Comparative analysis of transferrin and IgG N-glycosylation in two human populations.

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans' role in their distinct physiological functions.

Purification of transferrin by magnetic nanoparticles and conjugation with cysteine capped gold nanoparticles for targeting diagnostic probes.

Transferrin is an iron binding glycoprotein actively involved in the growth and maintenance of cell cycle. The transferrin receptors expression is increased on growing cancer/tumor cells for absorption of iron through transferrin and participation in biological activity. In this study, a novel method for the purification of transferrin by using magnetic nanoparticles (MNP) is developed and compared with reported method. Magnetic nanoparticles were synthesized by co-precipitation method under hydrothermal conditions in the presence of ammonium hydroxide. MNP were characterized by FTIR, VSM, DLS, TEM, and SEM. Purified transferrin was characterized by SDS-PAGE, MALDI-TOF, ELISA, Western blot, and its activity was further confirmed by iron binding assay and receptor binding assays. Purified transferrin was also conjugated with cysteine capped gold nanoparticles (GNP) and characterized by UV-Vis spectra, TEM, DLS, and fluorescent spectrophotometry. Transferrin conjugated cysteine capped GNP used as a targeted fluorescent probe on gastric cancer, tumor tissue and MDA-MB 231 cancer cells to confirm transferrin receptor binding activity and application as diagnostic probe. The purified transferrin showed stability and activity up to 36 months. The results indicated that the synthesized superamagnetic MNP are good for the purification of transferrin. A good yield of transferrin was purified by this method, good quality and showed active biological activity. GNP conjugated transferrin has a potential to be used in cancer diagnosis as targeted diagnosis probe in vivo and in vitro. Experiments are underway for utilizing transferrin as carrier for targeting drug delivery.

Synthesis of penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) microsphere and its HPLC application in protein separation.

In the present study, the narrow-dispersed penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EDMA)) microspheres were successfully synthesized based on the sacrificial support method. The poly(MAA-co-EDMA) microspheres mirrored the porous structure of the sacrificial support, i.e. penetrable silica, characteristic of copious mesopores and throughpores. In addition, they possessed large surface area, adjustable hydrophobicity and the cation-exchange ability. Owing to their multi functionalities, they were applied as chromatographic stationary phase to separate proteins in different separation modes, including reversed phase, hydrophobic interaction and weak cation exchange. Moreover, thanks to their throughpores, fast separation at low column backpressure could be achieved in these three modes. Both protein recovery and column stability were satisfactory. The penetrable poly(MAA-co-EDMA) microspheres were potential stationary phase matrix for fast protein separation.

Purification, characterization and expression of transferrin from rainbow trout seminal plasma.

Transferrin (TF) is recognized as a multifunctional protein and has been implicated in antioxidative, antimicrobial protection, growth, differentiation and cytoprotection effects. An efficient, original three-step isolation procedure for TF consisting in hydrophobic interaction chromatography, gel filtration and preparative electrophoresis was developed. Rainbow trout TF was found to be N-glycosylated (not O-glycosylated) and phosphorylated at all serine, threonine, and tyrosine residues. The protein consists of several proteoforms with an average molecular weight of 76.9kDa and isoelectric point ranging from 5.2 to 5.7. Rainbow trout TF has two functional iron-binding sites and appears to be quite distinct from carp TF regarding glycosylation and iron-binding properties. The highest gene expression of TF was detected in liver and testis, the lowest was detected in head kidney, spleen and efferent ducts. For the first time TF was identified in the semen of several salmonid species. TF was localized within testis, mainly in spermatozoa, Sertoli, Leydig cells, as well as in both columnar secretory and basal cells within the efferent duct. This work contributes to the existing knowledge information indicating significant variations in TF structure within teleost fish. The results obtained in this study provide valuable data on the TF from trout seminal plasma and the physiological role of this protein in the reproductive tract of salmonids. The results are important for our understanding of the role of TF in the antioxidant protection and resistance to pathogenic infections of reproductive cells. The protective role of TF against environmental pollution with heavy metals, especially during prolonged storage of spermatozoa in the spermatic duct, as well as regulation of spermatogenesis and providing Fe for developing germ cells is also postulated.

The immune properties of Manduca sexta transferrin.

Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 µM to 10 µM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 µM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 µM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.

Separation of iron-free and iron-saturated forms of transferrin and lactoferrin via capillary electrophoresis performed in fused-silica and neutral capillaries.

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.

A new method for the rapid diagnosis of protein N-linked congenital disorders of glycosylation.

The Congenital Disorders of Glycosylation (CDG) are a devastating group of genetic disorders that encompass a spectrum of glycosylation defects and are characterized by the underglycosylation of or the presence of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type I and II) are usually diagnosed in chemical pathology laboratories by an abnormal serum transferrin isoelectric focusing (IEF) pattern. Transferrin has been the protein of choice for CDG analysis because it is well characterized, highly abundant, and easily detected in plasma. However, IEF provides limited information on the glycosylation defect and requires a separate and extensive glycan analysis to diagnose CDG Type II. We have therefore developed a simple bead-based immunoaffinity and mass spectrometry-based assay to address these issues. Our method uses immuno-purified transferrin and proteolytic digestion followed by a rapid 30 min mass spectral analysis and allows us to identify both micro- and macroheterogeneity of transferrin by sequencing of peptides and glycopeptides. In summary, we have developed a simple, rapid test for N-linked glycosylation disorders that is a significant improvement on existing laboratory tests currently used for investigating defective N-linked glycosylation.

Contribution of CE to the analysis of protein or peptide biomarkers.

Biomarker analysis is pivotal for disease diagnosis and one important class of biomarkers is constituted by proteins and peptides. This review focuses on protein and peptide analyses from biological fluids performed by capillary electrophoresis. The various strategies that have been reported to prevent difficulties due to the handling of real samples are described. Innovative techniques to overcome the complexity of the sample, to prevent the adsorption of the analytes on the inner capillary wall, and to increase the sensibility of the analysis are summarized and illustrated by different applications. To fully illustrate the contribution of CE to the analysis of biomarkers from human sample, two detailed protocols are given: the analysis from CSF of five amyloid peptide, biomarkers of the Alzheimer disease, and the analysis of sialoforms of transferrin from human serum.

A biologically active vMIP-II-IgG3-TfN fusion protein, secreted from methylotrophic yeast Pichia pastoris.

The viral macrophage inflammatory protein II (vMIP-II) which showed a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus. To explore the feasibility of using TfN as a carrier moiety for delivery of therapeutic proteins, a genetically engineered vMIP-II-IgG3-TfN fusion gene was loaded into the yeast expression vector pPICZα. The linearized recombinant plasmid pPICZα-vMIP-II-IgG3-TfN was transformed into X33 competent cells. The recombinant protein was expressed in methylotrophic yeast Pichia pastoris and was confirmed to have expected molecular mass of 48 kDa by SDS-PAGE. Using methods combining ammonium sulfate precipitation, dialysis, ultrafiltration and affinity chromatography, the vMIP-II-IgG3-TfN fusion protein was successfully purified from the supernatant of the broth. Western-blotting analysis showed that 6× His antibody recognized the purified vMIP-II-IgG3-TfN. CD spectrum revealed a positive peak at 196.5 nm and a negative peak at 209 nm. MALDI-TOF MS analysis showed that the purified vMIP-II-IgG3-TfN was an intact and homogeneous protein. The pepsin digestibility assay showed that the vMIP-II-IgG3-TfN fusion protein could be digested into small fragments by pepsin after 2 min treatment. The vMIP-II-IgG3-TfN fusion protein was found to be stable in human plasma for up to 48 h. Furthermore, in vitro bioactivity assay indicated that the vMIP-II-IgG3-TfN fusion protein can block the chemotaxis of U937 cells induced by SDF1α. In total, this study illustrates the development of an active vMIP-II-IgG3-TfN fusion protein expressed in P. pastoris.

Dynamic evaluation of polypropylene capillary-channeled fibers as a stationary phase in high-performance liquid chromatography.

Polypropylene (PP) capillary-channeled polymer (C-CP) fiber stationary phases are investigated for applications in HPLC. Specifically, the roles that fiber size and shape, linear velocity, interstitial fraction, and column inner diameter play in separation efficiency were evaluated using a uracil and butylparaben mixture eluted under isocratic conditions. Four fiber types, having nominal diameters ranging from 30 to 65 µm, were used in 250 mm × 2.1 mm columns. Optimum flow characteristics, as judged by plate height and resolution, were observed for 40 µm diameter PP C-CP fibers packed at an interstitial fraction of ~0.63, over a broad range of linear velocities (~2 to 37 mm/s). The influence of column inner diameter was studied on 1.5, 2.1, and 4.6 mm columns packed at the optimal interstitial fraction. The best performing column in terms of plate height and resolution was the 2.1 mm inner diameter. C-CP columns were also evaluated for the separation of a protein mixture composed of ribonuclease A, cytochrome c, and transferrin. Results obtained with the biomacromolecules mixture validate the optimal structural and operative conditions determined with the small solutes, laying the groundwork towards biomacromolecule applications, focusing more on the chemical aspects of separations.

Beta-35 is a transferrin-derived inhibitor of angiogenesis and tumor growth.

An angiogenesis inhibitor named Beta-35 has been identified and purified from the conditioned medium of mouse pancreatic ß cells tumor cells. Beta-35 has a molecular weight of 35 kDa and inhibits DNA synthesis of bovine capillary endothelial cells at a half-maximal concentration of approximately 5 nM. It shows anti-angiogenic activity in the chick embryo chorioallantoic membrane at a dose of about 1 µg/embryo. Amino acid microsequencing and mass spectrometric analysis of the purified protein demonstrate that Beta-35 contains the first 314 residues of the N-terminal sequence of bovine transferrin. We have cloned and expressed this protein in Escherichia coli using the corresponding gene segment of Beta-35 contained in the cDNA of human transferrin. The recombinant protein of Beta-35 shows significant anti-tumor activity at a dose of 5mg/kg/day against human pancreatic cancer or melanoma implanted subcutaneously in SCID mice.

Carp transferrin can protect spermatozoa against toxic effects of cadmium ions.

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans.

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. ß1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human ß1,4-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-ß1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

A high-performance liquid immunoaffinity chromatography method for determining transferrin-bound iron in serum.

BACKGROUND: The analyzed values in the ICSH reference method for serum iron analysis are affected by non-transferrin(Tf)-bound iron such as ferritin. Also, non-Tf-bound plasma iron (iron citrate) is present in iron-overloaded specimens from patients with hemochromatosis, which was measured as serum iron in previous methods. We developed a specific determination method for serum transferrin-bound iron (serum t Fe) by high-performance liquid immunoaffinity chromatography (HPLAC), and compared it with the ICSH method and a fully automated (FA) method. METHODS: Tf and t Fe were isolated from interferents in serum by HPLC using an immunoaffinity column. The concentration of t Fe isolated was determined by a colorimetric reaction using a highly sensitive chromogen. RESULTS: Interferents, except iron saccharate (detected at 5%), do not affect t Fe determination. Within-run and between-run imprecisions were in the ranges of 0.2-0.4% and 0.4-1.0% CV. The results of the HPLAC method correlated well with those of the ICSH method (r=0.9993) and FA method (r=0.9984). CONCLUSIONS: In contrast to the ICSH and FA methods for determining serum iron, the HPLAC method is simple, highly precise and specific for serum t Fe, which can contribute to the measurement of iron status.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins.

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and ß-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.

Synthesis and application of novel phenylboronate affinity materials based on organic polymer particles for selective trapping of glycoproteins.

We report on synthesis concepts for the fabrication of various novel phenylboronate affinity materials based on polymethacrylate epoxy beads (Fractogel EMD Epoxy (M) 40-90 microm) and the testing of these functionalized polymer particles for selective trapping of a glycoprotein from a standard mixture containing a glycosylated and a nonglycosylated protein. Two inherently different approaches for the functionalization of the bare beads with boronate groups have been elucidated. In the first, the epoxy residues of the polymer particles were converted into reactive thiol groups which were subsequently used as anchor moieties for the immobilization of 4-vinylphenylboronic acid by radical addition or radical polymerization reaction. Three different ways for the generation of sulfhydryl groups have been examined leading to materials with distinct linker chemistries. In the second and more straightforward approach, the epoxy groups were reacted with 4-mercaptophenylboronic acid. The novel materials were thoroughly characterized by (i) quantitation of the sulfur content by elemental analysis, (ii) reactive sulfhydryls were determined in a photospectrometric assay, (iii) boron content was measured by inductively coupled plasma-atomic emission spectrometry, and (iv) the amount of reactive boronate groups was evaluated in a fast binding assay employing adenosine as test compound. A maximum concentration of 1.2 mmol boronate groups per gram dry beads could be achieved by the presented synthesis routes. Employing the novel phenylboronate affinity materials in capture and release experiments in the batch mode, a standard glycoprotein, viz. transferrin (Tf) from human serum was separated from a nonglycosylated protein, BSA. A commercial boronate affinity material based on 3-aminophenylboronic acid modified agarose gel was employed as reference material and was found to perform significantly worse compared to the herein presented novel polymethacrylate particles.

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1.

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis.

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.

Purification and identification of a 7.6-kDa protein in media conditioned by superinvasive cancer cells.

BACKGROUND: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. MATERIALS AND METHODS: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold up-regulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. CONCLUSION: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia.