Transferrin Molecules Research Articles

Transcytosis-Driven Treatment of Neurodegenerative Disorders by mRNA-Expressed Antibody-Transferrin Conjugates.

The recent setbacks in the withdrawal and approval delays of antibody treatments of neurodegenerative disorders (NDs), attributed to their poor entry across the blood-brain barrier (BBB), emphasize the need to bring novel approaches to enhance the entry across the BBB. One such approach is conjugating the antibodies that bind brain proteins responsible for NDs with the transferrin molecule. This glycoprotein transports iron into cells, connecting with the transferrin receptors (TfRs), piggybacking an antibody-transferrin complex that can subsequently release the antibody in the brain or stay connected while letting the antibody bind. This process increases the concentration of antibodies in the brain, enhancing therapeutic efficacy with targeted delivery and minimum systemic side effects. Currently, this approach is experimented with using drug-transferring conjugates assembled in vitro. Still, a more efficient and safer alternative is to express the conjugate using mRNA technology, as detailed in this paper. This approach will expedite safer discoveries that can be made available at a much lower cost than the recombinant process with in vitro conjugation. Most importantly, the recommendations made in this paper may save the antibodies against the NDs that seem to be failing despite their regulatory approvals.

Tailoring lipid nanoparticles for T-cell targeting in allergic asthma: Insights into efficacy and specificity

Asthma impacts over 300 million patients globally, with significant health implications, especially in cases of its allergic subtype. The disease is characterized by a complex interplay of airway inflammation and immune responses, often mediated by Th2 cell-related cytokines. In this study, we engineered lipid nanoparticles (LNPs) to specifically deliver therapeutic siRNA via the transferrin receptor to T cells. Strain-promoted azide-alkyne cycloaddition (SPAAC) was employed for the conjugation of transferrin ligands to PEGylated lipids in the LNPs, with the goal of enhancing cellular uptake and gene knockdown. The obtained LNPs exhibited characteristics that make them suitable for pulmonary delivery. Using methods such as nanoparticle tracking analysis (NTA) and enzyme-linked immunosorbent assay (ELISA), we determined the average number of transferrin molecules bound to individual LNPs. Additionally, we found that cellular uptake was ligand-dependent, achieving a GATA3 knockdown of more than 50% in relevant in vitro and ex vivo models. Notably, our findings highlight the limitations inherent to modifying the surface of LNPs, particularly with regard to their targeting capabilities. This work paves the way for future research aimed at optimizing targeted LNPs for the treatment of immunologic diseases such as allergic asthma.

Astrocytes and microglia-targeted Danshensu liposomes enhance the therapeutic effects on cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury (CI/RI) is the main cause of disability and death in stroke without satisfactory therapeutic effect. Inflammation mediated by activation of astrocytes and microglia is the main pathological mechanism of CI/RI. Danshensu (DSS) has been shown to exert anti-inflammatory effects against brain injury. However, limited by its poor cellular permeability and low bioavailability, it is still needed the new DSS preparations with the ability to cross the blood–brain barrier (BBB) and target inflammatory glial cells. In this study, we developed phosphatidylserine (PS) and transferrin (TF) modified liposomes carrying DSS (TF/PS/DSS-LPs) to improve the therapeutic efficacy against ischemic stroke. First, TF molecules targeted transferrin receptor (TfR) that is overexpressed in the BBB. Following the liposomes enter the brain, PS modification allowed the liposomes to target and bind to the overexpressed phosphatidylserine-specific receptors (PSRs) on the surface of astrocytes and microglia. Furthermore, it enhanced the uptake of TF/PS/DSS-LPs by astrocytes and microglia, while polarizing astrocytes from A1 to A2 and microglia from M1 to M2, reducing neuronal inflammation, and ultimately ameliorating cerebral ischemic injury. Thus, TF/PS/DSS-LPs could potentially serve as a promising strategy for the CI/RI treatment.

Immunoaffinity silica nanoparticles as efficient nanoprobes for selectively capturing transferrin molecules

In this study, silica nanoparticles were synthesized via the well-known Stöber method and subsequently decorated with amino functional groups before the immobilization of recognizing element, the anti-transferrin antibody. Antibody immobilization was achieved by following N-ethyl-N’-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) activation/coupling process while preventing protein denaturation as well as losing recognizing capability. After comprehensive characterization methods via scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDAX), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and zeta size/potential measurements, the factors affecting on target transferrin molecules were optimized by varying initial concentration, temperature, interaction time, and pH. According to the results, the size of silica nanoparticles was determined at around 100 nm in the coherency of SEM, TEM, and zeta size measurement. The surface modification and success of the antibody immobilization process were also confirmed with EDAX, zeta potential, and FTIR analyses. The amount of anti-transferrin antibody immobilized on the nanoparticles was calculated as 52.64 mg/g silica nanoparticles. The optimum conditions for transferrin recognition were found as 37 °C, 10 min, and 7.4 according to the parameters as temperature, interaction time, and pH, respectively. Meanwhile, the transferrin binding capacity increased from 125.00 mg/g to 686.54 mg/g when the initial concentration increased from 31.25 μg/mL to 250 μg/mL. In the end, the selectivity, in other word interfering effect of competing biomolecules, were confirmed against albumin, gamma-globulin, and ovalbumin. In the light of the results, not only the easy synthesis/modification route but also the efficient detection performance of the silica-based nanoprobe figured out a promising potential.

Investigating the interaction of CdTe quantum dots with plasma protein transferrin and their interacting consequences at the molecular and cellular level

This study investigated the interacting mechanism of CdTe quantum dots (QDs) with typical plasma protein transferrin (TF) as well as the impact of the formation of QDs-TF complex on the structure of TF and the cytotoxicity of mouse primary kidney cells. Dialysis experiments and cell viability assays revealed that the formation of QDs-TF complex reduced the contents of Cd released from CdTe QDs and thus counteracted the cytotoxicity of CdTe QDs. The assay of isothermal titration calorimetry found that CdTe QDs complexed with TF majorly through hydrophobic interaction. Multi-spectroscopic measurements showed that CdTe QDs caused the loosening of polypeptide chain, the changes of secondary and tertiary structures as well as the attenuated aggregation of TF molecule. Moreover, these structural and conformational changes were attributed to the nano-effects of CdTe QDs rather than the released Cd. This study is of great significance for fully evaluating the biocompatibility of Cd-QDs and comprehensively understanding the mechanism of Cd-QDs toxicity at the molecular and cellular level.

The many faces of transferrin: Does genotype modulate immune response?

In this study, the expression of pro-inflammatory and iron metabolism genes were analysed under Trypanoplasma borreli (T. borreli) challenge in common carp. Three transferrin (Tf) genotypic groups: two homozygous - DD, GG, and heterozygous DG were intraperitoneally infected with a dose of 2.16 × 105/100 μL parasites. Organ and blood samples were collected at weekly intervals. During the infection period, mortality and parasitaemia were assessed along with measurements of blood iron concentrations and antibody levels. Expression of Tf, Fer, IRP1 and 2, TfR 1a and 1b, Hep, TNF α1 and α2, and IL-1 β was measured in the peak of parasitaemia and the week preceding the peak.Study revealed, that changes in iron blood level induced by parasite were not correlated with the activities of iron homeostasis genes. Neither iron content nor the specific antibody response correlated with survival. We demonstrate that challenged carp, display three distinct, Tf genotype dependent activity patterns of iron homeostasis genes expression. The expected, “classical” way of up-regulation represented homozygous DD individuals. In contrast, GG individuals demonstrated downward trend, while gene expressions of heterozygous DG carp could be defined as an intermediate. We speculate, whether this phenomenon is related to the transferrin molecule itself or to Tf-genotypes being markers of other factors, that influence the iron homeostasis genes activities. We discussed the role of alarmins in triggering the immune response. Distinct genes activating patterns of homozygous genotypes DD and GG had no consequences in terms of mortality rates caused by T.borreli. The highest mortality was observed in the heterozygous group DG.In conclusion, this study suggest that transferrin variant, but not iron blood concentration, has a significant impact on carp immune response to blood parasite infection. This research sheds a new light on the inflammation process and interaction between a host and invaders.

High‐resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids

High-resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid, and serum treated with neuraminidase and N-glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo-transferrin, monosialo-transferrin, and disialo-transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High-resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo-transferrin, monosialo-transferrin and disialo-transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N-glycans of varying structure. Undersialylated disialo-transferrin is also observed in sera with high levels of trisialo-transferrin.

Off-Resonance SERS Nanoprobe-Targeted Screen of Biomarkers for Antigens Recognition of Bladder Normal and Aggressive Cancer Cells.

The discovery of different binding receptors to allow rapid and high-sensitivity detection via a noninvasive urine test has become the goal for urothelial carcinoma (UC) diagnosis and surveillance. In this study, we developed a new screening membrane receptor platform for bladder cancer cells by integrating surface-enhanced Raman spectroscopy (SERS) with 4-aminothiophenol (4-ATP)-modified AuAg nanohollows upon NIR laser excitation. AuAg nanohollows have an absorption band at ∼630 nm, and slightly off-resonance 785 nm laser excitation is used for minimal photothermal effect. Using the same carbodiimide cross-linker chemistry to conjugate anti-EGFR, transferrin (TF), 4-carboxyphenylboronic acid (CPBA), folic acid (FA), and hyaluronic acid (HA) molecules, by screening the 4-ATP SERS signals intensity, we demonstrated that the targeting efficiency with the cost-effective CPBA molecule is comparable with the conjugation of anti-EGFR antibody to aggressive T24 cancer cells (high-grade), while weak intensity 4-ATP SERS responses to targets were obtained by grade-I RT4 bladder cancer cells, NIH/3T3 fibroblast cells, and SV-HUC1 bladder normal cells. This SERS nanoprobe platform makes primary bladder carcinoma screening from in vitro to ex vivo more straightforward. Our demonstration offers exciting potential for SERS screening of specific receptors on cancer cells of different grades and facilitates new opportunities ranging from surface engineering of SERS material tags to SERS imaging-guided and targeted phototherapy of cancer cells by controlling the laser powers.

Abstract 3694: Measuring oncogene signaling with transferrin-based PET: From bench to bedside

Abstract Background: Non-invasive tools that measure the activity of central oncogenes could be broadly useful for cancer detection and management. We have shown that radiolabeled transferrin molecules can be used to measure mTORC1 and MYC signaling in prostate cancer models, because the transferrin receptor is a MYC target gene. The purpose of this study was to complete the preclinical assessment of 89Zr-transferrin, as well as demonstrate that clinical disease harbors avidity for transferrin using 68Ga-citrate. Methods: Human prostate cancer cell lines have been manipulated pharmacologically and genetically to alter mTORC1 and/or MYC signaling, and radiolabeled transferrin has been used to study the impact of aberrant oncogene signaling on transferrin biology. A first in man study with 68Ga-transferrin PET was also performed at UCSF using a GE SIGNA PET/MR. A dose escalation study was performed in eight patients with castration resistant prostate cancer to identify the optimal signal to noise ratio. Results: Activation of mTORC1 or MYC resulted in higher transferrin receptor expression and higher transferrin uptake into cells, as expected. Pharmacological inhibition of either oncogene suppressed transferrin uptake in vitro and in vivo. 68Ga-transferrin PET/MR resolved ~75% of lesions that were detectable by CT or bone scan. Approximately 4 mCi and >3.5 hours post injection was required to visualize tumor lesions. Conclusions: These experiments establish that like MYC, mTOR activity can be quantified in prostate cancer models with 89Zr-transferrin. Moreover, our first in human study with 68Ga-transferrin shows that human disease is generally avid for the radiotracer, but the uptake is variable among lesions, consistent with a molecularly diverse disease. Citation Format: Michael J. Evans, Charles Truillet, Davide Ruggero, Jason S. Lewis, Rahul Aggarwal, Spencer C. Behr. Measuring oncogene signaling with transferrin-based PET: From bench to bedside [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3694.

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.

Retargeting Polyomavirus-Like Particles to Cancer Cells by Chemical Modification of Capsid Surface.

Virus-like particles based on polyomaviruses (PVLPs) are promising delivery devices for various cargoes, including nucleic acids, imaging probes, and therapeutic agents. In biological environments, the major coat protein VP1 interacts with ubiquitously distributed sialic acid residues, and therefore PVLPs show a broad tropism. For selective targeting, appropriate engineering of the PVLP surface is needed. Here, we describe a chemical approach to retarget PVLPs to cancer cells displaying abnormally high levels of transferrin receptor. We created an array of transferrin molecules on the surface of PVLPs by combining a high-yielding bioconjugation approach with specific point modification of transferrin. This artificial surface protein architecture enables (i) suppression of natural VP1-specific interactions by blocking the surface conformational epitope on the VP1 protein, (ii) unusually high cellular uptake efficiency, and (iii) selective retargeting of PVLPs to osteosarcoma (U2OS) and lymphoblastoid leukemia (CCRF-CEM) cells.

Congenital Hypotransferrinemia, an Unusual Cause of Iron Deficiency Anemia: Report of Two Cases.

Hereditary hypotransferrinemia is a very rare cause of iron deficiency anemia in childhood characterized by microcytic hypochromic anemia refractory to iron therapy and concomitant iron overload. Regular plasma infusion to replace the deficient transferrin molecule is the therapeutic option. We report two cases; both presented with refractory anemia requiring blood transfusions and responded to monthly fresh frozen plasma replacement.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.

Transferrin variants: Pitfalls in the diagnostics of Congenital disorders of glycosylation

ObjectivesTransferrin variants can hinder the diagnostic process in cases of suspected Congenital disorders of glycosylation which affect N-Glycosylation. In addition they can impair the use of Carbohydrate deficient Transferrin as a biomarker for chronic alcohol abuse, in which Asialo-Transferrin and Disialo-Transferrin are increased. We present a novel transferrin variant as well as an overview of transferrin mutations found at our laboratory. Design and methodsBlood samples from patients with suspected CDG were analyzed using the standard diagnostic procedures of Isoelectric focusing and High-performance liquid chromatography as well as the additional procedures of neuraminidase digestion of glycans and Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). ResultsFour known and one previously unreported transferrin variants were identified. Neuraminidase digestion and ESI-TOF MS revealed changes in charge of the transferrin molecules while the glycosylation status was found to be normal. ConclusionTransferrin variants are pitfalls in the diagnostics of CDG. The found variants change the charge of the transferrin molecule, thus affecting the standard diagnostic procedures. Neuraminidase digestion as well as ESI-TOF MS can identify variants and mutations in a laboratory context.

The novel transferrin E592A variant impairs the diagnostics of congenital disorders of glycosylation

BackgroundThe analysis of serum transferrin either by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) is the standard diagnostic procedure in patients with the suspicion of a congenital disorder of glycosylation (CDG). Carbohydrate-deficient transferrin (CDT) is also analysed in monitoring programmes in cases of alcohol abuse. We report a novel transferrin variant that impairs the analysis using conventional methods and propose alternative forms of analysis. MethodsTransferrin samples were analysed using HPLC, immunoprecipitation followed by SDS-PAGE and IEF. Neuraminidase treatment followed by conventional IEF and electrospray ionization time of flight mass spectrometry (ESI-TOF MS) were applied before sequencing of the transferrin gene was performed. ResultsThe novel transferrin variant E592A, found both in homozygous and heterozygous form, causes an altered charge of the transferrin molecule, which changes the results of IEF and HPLC and mimics an increase in trisialo-transferrin. The change in charge can be detected either by neuraminidase digestion followed by IEF or by ESI-TOF MS. ConclusionConventional diagnostic methods for CDG are hindered by the novel transferrin E592A. Neuraminidase treatment followed by IEF and ESI-TOF MS can identify the mutation. The mutation appears to be functionally normal.

Scara5 involvement in retinal iron metabolism

AbstractPurpose Although transferrin is the principal source of iron in mammalian cells, recently, ferritin was proposed as a new iron transporter protein. Serum ferritin is composed mostly, but not exclusively, of L‐ferritin, and in the presence of even a single H‐ferritin chain, up to 4500 atoms of iron can be incorporated into one ferritin molecule. As each transferrin molecule only binds to two iron atoms, serum ferritin can be regarded as a potential deliverer of a considerable large amount of iron to tissues. Iron implication in oxidative damage has become clear, and it is well known that iron accumulation is associated in the retina with several retinopathies.Methods Scara5 and L‐ferritin expression in the retina were studied by means of laser confocal microscopy, rt‐PCR and Western Blotting in ICR adult mice and in a murine model of retinopathy. Horse spleen ferritin was intravenously injected in healthy ICR adult mice and alteration in iron handling proteins expression was analyzed.Results Scara5 receptors, that specifically bind to L‐ferritin, were found in retinal endothelial and perivascular cells. Intravenous injected ferritin crossed the blood‐retinal barrier (BRB), through Scara5 receptor binding, and accumulated in these cells, suggesting that serum ferritin, mostly composed by L‐ferritin, can be transported across the BRB into the retinal parenchyma. During retinopathy, alterations in L‐ferritin levels and Scara5 expression were evaluated.Conclusion Serum ferritin uptake could represent a new pathway of iron delivery to the retina and points out perivascular cells as a key element in retinal iron traffic and during retinal iron accumulation associated with some retinopathies.

Fast 3D Single Molecule Tracking with Multifocal Plane Microscopy in Polarized Epithelia Reveals a Novel Cellular Process of Intercellular Transfer

The study of intracellular trafficking processes represents a fundamental problem in many areas of biomedical research. Single molecule imaging approaches are well suited to study heterogeneous processes in live cells. However, 3D single molecule imaging of intracellular trafficking events in a thick sample such as an epithelial-cell monolayer poses several technical challenges. Specifically, we require a methodology that not only enables fast 3D tracking of single molecules across a cell monolayer, but also enables the imaging of the cellular environment with which the single molecule interacts. Current approaches that are widely used for 3D single molecule imaging/tracking are not well suited for studying the intracellular trafficking pathways due to restricted imaging depth, poor temporal resolution, and the ability to track only a few molecules at one time. Here we show that multifocal plane microscopy (MUM), a 3D imaging modality developed by our group, provides the much needed solution to this longstanding problem by demonstrating fast 3D tracking of quantum dot labeled transferrin molecules in a ∼10 micron thick epithelial cell monolayer.The use of MUM led to the unexpected discovery of a novel cellular process, intercellular transfer, that involves the rapid exchange of Tf molecules between two adjacent cells in the monolayer. We also report 3D single molecule tracking of endocytosis and exocytosis at the lateral plasma membrane of cells in the monolayer. This lateral membrane has been notoriously difficult to image with other cellular imaging modalities. A detailed characterization of these events based on the temporal and 3D intracellular spatial behavior of Tf molecules has been made. The methods and approaches used in this study have broad applicability to investigate 3D trafficking pathways in other cell systems and models.

Chemical coupling as a potent strategy for preparation of targeted bacteriophage‐derived gene nanocarriers into eukaryotic cells

The ability to direct efficiently and specifically carriers toward target cells and express the transgene of interest is a critical step in gene therapy trails. The display of targeting molecules on the surface of phage particles might represent a potent solution. In the present study, we evaluated a chemical coupling strategy for displaying human holotransferrin as a targeting molecule on the surface of phage lambda particles for specifically delivering green fluorescent protein (GFP) encoding gene into a human cell line. Human holotransferrin was coupled on the phage lambda particles bearing a GFP-expression cassette by a chemical coupling strategy to formulate transferrin-targeted lambda-GFP (Tf-targeted-λ-GFP) gene nanocarrier. The carrier was then characterized by phage-enzyme-linked immunosorbent assay experiments and used for transfection of the human 293T cell line. Particle internalization into the cells was evaluated by immunocytochemical staining and transfection efficacy was studied using fluorescence-activated cell sorting (FACS) analysis. Characterization of the nanocarrier showed a rather high copy number (274 molecules) of transferrin molecules coupled per phage particle. Immunocytochemical staining revealed efficient internalization of the Tf-targeted-λ-GFP compared to wild lambda-GFP (λ-GFP) particles. FACS analysis showed 6.72% GFP positive cells for transfections mediated by Tf-targeted-λ-GFP, whereas the value was 0.61% for wild lambda-GFP particles. Our findings highlight chemical coupling as an efficient and straightforward strategy for displaying a targeting molecule at high density on the phage surface, which, in turn, may improve the efficiency of phage-mediated gene transfer and expression.

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH.

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.