The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type i by capillary zone electrophoresis

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.Abbreviations CDGS Carbohydrate Deficient Glycoprotein Syndrome - CZE Capillary Zone Electrophoresis - hTf human transferrin - gu HPLC glucose units - EOF electroosmotic flow. Nomenclature: for describing oligosaccharide structures: A(1,2,3,4) indicates the number of antennae linked to the t trimannosyl core - G(0–4) indicates the number of terminal galactose residues in the structure - F core fucose - B bisecting N-acetyl glucosamine (GlcNAc) - S sialic acid - Gal galactose; M - Man mannose     

Online single-step analysis of blood proteins: the transferrin story

The serum iron transport protein human transferrin (hTf) is a glycoprotein (MW approximately 79.6 kDa) containing two Asn-linked sites of glycosylation. The presence of specific glycoforms of hTf has been used as an indicator of carbohydrate-deficient glycoprotein syndrome (CDGS) or an indicator of alcohol abuse. The exact nature of the glycoforms described in the literature is controversial. In this work we demonstrate that the altered hTf glycoforms have lost one or both complete glycan side chains. Furthermore, we demonstrate using a combination of online immunoaffinity-postconcentration-mass spectrometry in conjunction with a blood spot cartridge that we can determine the relative quantities of the hTf glycoforms using <5 microL blood in under 30 min. This is in contrast to previous methods that used 1 mL and took 4 days. We show that this method can be useful to analyze hTf from CDGS and alcoholic patients.     

Influence of transferrin glycans on receptor binding and iron-donation

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, β-galactosidase or N-Acetyl-β-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions. This revised version was published online in November 2006 with corrections to the Cover Date.     

Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS

Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf baseline reference range for male (1.42-2.35 g L(-1)) and female German North Sea seals (1.93-2.74 g L(-1)) as well as a CDT level of 0.00-0.10 g L(-1), respectively, which provides valuable further diagnostic information regarding the health status of these specific marine mammals. Compared to assay based quantification approaches the proposed technique indicates great potential to obtain comparable and traceable absolute quantitative results, which are in particular important for long term investigations. This absolute quantification is based on an accurate, traceable element standard, while assay based approaches often show variations depending on the kit quality or changing activities of the used antibodies.     

Molecular basis of carbohydrate-deficient glycoprotein syndromes type I with normal phosphomannomutase activity

Carbohydrate deficient glycoprotein syndromes (CDGS) are inherited disorders in glycosylation. Isoelectric focusing of serum transferrin is used as a biochemical indicator of CDGS; however, this technique cannot diagnose the molecular defect. Even though phosphomannomutase (PMM) deficiency accounts for the great majority of known CDGS cases (CDGS type Ia), newly discovered cases have significantly different clinical presentations than the PMM-deficient patients. These differences arise from other defects affecting the biosynthesis of N-linked oligosaccharides in the endoplasmic reticulum and in the Golgi compartment. The most notable is the loss of phosphomannose isomerase (PMI) (CDGS type Ib). It causes severe hypoglycemia, protein-losing enteropathy, vomiting, diarrhea, and congenital hepatic fibrosis. In contrast to PMM-deficiency, there is no developmental delay nor neuropathy. Most symptoms in the PMI-deficient patients can be successfully treated with dietary mannose supplements. Another defect is the lack of glucosylation of the lipid-linked oligosaccharide precursor. The clinical features of this form of CDGS are milder, but similar to, PMM-deficient patients. Yeast genetic and biochemical techniques were critical in unraveling these disorders since many of the defective genes were known in yeast and corresponding mutants were available for complementation. Yeast strains carrying mutations in the homologous genes are likely to provide conclusive identification of the primary defects in novel CDGS types that affect the synthesis and transfer of precursor oligosaccharides.     

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.     

beta-Trace protein in human cerebrospinal fluid: a diagnostic marker for N-glycosylation defects in brain.

As carbohydrate-deficient glycoprotein syndromes (CDGS) are multisystemic disorders with impaired central nervous function in nearly all cases, we tested isoforms of beta-trace protein (beta TP), a 'brain-type' glycosylated protein in cerebrospinal fluid (CSF) of nine patients with the characteristic CDGS type I pattern of serum transferrin. Whereas the serum transferrin pattern did not discriminate between the various subtypes of CDGS type I (CDGS type Ia, type Ic, and patients with unknown defect), beta TP isoforms of CDGS type Ia patients differed from that of the other CDGS type I patients. The percentage of abnormal beta TP isoforms correlated with the severity of the neurological symptoms. Furthermore, two patients are described, who illustrate that abnormal protein N-glycosylation can occur restricted to either the 'peripheral' serum or the central nervous system compartment. This is the first report presenting evidence for an N-glycosylation defect restricted to the brain. Testing beta TP isoforms is a useful tool to detect protein N-glycosylation disorders in the central nervous system.     

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.     

Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS

This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and alpha1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.     

Carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse: a critical review of the literature 2001-2005

The diagnosis of alcohol abuse based on objective data is a necessary requirement in both clinical and forensic environments. Among the different biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is world wide recognized as the most reliable indicator. However, several problems about the real meaning of CDT and the reliability of its use for the diagnosis of alcohol abuses are still open, as reported by numerous research articles and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2001 to 2005 (included). The article is organized in the following sections: (1) introduction, (2) definition and structure of human serum CDT, (3) pathomechanisms of the ethanol-induced CDT increase, (4) preanalysis, (5) analysis, (6) data interpretation, (7) review papers, (8) conclusions. As many as 127 papers appeared in the international literature and retrieved by the search engines PubMed and Scopus are quoted.     

Abnormal synthesis of dolichol-linked oligosaccharides in carbohydrate-deficient glycoprotein syndrome

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a raremetabolic disorder presenting in infancy with severe neurologicinvolvement and variable multisystemic abnormalities. Diagnosisrelies upon the detection of abnormal serum glycoprotein isoformson isoelectric focusing (IEF) gels. Carbohydrate structuralanalyses were performed on the N-linked oligosaccharides ofserum     

Different Binding Affinities of Pb2+ and Cu2+ to Glycosylation Variants of Human Serum Transferrin Interfere with the Detection of Carbohydrate-Deficient Transferrin

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse, and for diagnosis of chronic alcohol, abuse is often determined using isoelectric focusing (IEF) and chromatographic techniques. To allow this method to be used for the diagnosis of alcohol abuse, inferences of various physical and chemical factors with the detection of CDT have been investigated. However, few reports have focused thus far on whether different metal ions have different binding affinities to CDT and HTf variants or further interfere in the detection of CDT. Here, in order to figure out whether and how metal ions such as Pb(2+) and Cu(2+) bind to holo-human serum transferrin (holo-HTf) and further interfere in CDT detection, the binding characteristics and the binding parameters of holo-HTf with metal ions such as Pb(2+) and Cu(2+) were investigated using UV-visible spectroscopy, Fluorescence spectroscopy, and ICP-MS. Moreover, whether the metal ions such as Pb(2+) and Cu(2+) will reduce the diagnostic accuracy of CDT in clinic was investigated using IEF. The present study demonstrates that Pb(2+) and Cu(2+) have different binding affinities to holo-HTf variants and produce different changes in the relative amounts of each glycosylation isoforms of HTf. Accordingly, the glycosylation chains of HTf will affect the binding affinities of glycosylation isoforms with Pb(2+) and Cu(2+), causing further interferences in CDT detection.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti₂Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti₂Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and ¹³C NMR spectroscopies, and shown to be stable within the pH range 5.5–9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti₂Tf, even after long incubation times, although some weak interactions of Ti(IV)NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

Transferrin in cultured human term cytotrophoblast cells: Synthesis and heterogeneity

Transferrin (Tf) mRNA was recently demonstrated in rat and mouseplacental tissue. Rat placental cells were shown to secrete transferrin. Thecell type with which Tf mRNA was associated was not investigated. Wetherefore studied the ability of immunopurified human term cytotrophoblastcells in culture to synthesize Tf, by means of pulse-label experiments with35S-methionine and report that these cells do synthesize Tf. Tf mRNA wasdemonstrated in the cell lysates by means of RT-PCR. Tf isolated fromcytotrophoblast and syncytiotrophoblast cells was shown to be different fromboth maternal and fetal serum Tf with respect to the distribution ofisoforms as demonstrated by means of iso-electric focusing. The iso-electricpoints were found at lower pH values (pH 5.0-5.4), compared to theiso-electric points of maternal and fetal serum Tf, suggesting a higherdegree of sialylation and glycan chain complexity.     

Carbohydrate-deficient glycoprotein syndrome type II

The carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans. This review describes recent findings on two patients with CDGS type II. In contrast to CDGS type I, the type II patients show a more severe psychomotor retardation, no peripheral neuropathy and a normal cerebellum. The CDGS type II serum transferrin isoelectric focusing pattern shows a large amount (95%) of disialotransferrin in which each of the two glycosylation sites is occupied by a truncated monosialo-monoantennary N-glycan. Fine structure analysis of this glycan suggested a defect in the Golgi enzyme UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) which catalyzes an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. GnT II activity is reduced by over 98% in fibroblast and mononuclear cell extracts from the CDGS type II patients. Direct sequencing of the GnT II coding region from the two patients identified two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) and H262R (CAC to CGC). Either of these mutations inactivates the enzyme and probably also causes reduced expression. The CDG syndromes and other congenital defects in glycan synthesis as well as studies of null mutations in the mouse provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms.     

Clear cell carcinoma of the human ovary synthesizes and secretes a transferrin with microheterogeneity of lectin affinity

Human ovarian clear cell carcinoma cell line (transferrin (Tf)-non-producer), HAC 2, cells were adapted to grow in chemically defined synthetic medium when the cells were cultured with medium containing 10 μg/ml of insulin at least for 6 months. They synthesized and secreted constantly the 80 kDa protein immunologically similar to human serum Tf(15 ± 12 ng/ml/10⁷ cells/3 days). By sensitive lectin-affinity electrophoresis followed by antibody-affinity blotting technique, a concanavalin A weakly bound or unbound, lentil lectin, a strongly reactive abnormal band, which was rarely found in human serum Tf, was detectable in the Tf synthesized by HAC 2 cells (HACTf). These findings suggest that the HACTf may act as one of the autocrine growth factors and that this heterogeneity of HACTf for lectin affinity is ascribed to differences in the carbohydrate moiety of the Tf.     

Haptoglobin glycoforms in a case of carbohydrate-deficient glycoprotein syndrome

Alterations in haptoglobin (Hp) glycosylation were examined in the plasma of the first patient with carbohydrate-deficient glycoprotein syndrome (CDGS) who was described in Poland. Hp concentration in the CDGS patient plasma was low (240mg/l) and the Hp phenotype was shown to be 2-2. Three glycoforms of the Hp subunit were observed in SDS-PAGE in CDGS. The densitometric analysis and molecular weight determinations suggested that 50% of glycoforms were fully glycosylated; 30% contained three out of four and 20% only two out of four glycan units compared to those that are present in Hp derived from healthy people. Results with lectins (concanavalin A and Sambucus nigra, Maackia amurensis and Alleuria aurantia agglutinins) indicate that all three glycoforms of subunit of CDGS-Hp contained biantennary complex glycans terminated with 2,6 bound sialic acid, but without fucose or 2,3 linked sialic acid. Hp glycosylation abnormalities described in this work suggest that this case was a type I carbohydrate-deficient glycoprotein syndrome.