respectively. Moreover, the results of circular dichroism spectra (CD), ultraviolet visible (UV-vis) absorption spectra, fluorescence Excitation-Emission Matrix (EEM) spectra, and molecular docking verified bLf indeed combined with TCH, and caused the changes of conformation of bLf. The influence of TCH on the functional changes of the protein was studied through the analysis of the change of the bLf surface hydrophobicity and research of the binding forces between bLf and iron ion. These results indicated that change in the structure and function of bLf were due to the interaction between bLf and TCH.

Silver decahedral nanoparticles empowered SPR imaging-SELEX for high throughput screening of aptamers with real-time assessment.

A highly efficient method for aptamer screening with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles (Ag10-NPs) enhanced surface plasmon resonance imaging (SPRI). A microarray chip was developed by immobilization of target protein (Lactoferrin (Lac)) and control proteins (α-lactalbumin (α), ß-lactoglobulin (ß), casein, and bovine serum albumin (BSA)) on the biochip surface. Ag10-NPs were conjugated with an ssDNA library (lib) (Ag10-NPs-library) that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence. Introduction of the Ag10-NPs-library to the SPRI flow channels drastically increased the sensitivity of SPRI signal for real-time monitoring of SELEX. The work allows rapid screening of potential targets, and yields nine aptamers with high affinity (nanomolar range) for Lac after only six-rounds of selection. The aptamer Lac 13-26 was then further tested by SPRI, and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac. The novel SPRI-SELEX method demonstrated here showed many advantages of real-time evaluation, high throughput, and high efficiency.

Balancing Accuracy and Cost of Confinement Simulations by Interpolation and Extrapolation of Confinement Energies.

Improvements to the confinement method for the calculation of conformational free energy differences are presented. By taking advantage of phase space overlap between simulations at different frequencies, significant gains in accuracy and speed are reached. The optimal frequency spacing for the simulations is obtained from extrapolations of the confinement energy, and relaxation time analysis is used to determine time steps, simulation lengths, and friction coefficients. At postprocessing, interpolation of confinement energies is used to significantly reduce discretization errors in the calculation of conformational free energies. The efficiency of this protocol is illustrated by applications to alanine n-peptides and lactoferricin. For the alanine-n-peptide, errors were reduced between 2- and 10-fold and sampling times between 8- and 67-fold, while for lactoferricin the long sampling times at low frequencies were reduced 10-100-fold.

Comprehensive assessment of milk composition in transgenic cloned cattle.

The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk.

Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

The blind search for the closed states of hinge-bending proteins.

The hinge-bending proteins provide the most pronounced example of the large-amplitude slow motions in a number of proteins, which are critical for their functioning. They are often used as the test ground for developing novel approaches to the simulation of slow protein dynamics. In the present study, we present the algorithm, which allows physically-consistent simulations of slow protein dynamics in globular proteins. Our algorithm is based on the hierarchical clustering of the correlation patterns (HCCP) technique of domain identification, which allows subdividing the protein into the hierarchy of the rigid-body-like clusters. The clusters are allowed to rotate relative to one another on the automatically identified hinges. The clusters are found in the course of automated, objective and well-tested procedure. In the present communication, our technique is applied to 10 hinge-bending proteins. For each of the proteins, we performed the blind search for the closed conformation, staring from the open one. Resulting closed conformations are compared with the closed states observed in crystallographic structures. It is shown that our technique produces realistic closed conformations for 8 out of 10 studied proteins. This demonstrates that HCCP technique can be used for finding alternative protein conformations and for sampling the slow motions in proteins.

Normal mode analysis of proteins: a comparison of rigid cluster modes with C(alpha) coarse graining.

The ability to infer dynamic motions from an equilibrium (static) conformation of a protein can be essential in establishing structure-function relationships. In particular, the low-frequency motions are of functional interest because statistical mechanics predicts these motions will have the largest amplitudes. In this paper, we address the computational cost of normal mode analysis (NMA) applied to a C(alpha)-based elastic network model (C(alpha)-NMA) and present a new coarse-grained rigid-body-based analysis (cluster-NMA). This new method represents a protein as a collection of rigid bodies interconnected with harmonic potentials. This representation produces reduced degree-of-freedom (DOF) equations of motion (EOMs) which, even in the case of large structures (10(3+) residues), enables the computation of normal modes to be done on a desktop PC. We present the complete theory and analysis of cluster-NMA and also include its application to a variety of structures. The results of the new method are compared with C(alpha)-NMA and it is shown that cluster-NMA produces very good approximations to the lowest modes at a fraction of the computational cost.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

Tertiary structural changes and iron release from human serum transferrin.

Iron release from human serum transferrin was investigated by comparison of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solution X-ray scattering (SAXS) for a series of equilibrated samples between pH 5.69 and 7.77. The phosphate buffers used in this study promote iron release at relatively high pH values, with an empirical pK of 6.9 for the convolved release from the two sites. The spectral data reveal that the N-lobe release is nearly complete by pH 7.0, while the C-lobe remains primarily metal-laden. Conversely, the radius of gyration, Rg, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of Rg between its value observed for the diferric protein at pH 7.77 (31.2+/-0.2 A) and that of the apo protein at pH 5.69 (33.9+/-0.4 A) exhibits an empirical pK of 6.6. While Rg is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, Rc, increases from 16.9+/-0.2 A to 17.6+/-0.2 A. Model simulations suggest that two different rotations of the NII domain relative to the NI domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of the iron-binding site, can be significantly advanced relative to their holo protein positions while yielding constant Rg and increased Rc values consistent with the scattering data. Rotation of the CII domain parallel with the C-lobe iron-binding site plane can partially account for the increased Rg values measured at low pH; however, no reasonable combined repositioning of the NII and CII domains yields the experimentally observed increase in Rg.