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.

A transferrin-like homolog in amphioxus Branchiostoma belcheri: Identification, expression and functional characterization.

Previous studies have shown the universal presence of transferrin (Tf) in both invertebrates and vertebrates, but little information is available regarding Tf in amphioxus, a protochordate on the evolutionary boundary between invertebrates and vertebrates. Here we isolated a Tf-like homolog from Branchiostoma belcheri, which encodes a deduced protein, BbTfl, of 1256 amino acids containing a N-terminal signal peptide, a conserved transferrin domain in its N-terminal lobe, with a putative iron-binding site consisting of Asp63 and Try188 and another transferrin domain in its C-terminal lobe with an long intervening sequence of 305 amino acids. Phylogenetic analysis shows BbTfl is grouped together with all the invertebrate Tfs and located at the base of melanotransferrins and other Tfs. Quantitative PCR analysis reveals that exposure to Escherichia coli and Vibrio anguillarum causes a significant increase in BbTfl expression mainly in the gut within 12-24h, suggesting that BbTfl is a positive acute phase reactant involved in the immune defense of B. belcheri. The recombinant N-terminal lobe, BbTflN, is able to bind iron and to inhibit E. coli and Staphylococcus aureus growth. Moreover, the antibacterial activity of BbTflN markedly decreases in the presence of excess iron. All these results provide a direct empirical evidence establishing a definitive link between binding to iron and bacterial growth-inhibiting activity. It is also shown that BbTfl is expressed in a tissue-specific manner, with the most abundant expression in the hepatic caecum, hind-gut and ovary, supporting the idea that the digestive system including the hepatic caecum of amphioxus is the primary tissue involved in acute phase response.

Alignment-free comparison of protein sequences based on reduced amino acid alphabets.

Protein sequences are treated as stochastic processes on the basis of a reduced amino acid alphabet of 10 types of amino acids. The realization of a stochastic process is described by associated transition probability matrix that corresponds to the process uniquely. Then new distances between transition probability matrices are defined for sequences similarity analysis. Two separate datasets are prepared and tested to identify the validity of the method. The results demonstrate the new method is powerful and efficient.

Evolution of the transferrin family: conservation of residues associated with iron and anion binding.

The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins.

Subtyping of transferrin by isoelectric focusing in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients ranging from pH 5.05 to 5.60 was used to study the distribution of transferrin (Tf) subtypes and their gene frequencies from 188 unrelated healthy donors of the Han population in Beijing. Six phenotypes (TfC1, TfC2, TfC1C2, TfC1C3, TfC1Dchi and TfC2Dchi) were detected and Tf*C3 has never before been reported in the Han population. Gene frequencies were as follows: Tf*C1 = 0.7420, Tf*C2 = 0.2420, Tf*C3 =0.0027, Tf*Dchi = 0.0133. There is good agreement between the observed and expected values corresponding to the Hardy-Weinberg equilibrium (sum (chi2) = 0.9183, df = 2, 0.5 < P < 0.75). The allele frequencies for Tf*C1, Tf*C2 and Tf*Dchi agreed with those previously reported for the Chinese Han population.

Genetic variability of transferrin subtypes in the populations of India.

Five hundred fifteen samples from five populations of India (Brahmins, Marathas, Patels, and Parsees of western India and Hindus of Andhra Pradesh) were analyzed for transferrin subtypes using the isoelectric focusing technique. The allele frequencies of 8444 samples belonging to 93 populations of India have been tabulated, and data were analyzed for genetic diversity among geographic, regional, and socio-cultural groups. Three relatively common alleles, TF*C1, TF*C2, and TF*C3, showed wide variation in various populations of India. Compared with western India, a high frequency of the TF*C2 allele was observed in eastern India. This variation of the TF*C2 allele showed a geographic cline increasing from west to east, giving a significant positive correlation between the TF*C2 allele frequency and longitude. Various genetic processes that possibly maintain TF polymorphism are selection, admixture, genetic drift, and isolation by distance. The possible influence of various genetic processes is discussed.

A new approach to quantitate carbohydrate-deficient transferrin isoforms in alcohol abusers: partial iron saturation in isoelectric focusing/immunoblotting and laser densitometry.

Carbohydrate-deficient transferrin (Tf) represents a significant advance over previous markers of alcohol abuse. Isoelectric focusing (IEF) analysis of affinity-purified Tf, under conditions of total iron saturation, identifies a major isoform at pI 5.4 in both normal consumers and alcohol abusers; three additional Tf isoforms (pI 5.6, 5.7, and 5.8) are associated with alcohol abuse. Under conditions of partial iron saturation, IEF analysis of affinity-purified Tf reveals up to seven isoforms (pI range 5.3-6.0) common to normal consumers and alcohol abusers; three additional transferrin isoforms (pI range 6.1-6.3) are present in 68% (15/22) of the alcohol abuser specimens, but in only 8% (1/12) of the specimens from normal consumers and in none of the three specimens from abstainers. These three diagnostic bands comigrate with a set of defined Tf isoforms: human iron-free Tf containing two sialic acid residues, human sialic acid-free Tf with one iron molecule, and human sialic acid-free, iron-free Tf. Serum specimens from normal consumers and alcohol abusers, analyzed for Tf isoforms by an IEF-immunoblot method under conditions of partial iron saturation, expressed Tf isoforms similar to those found using affinity-purified Tf in standard IEF. Visual examination of the immunoblots reveals the diagnostic bands in 67% (32/48) of patients with histories of sustained alcohol abuse compared with only 17% (8/48) of the normal consumers. Scanning densitometry and volume integration analysis of the immunoblots representative of normal consumer and alcohol abuser populations results in mean (+/- SE) values of 4.1 +/- 0.8 and 19.3 +/- 3.6 units, respectively (p < 0.0002).

Transferrin types, iron-binding capacity and body iron stores.

Increased body iron stores and transferrin (TF) variants have been found to be associated with adverse health effects believed to be caused by oxygen free radicals. Previous attempts to establish a relationship between TF types, serum TF concentrations and iron-binding have been inconclusive. We have studied serum iron, total iron-binding capacity (TIBC), TF saturation and serum ferritin in relation to genetic TF types in a population sample (691 females and 639 males) from northern Sweden in an attempt to elucidate whether individuals with TF variants associated with adverse somatic and reproductive effects (TFC2 and C3) have increased body iron stores. As expected there was a highly significant sex difference, males manifesting increased body iron stores viz. increased levels of serum iron, TF saturation and serum ferritin, and a lower TIBC. There was no consistent and statistically significant association between the TFC2 variant and the parameters that indicate iron binding and storage. Thus the associations between TFC2 and somatic and reproductive damage appear to be independent of iron binding and body iron stores. TIBC (and TF levels) showed significant differences between TF types in females (p = 0.0015) but not in males. In females the TFC3 variant was associated with a significantly lower (p = 0.002) TIBC value. This decreased TIBC value was, however, not accompanied by an increased ferritin value, thus there was no unequivocal evidence for an association between TFC3 and increased body iron stores.

Transferrin subtyping in dental pulps.

Serum transferrin (TF) subtypes were also found in dental pulps by isoelectric focusing and immunoblotting. The types observed in dental pulps completely agreed with those in serum samples from the same individuals. The allele frequencies in 105 samples were TF*C1 = 0.757 and TF*C2 = 0.243. Reliable subtyping was possible for 4 weeks following extraction of the teeth. The TF system can provide a useful genetic marker for the medicolegal individualization of teeth.

[Transferrin subtypes in 11 south China minority populations].

Transferrin subtypes were determined by isoelectric focusing (IEF) and a special treatment of sera with Rivanol- ferrous ammonium sulfate (FAS) in a total of 2121 individuals from 11 southern minority populations in China, including Hani minority (217), Buyi minority (209), Tujia minority (168), Miao minority (197), Zhuang minority (171), Yao minority (210), Bai minority (178), Dong minority (157), Yi minority (206), Qiang minority (219) and Li minority (189). TfC1, TfC2 and TfDChi were present in all the populations, whereas TfB was totally lacking. TfC4 was found in only 3 populations and TfC3 in 6; both were present at frequencies of less than 0.01 in all the populations. We found rare phenotypes TfC4-2, TfC4-4, TfC4-1, TfC3-2 and TfD Ching in these populations. The distributions of TfC2 and TfC4 frequency were notable. In these minority groups, the frequencies of the C2 gene were higher (0.25-0.38) than those of the majority Chinese Han nationality (0.18-0.25). In the Bai minority, the TfC2 gene frequency was as high as 0.38, the highest value found in the world to date. Besides being found in US and South American, the TfC2 allele is also present in both Han and minority nationalities in China, although at a frequency less than 0.01, suggesting that the C4 allele probably to ancestral already existed in the ancestral Mongoloid population at a low frequency prior trial separation and increased in frequency in Amerindians due to genetic drift or selection.

Transferrin polymorphisms in Japanese populations: north-south cline in the distribution of the TF*C2 allele.

Allele frequencies for human transferrin (TF) subtypes were determined using serum samples from Japanese subjects living in Fukui prefecture, Japan, and compared with other Japanese populations using isoelectric focusing (IEF) and immunoblotting. The application of IEF revealed considerable heterogeneity in the TF system, enhancing its potential value for anthropologic and genetic studies. So far, TF subtypes of about 27,000 Japanese individuals from 35 population groups have been analyzed to evaluate the degree of genetic variation at the TF locus. Possible geographic and biologic factors are discussed.

Group-specific component (GC) and transferrin (TF) subtypes in psoriatic patients.

Frequency of occurrence of TFC and GC subtypes and determining genes was evaluated in 90 and 68 nonrelated psoriatic patients, respectively. The obtained distributions of frequencies were compared with those recorded in healthy population. No statistically significant differences were observed.

Lack of influence of maternal and fetal transferrin phenotypes and concentrations on normal fetal growth.

Influence of maternal and fetal transferrin types and concentrations on the fetal growth has been investigated in 1,174 normal full-term singleton newborns, including 352 mother-newborn pairs. No significant effect of these parameters was observed on the weight and length of the newborns. Newborn plasma transferrin concentration was not correlated with maternal plasma transferrin concentration.

Transferrin subtypes and spontaneous abortion in a Chinese population.

A series of Chinese newborns of consecutive normal vaginal deliveries were investigated for the distribution of serum transferrin subtypes by polyacrylamide gel iso-electric focusing at pH 3.5-9.5. Newborns whose mothers had a history of previous spontaneous abortion (n = 189) had a significantly higher frequency of the C2 variant and the C2 gene compared to those (n = 864) without a history of spontaneous abortion. There was no significant difference in the frequency of transferrin alleles between newborns with normal and low birth weight (n = 147).

Occurrence of degradation products of chicken transferrin in murine circulating blood.

To determine the true survival of an exogenous substance in the circulation, it is requisite to assess that of the intact molecules. We studied the survival of chicken transferrin (Tf) in murine circulating blood after i.v. injection. With the aid of polyacrylamide-gel isoelectric focusing, direct immunofixation and densitometry, degradation products of chicken Tfs were found, and the disappearance rate of intact molecules was rather higher than that obtained by the single radial immunodiffusion method. These results strongly suggest that the degradation products must be taken into consideration in studies on the clearance of proteinous substances.

[Examination of the correlation between transferrin C and atopic dermatitis]. / Badanie korelacji pomiedzy podtypami transferyny C a atopowym zapaleniem skóry.

Transferrin subtypes are considered to be some of genetic markers which can correlate with the incidence of certain diseases. The present study was designed to find out whether such a correlation is true for atopic dermatitis. Transferrin C subtypes (Tf CI, Tf C2, Tf C3) in blood serum were determined by the method of isoelectric focussing in polyacrylamide gel. Examinations were made in 30 atopic dermatitis patients. The control group was made up by a Polish population sample of 728 normal persons of both sexes. The results obtained were analysed statistically by means of test chi 2. There was no correlation between Tf C and atopic dermatitis (chi 2 = 0,638, d.f. = 2, 0,750 greater than p greater than 0,500).

[Study on application of Tf subtyping in forensic medicine].

Tf is an important genetic marker for both parentage testing and personal identification in forensic medicine. It is valuable in anthropology, genetics and clinical medicine. We examined the Tf subtypes in the human serum and dried bloodstain using the ULPAGIF method. Tf subtyping was performed in four disputed parentage cases. One of the alleged fathers was excluded. Tf subtypes were successfully demonstrated from dried bloodstains kept at 4 degrees C for up to four weeks. In bloodstains kept at room temperature and 37 degrees C, the 100% of Tf subtyping was no longer possible after two weeks (or one day). It was suggested that the bloodstains should be examined no longer than seven weeks if the bloodstains were kept at room temperature; or otherwise they should be stored below 4 degrees C as soon as they are collected in forensic practice.

Occupancy of the iron-binding sites of human transferrin in sera obtained from different anatomical sites.

Transferrin is the major iron-transport protein in serum. Four molecular species (apo-, two mono-, and diferric transferrins) can be distinguished on the basis of their occupancy with iron. These species differ physicochemically and in the affinity with which they bind to the transferrin receptor. To elucidate the possible role of the four molecular species in directing the flow of iron between the major anatomical sites of iron release and utilization we have analyzed sera from eight stable multiorgan donors. The samples were obtained from veins draining the spleen, gut, and liver, and from the periphery. Employing polyacrylamide-gel electrophoresis in combination with crossed immunoelectrophoresis we were able to identify the four molecular species in all samples. Apo-transferrin was the predominant molecular species while diferric transferrin was the least abundant (P less than 0.01). The monoferric species were dominated by the acid-stable form (iron loading on the C-terminal end of the molecule) with ratios C/N from 1.2 (splenic and posthepatic serum) to 1.5-1.6 in mesenteric or peripheral samples respectively. We conclude from our study that it is unlikely that the monoferric transferrin species play a role in directing internal iron exchange and that in accordance with most of the literature the acid-stable form is preferentially loaded under physiological conditions of iron metabolism.

The N-acetylneuraminic acid content of five forms of human transferrin.

Five isoforms of human serum transferrin were separated by isoelectric focusing and their N-acetylneuraminic acid content was determined. The forms differed in isoelectric point by about 0.1 of a pH unit with the structural differences situated in the carbohydrate parts. Each form had one sialic acid molecule (NANA) less than the next most acidic form. GLC-MS showed that the most abundant form with isoelectric point 5.5 had two two-branched carbohydrate chains, each having the galactoses covered by terminal sialic acid. The form with isoelectric point 5.4 had one three-branched and one two-branched carbohydrate chain, and all branches terminated with a sialic acid residue. The form with isoelectric point 5.6 had a terminal galactose on one of its two two-branched carbohydrate chains. Comparison of the sialic acid content of the five transferrin forms and their carbohydrate structures showed that some of the forms expose terminal galactose without attracting the asialoglycoprotein receptors on hepatocytes.