Polymorphism of transferrin in carp (Cyprinus carpio L.): Genetic determination,isolation, and partial characterization

Seven transferrin variants (A, B, C, D, E, F, and G) have been found in carp sera (Cyprinus carpio L.). Genetic analysis involves five variants and agrees with the hypothesis of simple codominant autosomal inheritance at one transferrin (Tf) locus in spite of the fact that the carp is a tetraploid in relation to other species of the same family. Carp populations from three regions were studied which differed in gene frequencies. Individual populations were in Hardy—Weinberg equilibrium. The polymorphism of carp transferrins can be used for the identification of offspring of single parent pairs, stocked in one pond. Transferrins have been isolated and characterized. Homozygous phenotypes comprised four iron-binding components differing in electrophoretic mobility. This heterogeneity is not caused by sialic acid, which is absent. Amino acid composition, content of hexoses (1 mole/mole of protein) and hexosamines (1 mole/mole of protein), molecular weight (70,000), and the isoelectric point (5.0) have been determined. No N-terminal amino acid could be detected.     

Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

THE LIMITED PROTEOLYSIS OF BOVINE NEUROPHYSINS BY CATHEPSIN D

Abstract— The limited proteolysis of the bovine neurophysins at acid pH has been studied and the enzyme responsible has been characterized. Only 15 per cent of the catheptic activity in 4-year-old acetone-dried posterior pituitary lobe powder is soluble at pH 4.0. Solubility increases as the age of the powder decreases and the cathepsin is completely soluble in the presence of 1% Triton X-100. Acid proteinase activity in the neurohypophysis is not thiol activated and is inhibited by 3-phenylpyruvic acid. Bovine serum albumin was degraded at only 1 per cent of the rate of haemoglobin but with the same pH optimum (3.7). On this basis the enzyme was identified as cathepsin D. Neurophysin-I is degraded in two stages by cathepsin D; the first product (neurophysin-I′) runs faster and the second product (neurophysin-I″) runs slower than the native protein on starch-gel electrophoresis at pH 8.1. Neurophysin-II is also degraded in two stages; the first product has a higher electrophoretic mobility than the native protein and is identical in mobility with the faster-running component of the so-called neurophysin-M of Hollenberg and Hope (1967b). Prolonged incubation with the cathepsin gives rise to a slower-running component. Neurophysin-C is not attacked by the acid proteinase. Neurophysin-I′ and I″ have been isolated by ion-exchange chromotography. They have the same N-terminal amino acid (alanine) and C-terminal sequence (Ala-Phe-Ser) as the native protein and both bind 8-argininevasopressin. Neurophysin-I′ is identical in amino acid composition with the native protein but neurophysin-I″ has lost one leucine and two aspartic acid residues. Reduction, ¹⁴C-alkylation and separation of the fragments by starch-gel electrophoresis shows that the structural and functional integrity of neurophysin-I″ is maintained by the disulphide bonds, even though a tripeptide has been split out of the interior of the molecule. The low molecular weight material produced by catheptic digestion of neurophysin-I has been purified and shown to have a composition of one leucine and two aspartic acid residues. It is suggested that extensive in vivo proteolysis of neurophysin by lysosomal cathepsin, with consequent abolition of hormone-binding ability, is unlikely.     

Structural studies on individual components of bovine transferrin

The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ;maps', although differences in composition and peptide ;maps' occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain differences in electrophoretic mobility between bands of the same variant which are not produced by differing sialic acid content.     

The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the `hinge' region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the `hinge' region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of `hinge' peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the `hinge' region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the `hinge' region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.     

Obtaining of pure transferrins D, M and R from equine serum and determination of transferrin level in relation to phenotype

By the method of precipitation with Rivanol (2-ethoxy-6,9-diaminoacridine lactate) and ammonium sulphate followed by chromatography on DEAE cellulose three genetic variants of transferrin were purified from equine serum: D, M and R. Their molecular mass determined in this study was 80 000, and it was identical for all three variants, which differed slightly in their amino acid composition. The protein level was determined in the serum of 535 two-year-old thoroughbred English horses by the method of rocket immunoelectrophoresis using antibodies obtained against three transferrins. The individual variability of the protein level in horses of the same phenotype was fairly high (variability index 9-15%). No differences were observed in the transferrin level related to sex. It was found that the presence of D, F and H alleles was connected with a higher serum transferrin level, while O and R alleles were connected with a lower level.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

Comparison of bovine serum transferrin A and D2. II. Glycopeptides

Glycopeptides are isolated from subtilisin and pronase digests of whole bovine serum transferrin A and D2. The two variants yield glycopeptides with identical amino acid composition. Hence, there is probably no amino acid substitution in this region of the peptide chain. Amino acid sequence determination of one glycopeptide (subtilisin glycopeptide 8) gives the sequence: (CHO)Asn-Ser-Ser-Leu-Cys. This sequence is identical with that of residues 491-495 of the sequence for human serum transferrin (MacGillivray et al., 1982) except that in the bovine transferrin, Asp is replaced by Asn, enabling carbohydrate attachment. A second glycopeptide sequence Arg-(CHO)Asn-Ala-Thr-Tyr is observed, and the significance discussed in relation to carbohydrate moieties of serum glycoproteins.     

Studies on equine transferrin--I. The isolation and partial characterization of the D and R variants

Each of two genetic variants of equine transferrin, D and R, is isolated from the blood of the heterozygote by a gentle fractionation procedure at pH 7.2. It is shown by step gradient polyacrylamide gel electrophoresis at pH 7.9 that each of these phenotypes exhibits two major bands (designated F, fast, and S, slow) and several minor bands. Components corresponding to these bands are separated by ion-exchange chromatography at pH 6.6 and 6.9 respectively for the D and R variants. The F and S components of each variant contain respectively four and two sialic acid residues. The nature of their heterogeneity is, at least in part, due to their varying sialic acid contents. It has not been possible to desialylate them completely by neuraminidase. On the basis of comparative studies of the tryptic and chymotryptic peptide maps of transferrins D and R it is concluded that there are at least two amino acid substitutions--D:R:Asp:Gly and Glu:Gly. These two substitutions are qualitatively in accordance with the difference in the electrophoretic mobility between the two variants at alkaline pH.     

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10–25th and C-terminal 1–9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1–7th and C-terminal 1–3rd residues were not necessary to maintain enzymatic activity.     

Bimolecular fluorescence complementation based on the red fluorescent protein FusionRed

The aim of this work is to construct split variants of the red fluorescent protein FusionRed, each of which consists of two separate polypeptides, nonfluorescent parts of FusionRed, that can form functional fluorescent proteins upon reassociation. At the first stage, various circularly permuted FusionRed variants have been created (in circular permutants the protein polypeptide chain is divided into two parts, which change places so that the C-terminal part is followed by the N-terminal part). Two variants with the highest rate of chromophore maturation (fluorescence development) have been selected out of 23 tested permutation points. These proteins called cpFR76-73 and cpFR189-188 (the first number indicates the last amino acid residue of the N-terminal part; the second number, the first residue of the C-terminal part) are spectrally similar to parental FusionRed but possess lower fluorescence quantum yields. Split variants corresponding to these two circular permutants have been tested in mammalian cells. For reassembly of the fluorescent protein fragments, heterodimerizing leucine zippers have been used. It has been shown that split variant FR189-188 matures at 37°C and possesses fluorescence brightness similar to that of FusionRed. Consequently, FR189-188 is potentially suitable for a wide range of applications, for example, the study of protein–protein interactions or visualization of cell populations, in which two target gene promoters are simultaneously active.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Characterization of the isoforms of phospholipase A2 from honeybee venom

Phospholipase A₂ from the venom of the European honeybee (Apis mellifera) consists of three isoforms with approximate molecular masses of 16, 18, and 20 kDa, respectively, as deduced from SDS-PAGE. These variants, termed PLA-16, PLA-18, and PLA-20, were isolated by lectin affinity chromatography and preparative polyacrylamide gel electrophoresis. The amino acid sequences of the N-terminal peptide portions of all three isoforms, as assessed by automated Edman degradation, were identical with that expected for honeybee phospholipase A₂. Sequencing data suggest that, while PLA-18 and PLA-20 carry oligosaccharide residues at asparagine-13, PLA-16 has escaped glycosylation during biosynthesis. Release of the carbohydrate from PLA-18 and PLA-20 with peptide: N-glycosidase F abolished the molecular mass differences between the three isoforms of phospholipase. Differences in sensitivity to α-mannosidase and monosaccharide composition of PLA-18 and PLA-20 further indicate that their electrophoretic separation is based on structural features of the N-glycosidically linked oligosaccharide. Noticeably, PLA-20 contains N-acetylgalactosamine, a sugar not having yet been described as a constituent of insect glycoproteins.     

The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C-lobe

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

Characterization of lamprey fibrinopeptides

1. Lamprey fibrinopeptide B is a relatively large peptide made up of about 40 amino acid residues. The peptide is highly electronegative, containing a large number of aspartic acid residues and a tyrosine O-sulphate residue. 2. The amino acid sequence of the first 18 residues from the N-terminal end of fibrinopeptide B has been established. The C-terminal ends with the sequence Val-Arg. Fibrino-peptide B is released by both lamprey and bovine thrombins. 3. Lamprey fibrino-peptide A is a short peptide containing only eight residues. The proposed amino acid sequence is: Asp-Asp-Ser-Ile/Leu-Asp-Ser-Leu/Ile-ArgThis peptide is released by lamprey thrombin but not by bovine thrombin.     

Geographical Distribution of Twelve Transferrin Alleles in Black Rats of Asia and Oceania

About 450 black rats (Rattus rattus) were collected from 25 localities in Asia and Oceania. Their serum transferrins were analyzed by a newly developed thin layer acrylamide gel electrophoresis accompanied with acrinol pretreatment, exhibiting 12 transferrin bands. Generally, Asian type rats (2N=42) had fast-moving transferrins (R-series), Ceylon type (2N=40) moderately moving ones and Oceanian type (2N=38) slowly moving ones (C-series). Exceptionally, in northern India and Pakistan all Asian-type rats had C-series Tf. The possibility that divergence of R-series Tf and C-series Tf had preceded the karyotypic differentiation from 42 to 38 is proposed. In combination with the previous molecular data, the time of the divergence is roughly estimated between the order of a million years and ten thousand years.     

Synthesis and antiproliferative activity of C- and N-terminal analogues of culicinin D

Culicinin D (1), a 10 amino acid peptaibol containing several unusual residues, has been shown to exhibit potent anticancer activity. Previous work in our group towards developing a structure-activity relationship (SAR) for this peptaibol has concentrated on replacement of the synthetically challenging AHMOD (3) and AMD (4) residues, resulting in the discovery of analogues with equivalent or better potency and simplified synthesis. The SAR of this peptaibol is extended in this work by investigating the effect of the N-terminal lipid tail and C-terminal amino alcohol, revealing the key contribution of each of these moieties on antiproliferative activity in a panel of breast and lung cancer cell lines.     

Role of the C-terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.