Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.     

A new proalbumin variant: albumin Jaffna (-1 Arg----Leu)

Albumin Jaffna is an electrophoretically slowly moving genetic variant of human serum albumin found in two members of a Tamil family from Jaffna (Northern Sri Lanka), both heterozygous for the abnormal protein. Sequential analysis of albumin Jaffna, purified from serum by ion exchange chromatography on DEAE Sephadex and Mono Q columns, revealed that this variant is a new abnormal proalbumin, arising from a -1 Arg----Leu substitution, which prevents the proteolytic removal of the N-terminal hexapeptide and allows the mutated proalbumin to enter the circulation. The presence of two additional positive charges is in keeping with the decreased electrophoretic mobility of albumin Jaffna, as well as with its isoelectric point of 5.01, determined by chromatofocusing on a Mono P column. The variant is selectively cleaved by trypsin in vitro, leaving leucin -1 as N-terminal residue.     

Structural differences between albumin A and albumin C of the house mouse,Mus musculus

Two albumins, albumin A from C3H mice and albumin C isolated from descendents of the wild mice in which the variant was first uncovered, were found to differ in their electrophoretic properties. Albumin C was shown to bind two more H⁺ ions than albumin A at pH 5.4. Peptide mapping after trypsin digestion revealed that albumin C had three peptides (TP-C1, TP-C2, and TP-C3) which were missing in albumin A. The latter likewise had a peptide (TP-A1) which was not found in albumin C. An amino acid analysis of the variant peptides suggests that TP-A1 had been split into TP-C1 and TP-C2 on digestion with trypsin, because a glutamic acid in TP-A1 was replaced by a lysine. This change would also appropriately alter the electrophoretic properties of albumin C. No obvious counterpart was discovered for TP-C3 of albumin C in albumin A.This work was supported by a grant from the National Research Council of Canada.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Study of a further case of bisalbuminemia in Brittany]

A rapidly migrating variant of albumin has been discovered in a 28-year-old breton woman. At least eight relatives also bear the trait. This new variant is described in terms of its electrophoretic mobilities, immunological properties and heat- and storage stability. Two other reports of the slow type are presented, but no family study was performed in these cases. It is suggested that bisalbuminaemia could be a relatively common inherited condition in Brittany.     

Studies on a genetically determined albumin dimer

An electrophoretic variant of human albumin has been characterized as a dimer involving both disulfide and noncovalent bonds. The variant protein was isolated by starch block electrophoresis and gel filtration and extensively investigated by gel electrophoresis and immunoelectrophoresis under a variety of conditions. No significant differences were found between normal albumin and the propositus's albumin in sulfhydryl reactivity or content or in the tryptic fingerprint.Contribution No. 140 from the Blood Research Laboratory, American National Red Cross.     

Genetic variants of human albumin: structural characterization of allotypes used as references for electrophoretic classification

Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.     

A new glucose-6-phosphate dehydrogenase variant (G-6-PD Kalyan) found in a Koli family

Summary A new Indian variant of erythrocytic glucose-6-phosphate dehydrogenase (G-6-PD) has been detected in a Koli male subject during population genetic studies. The enzyme variant is characterized by mild enzyme deficiency, slow electrophoretic mobility, low K_m for G-6-P, increased utilization of substrate analogues, heat instability and a normal pH optimum curve. From these results this was considered to be a new variant and was designated G-6-PD Kalyan. The family history and routine hematological studies did not reveal any evidence that the G-6-PD Kalyan is associated with any hematological abnormalities or clinical symptoms.     

Linkage of the locus for serum albumin in the house mouse,Mus musculus

An electrophoretic variant for serum albumin in Mus musculus has been used to map the structural gene for this protein to chromosome 5.     

Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja).

A novel variant of soybean Kunitz trypsin inhibitor (SKTI) was detected in 530 lines of wild soybean (Glycine soja). This variant showed an intermediate electrophoretic mobility between the Tia and Tic types. In isoelectric focusing polyacrylamide gel electrophoresis gels containing urea, this variant had a similar isoelectric point as that of Tia. The genetic analysis of SKTI bands in F2 seeds from crosses of the new variant type with Tia or Tic type showed that this variant type is controlled by a codominant allele at the SKTI locus. We propose the genetic symbol Tif for this novel variant. When the nucleotide sequence of the Tif gene was compared with those of other types of SKTI genes (Tia, Tib, and Tic), the sequence of Tif was identical to that of Tib with the exception of one A-->G transitional mutation occurring at position 676 of Tif. This mutation resulted in an amino acid change from Lys to Glu at the 178 residue. These results suggest that this variant is derived from Tib through a point mutation. In addition, we settled an inconsistency in the number of amino acid differences between Tia and Tib (eight or nine). Analysis of nucleotide and amino acid sequences revealed that Tib was different from Tia by nine amino acids.     

A new transferrin variant from Iran (Tf B-Iran): review of 36 variant alleles

A total of 2581 serum samples collected from five population groups of Iran was studied for electrophoretic variations of the transferrin (Tf). Besides the common phenotype Tf C the authors could observe 41 individuals with rare Tf types: CB1, CB2, CD1, CDChi, CD2. In addition to these Tf types two individuals with a new Tf B variant were observed. This new variant was found in the Dezfooli sample and was designated as Tf B-Iran. The electrophoretic position of this variant is described, and all the hitherto known Tf variants are reviewed.     

Detection of genetic variation with radioactive ligands. I. Electrophoretic screening of plasma proteins with a selected panel of compounds.

To detect new genetic variation in human plasma proteins, a panel of 63 radioactive substances were screened as potential radioligands using polyacrylamide gel electrophoresis (PAGE) and autoradiography. Vitamins, hormones, drugs, amino acids, purines, pyrimidines, sugars and lipids labeled with 14C or other radionuclides were among those substances tested. A majority bound to albumin and a smaller fraction to prealbumins and lipoproteins. Several vitamins and hormones bound to specific alpha and beta globulins. (1) Electrophoretic polymorphisms of vitamin D-binding protein (group-specific component), a vitamin B12-binding protein (transcobalamin II), and thyroxine-binding alpha-globulin are described elsewhere. (2) Testosterone-binding beta-globulin (TeBG) showed an electrophoretic polymorphism in Caucasians and a possible deficiency allele. (3) Transcortin showed an electrophoretic doublet in all persons tested but no electrophoretic variation. (4) A protein binding derivative of norepinephrine or epinephrine was identified as transferrin. (5) A nonpolymorphic protein running cathodal to albumin and binding a derivative of riboflavin was tentatively identified as a fraction of albumin with mobility altered as a result of interaction with the ligand.     

Plasma cholinesterase polymorphism in Down's syndrome.

The plasma cholinesterase variants in 80 patients having Down's syndrome have been examined and compared with a random control sample of individuals domiciled in the same area. Neither a change in frequency of known C5+ variant nor any new variant has been found in the electrophoretic studies. A highly significant increase in frequency of the fluoride resistant gene as well as a significant decrease in frequency of the atypical gene has been found in the Down's syndrome individuals. This altered distribution of the rare phenotype of plasma cholinesterase in mental illness may lead to a useful diagnostic acid in genetic counselling.     

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.     

Variants of the group-specific component system as demonstrated by immunofixation electrophoresis. Report of a new variant, Gc Boston (Ge B).

Immunofixation electrophoresis is a relatively simple and reliable method for the genetic phenotyping of the group-specific component (Gc) of serum. This method permits direct comparison of electrophoretic mobilities and band concentrations, with no interference by other proteins. The variants Gc Ab and Gc Y appear identical by this technique; the Eskimo variant appears to be similar to Gc D but not to Gc Ab as previously reported. Gc Norway, also designated Gc 1C, is electrophoretically cathodal to the slower band of Gc 1 and therefore appears to be a distinct variant. A new variant, Gc Boston, is single banded with mobility between the two bands of Gc 1.     

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.     

Distribution of albumin variants Naskapi amd Mexico among Aleuts, Frobisher Bay Eskimos, and Micmac, Naskapi, Mohawk, Omaha, and Apache Indians

In order to help define the boundaries of the distribution of the albumin variants Naskapi and Mexico which are polymorphic among several American Indian groups, we examined sera from Micmac, Mohawk, Northwest River Naskapi, Omaha and Apache Indians, and from Aleuts and Eskimos. Sera from a total of 1,524 individuals were examined. Using a cellulose acetate membrane electrophoretic system with Tris-Citric acid at pH 5.4 we were able to distinguish normal albumin and both variants in the same run. Naskapi and Mexico variants were absent from Aleut, Eskimo, Micmac, Mohawk and Omaha samples. The albumin Naskapi variant was present in an allele frequency of 0.03 in the Naskapi Indian sample. Albumin variants Naskapi and Mexico were found in the Apache sample at frequencies of 0.016 and 0.037, respectively. This report supersedes that previously published by Schell and Agarwal ('76). Generally, within an area there is a correspondence between changes in the frequency of albumin variants and changes in the ethnic background and history of the area's populations. At the same time, when viewing widely separated areas, relationships between distant groups based on linguistic and cultural similarities are paralleled on a biologic level by the distribution of normal albumin and variant albumins.     

Genetic polymorphism of human erythrocyte glyoxalase II.

A new method for the detection of glyoxalase II (hydroxyacylglutathione hydrolase) after starch gel electrophoresis is described. A survey of Asian-Pacific populations indicates that genetic polymorphism of glyoxalase II is extremely rare. Polymorphism was observed only in a Micronesian population from the island of Nauru, in which the frequency of the new variant allele HAGH2 was found to be .016. The electrophoretic pattern in heterozygotes was a double band, suggesting that the structure of glyoxalase II is monomeric.     

Albumin Hawkes Bay; a low level variant caused by loss of a sulphydryl group at position 177

A slow migrating minor albumin component, representing 5% of total circulating albumin, was detected by routine serum protein electrophoresis and immunofixation. After treatment with 5 mM dithiothreitol the abnormal component was found to migrate normally suggesting the attachment of some component to the free thiol at position 34. However, purification and analysis by SDS-PAGE showed that the abnormal component had a slightly lower apparent molecular weight than normal albumin. Limited tryptic cleavage indicated the abnormal site to be in the N-terminal third of the molecule. HPLC analysis of tryptic peptides from this domain showed the presence of a new peptide of sequence Ala-Ala-Phe-Leu-Leu-Pro-Lys, indicating either a point mutation of 177 Cys → Phe or the deletion of residues 166–177. DNA sequencing of PCR-amplified DNA confirmed the former Cys → Phe substitution by indicating a point mutation of C to A at nucleotide position 5185. It appears that the aberrant electrophoretic mobility of the variant might be due to a gross conformational change associated with the formation of a new disulphide bond between Cys-168 and Cys-124.