Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 45: 21-28, 1999;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paleari, R.
Right arrow Articles by Galanello, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paleari, R.
Right arrow Articles by Galanello, R.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Proteomics and Protein Markers
Right arrow Hematology
(Clinical Chemistry. 1999;45:21-28.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Posttranslational Deamidation of Proteins: The Case of Hemoglobin J Sardegna [{alpha}50(CD8)His->Asn->Asp]

Renata Paleari1, Elisabetta Paglietti2, Andrea Mosca1,a, Michele Mortarino3, Liliana Maccioni2, Stefania Satta2, Antonio Cao2 and Renzo Galanello2

1 Dipartimento di Scienze e Tecnologie Biomediche, Laboratorio Interdisciplinare Technologie Avanzare (L.I.T.A.), Università degli Studi, I-20090 Segrate, Milan, Italy.

2 Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi, I-09131 Cagliari, Italy.

3 Istituto di Fisiologia Veterinaria e Biochimica, Università degli Studi, I-20133 Milano, Italy.
a Address correspondence to this author at: L.I.T.A., Via Fratelli Cervi 93, I-20090 Segrate, Milano, Italy. Fax 39 2 2642 3302; e-mail andrea.mosca{at}unimi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hemoglobin J Sardegna [{alpha}50(CD8)His->Asn ->Asp] is a human Hb variant in which a posttranslational deamidation process takes place, transforming an Asn to an Asp residue. This variant, particularly widespread in northern Sardinia, has for the first time been characterized at the DNA level (codon 50 C->A) on the selectively amplified {alpha}2-globin gene. We determined the protein and DNA sequences and performed cellulose acetate electrophoresis, isoelectric focusing, globin chain separation, stability tests with isopropanol and heat precipitation, and oxygen affinity analyses on whole blood to fully characterize the variant. A comprehensive review of the deamidation processes involving Asn and Gln residues in mutant proteins is reported, together with a discussion of the molecular mechanisms of such deamidations. Finally, examples of other proteins of clinical importance in which Asn or Gln residues have been implicated by DNA analysis alone are presented. These findings point out the importance of the complete characterization of variant proteins by use of both DNA and protein analyses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemoglobin (Hb) J Sardegna is a well-known Hb variant, being one of the most common {alpha}-globin structural mutants. Hb J Sardegna was first reported in 1968 in members of a family from the island of Sardinia, Italy (1). At that time, the variant was described as a fast-moving Hb and characterized by the classic techniques of protein chemistry, which demonstrated the substitution of a His with an Asp residue at position 50 of the {alpha} chain. Since then, large screening programs performed by means of isoelectric focusing and reverse-phase HPLC have produced detailed information on the prevalence of this variant and its association with {alpha}- and ß-thalassemia.

Researchers have discovered that Hb J Sardegna has a relatively high frequency in Sardinia, where it occurs in ~0.09% of the population. The geographic distribution is not uniform: some variations of incidence are found in different areas of the island. A higher incidence has been reported in the north, where it reaches 0.25% (2)(3).

Because thalassemic diseases are also widespread in the Sardinian population, the association of Hb J Sardegna with {alpha}- or ß-thalassemia is not rare (4)(5). Studies that correlate the Hb pattern with the {alpha}- and ß-globin genotype have shown that in double heterozygotes for Hb J Sardegna and {alpha}-thalassemia, the quantity of the abnormal Hb is substantially higher than in heterozygous carriers of the Hb variant alone (6). On the other hand, the concomitant presence of ß-thalassemia leads to a slight decrease in the amount of the variant, probably because of a lower affinity of the abnormal {alpha} chain for the ß chain. Hb J Sardegna has also been described in association with Hb G Philadelphia, another {alpha}-chain variant that occurs rather frequently in the Sardinian population (2)(5).

The presence of Hb J Sardegna does not seem to produce any particular clinical manifestation. Appreciable clinical complaints have never been reported in heterozygous carriers of this abnormal Hb, even if it occurs in combination with {alpha}- or ß-thalassemia. To date, no data are available on the DNA molecular defect associated with Hb J Sardegna; information on the stability of this variant and its functional properties is also not available.

In the past few years, methods based on molecular biology techniques have become reliable and easy tools for identifying Hb variants. Recently, we used a DNA sequencing approach for additional characterization of Hb J Sardegna. Surprisingly, the results obtained by DNA sequencing analysis appeared to disagree with the results of protein analysis reported originally (1). The mutation found at the genomic level indicated a His->Asn substitution, whereas protein structural analysis demonstrated that it was His->Asp. This enigmatic finding prompted us to reinvestigate this abnormal Hb. We report here on the characterization of Hb J Sardegna performed at both the DNA and protein levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
blood samples
Blood samples were obtained from different heterozygous carriers of Hb J Sardegna, all coming from Cagliari on the island of Sardinia, Italy. None of them were affected with any clinical symptoms or hematological abnormalities. For Hb studies, blood samples were collected in EDTA, shipped by fast air mail, and analyzed within 2 days. Twenty-six different carriers of Hb J Sardegna were studied by DNA sequencing as well as by HPLC analysis.

dna analysis
DNA was obtained from white blood cells by standard methods. Gene mapping of the {alpha}-globin genes was carried out by PCR (7)(8). The {alpha}2-globin gene was selectively amplified by choosing the upstream primer in the 5' promoter region of both {alpha}-globin genes and the downstream primer in the 3' noncoding {alpha}2-specific region. Sequencing reactions were performed on amplified single-stranded DNA by the dideoxy chain termination method of Sanger et al. (9). The sequencing reverse primer was located at positions 355 to 375 upstream from the Cap site (5'-ACGTGCGCCACGGCGTTGGT-3'). The detection of codon mutation by use of restriction enzymes was performed with primers to amplify the 127-bp sequence encompassing codon 50 of the {alpha}2-globin and {alpha}1-globin genes. The forward and reverse primers (5'-CCCTCACTCTGCTTCTCCCC-3' and 5'-GGGCGTCGGCCACCTTCTT-3', respectively) are located at positions 226 and 353 upstream from the Cap site of the {alpha}-globin genes. The PCR conditions were as follows: denaturation at 94 °C for 1 min, annealing at 65 °C for 2 min for 30 cycles after an initial incubation at 95 °C for 5 min. The product of amplification was digested by restriction enzyme DsaI, which recognizes the sequence C{downarrow}C(A or G)(T or C)GG.

rna analysis
RNA was extracted from lymphocytes by the acid guanidinium thiocyanate-phenol-chloroform method (10). The cDNA fragment, obtained by reverse transcription-PCR, was sequenced with the same primer used for DNA sequencing, as described above.

Hb CHARACTERIZATION
Hemolysate was prepared from washed erythrocytes by the conventional CCl4-water method. Hb stability was assessed by isopropanol and heat denaturation tests (11). Methemoglobin and carboxyhemoglobin were measured by standard spectrophotometric techniques (12). Oxygen affinity was studied on fresh blood at pH 7.1 and 37 °C by a continuous recording method, using a Hemox Analyzer (TCS) as described previously (13). The measured venous P50 was corrected to standard values for the most important factors that affect oxygen affinity, i.e., pH, PCO2, 2,3-diphosphoglycerate, carboxyhemoglobin, and methemoglobin (14). Red cell 2,3-diphosphoglycerate was measured with an enzymatic test (Boehringer Mannheim), according to the manufacturer's instructions.

Electrophoresis of the hemolysate was performed at pH 8.4 in Tris-EDTA-borate buffer on cellulose acetate plates. Isoelectric focusing was performed on 0.5 mm thick polyacrylamide gel containing 20 mL/L ampholine, pH 6–8. The matrix concentration was 6% T (6 g monomers/100 mL) and 4% C (4 g cross-linker/100 g monomers). Hemolysate samples were applied in a 10-µL volume corresponding to 100 µg of total Hb loaded per track. Running conditions included a prerun at 400 V for 15 min and, after sample application, separation at 750 V for 2 h. Focusing was performed at 10 °C.

Additional characterization of Hb fractions was carried out by HPLC analysis using a Kontron 400 system (Kontron Instruments) composed of two pumps (model 422), an autosampler (model 360) with a 10-µL injector and a ultraviolet-visible detector set at 415 nm. The system was equipped with a cation-exchange polyCAT A column (0.46 x 20 cm, 5 µm particle size, 300 Å pore size; PolyLC). The flow rate was 2 mL/min. Two buffers of the following composition were used: 35 mmol/L bis-Tris, 3.0 mmol/L ammonium acetate, 0.1 g/L KCN, pH 6.47 (buffer A); and 35 mmol/L bis-Tris, 16.9 mmol/L ammonium acetate, 150 mmol/L sodium acetate, 1 g/L KCN, pH 7.0 (buffer B). Samples (10 µL, containing 10 µg of Hb) were applied to the column. The chromatographic separation was achieved by the following step gradient: 20–70% buffer B (20-min step), 70–100% buffer B (1-min step), and 100% buffer B (3-min step). The original conditions were then rapidly restored, and the column was equilibrated with 20% buffer B:80% buffer A for 3 min. Integration of the peaks was performed with the Kontron 450 Data System, Ver. 3.4.

Globin chain analysis was performed by reverse-phase HPLC using a Vydac 218TP54, C18 column (0.46 x 25 cm, 5 µm particle size). The hemolysate was diluted with water to deliver an Hb concentration of 5 g/L, and 10-µL samples were injected in the column. Buffer A was acetonitrile–methanol–155 mmol/L sodium chloride, pH 2.7 (50:20:30, by volume); and buffer B was acetonitrile–methanol–155 mmol/L sodium chloride, pH 2.7 (25:40:30, by volume). The elution was performed at a flow rate of 1.4 mL/min for 65 min, with the following gradient: 50–20% buffer B (55-min step) and 20% buffer B (5-min step). The detector was set at 215 nm.

structural studies
For preparative purposes, the heme was removed from the globins by precipitation of the total hemolysate in cool acetone (-20 °C) containing 0.3 mol/L HCl. Precipitated globins were washed with cold acetone, dried at room temperature, and then redissolved in 8 mol/L urea buffer. Globin chain separation was obtained by preparative chromatography on carboxymethyl cellulose (Whatman CM-52) in the presence of urea and dithiothreitol (15). After extensive dialysis to remove all traces of urea and salts, the abnormal {alpha} chains were digested with trypsin (N-methyl-tosyl-Lphenylalanine chloromethyl ketone-treated) in 50 mmol/L ammonium acetate, pH 8.3, using 20 mg of enzyme per 1 g of substrate. The reaction was performed at 37 °C for 5 h and stopped by acidification.

Tryptic peptide separation was performed by reverse-phase HPLC essentially as described (16). Briefly, 1 mL of the tryptic digest from native and mutant {alpha} chains (1 mg of starting protein) was centrifuged, and the supernatant was applied to an Aquapore RP-300 C8 column (25 x 0.46 cm; Applied Biosystems), equilibrated in 0.01 mol/L ammonium acetate, pH 5.7. Peptides were eluted with a 125-min linear gradient (0–30%) of 0.01 mol/L ammonium acetate, pH 5.7, in 500 mL/L acetonitrile at a flow rate of 1 mL/min. Approximately 60 mL of eluted native and mutated {alpha}T-6 peptides were N-terminal sequenced on a 477/A protein sequenator (Applied Biosystems) according to the manufacturer's instructions.

BFU-e STUDIES
Mononuclear cells were isolated from peripheral blood on a gradient of Histopaque 1077 (Sigma) and seeded at a density of 5 x 109 cells/L in {alpha} minimum essential medium supplemented with 100 g/L fetal calf serum (both from Life Technologies), 1 mL/L cyclosporin A (Sandoz), and 100 mL/L conditioned medium collected from cultures of the 5637 bladder carcinoma cell line (17). The cultures were incubated at 37 °C in an atmosphere of 5% CO2. After a 5-day incubation period in this phase I culture, the nonadherent cells were harvested, washed, and recultured for 10–15 days in a fresh medium composed of {alpha} medium, 300 mL/L fetal calf serum, 10 g/L deionized bovine serum albumin, 10 µmol/L ß-mercaptoethanol, 1.5 mmol/L L-glutamine, 1 µmol/L dexamethasone, and 1 kilounit/L human recombinant erythropoietin.

The Hb composition was studied by ion-exchange HPLC as described for hemolysate obtained from mature red blood cells. Globin chain biosynthesis was determined in mature erythroid colonies incubated with 3H-leucine (TRK 754; Amersham). Separation was carried out on a Beckman HPLC System Gold, and the total area under the chromatogram was measured. The percentage of globin synthesis was calculated according to the following equation: [{alpha}-globin/({alpha}-globin + ß-globin)] x 100 (18).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct sequencing of DNA and cDNA from patients with Hb J Sardegna detects a single nucleotide substitution C->A in the first position of codon 50 (Fig. 1 ). The result was the same for all 26 subjects studied. The CAC->AAC mutation is expected to produce a change of His to Asn. In the patient with Hb J and -{alpha}/{alpha}{alpha} (-3.7) genotypes the nucleotide usually at the first position of codon 50 is not evident because only one {alpha} gene (i.e., the {alpha}2 gene) carrying the C->A mutation is present. The C->A mutation was also confirmed by use of a restriction enzyme (Fig. 2 ). DsaI digestion of amplified DNA (127 bp) from a healthy subject produces three DNA fragments of 78, 24, and 25 bp (Fig. 2 , lane 1). The codon 50 mutation (C->A) removes a recognition site for this enzyme, generating a novel DNA fragment of 102 bp (Fig. 2 , lane 2). The faint band at the position corresponding to a 102-bp fragment in the DNA from a healthy subject represents a small amount of amplified partially digested DNA.



View larger version (87K):
[in this window]
[in a new window]
  		Figure 1. Sequence analysis of the {alpha}2-globin gene in a carrier of Hb J Sardegna and -{alpha}/{alpha}{alpha} genotype.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 2. Identification of the mutation by DsaI digestion of the amplified DNA.

The 127-bp sequence encompasses the codon 50 of the {alpha}2 and {alpha}1 genes. The forward and reverse primers are located at positions 226 and 353 upstream from the Cap site of the {alpha} genes. Lane 1, control DNA from a healthy subject; lane 2, DNA from an Hb J Sardegna carrier; lane 3, size markers; lane 4, amplified undigested DNA.

The results of both isopropanol and heat precipitation tests for Hb stability were within reference values. Methemoglobin and carboxyhemoglobin were within reference values (0.5% and 2.5%, respectively). The whole blood of the Hb J Sardegna carrier displayed an oxygen equilibrium curve of standard sigmoidal shape and shifted towards the left side of the health-related reference range. After standardization, the P50 value was found to be 24.9 mmHg (reference range, 24.6–28.9 mmHg). The red cell 2,3-diphosphoglycerate concentration was slightly decreased (0.68 mol/mol Hb; reference range, 0.77–1.07 mol/mol Hb).

Cellulose acetate electrophoresis revealed the presence of an abnormal band on the anode side of Hb A0 and migrating in a position consistent with a difference of two negative charges compared with normal Hb. Isoelectric focusing (Fig. 3 ) demonstrated the presence of two abnormal Hb components. The first component, amounting to 23% of the total Hb, displayed a much more acid isoelectric point than Hb A0; the second component focused slightly to the anode side of Hb A0 and was 5% of the total Hb.



View larger version (66K):
[in this window]
[in a new window]
  		Figure 3. Isoelectric focusing pattern at pH 6–8.

The anode is at the top. Lane 1, hemolysate from an Hb J Sardegna carrier; lane 2, hemolysate from healthy control; NHP, non-hemoglobin proteins.

The separation of Hb by cation-exchange HPLC is shown in Fig. 4 . The elution profile showed an anomalous Hb fraction that eluted just after the Hb A1c peak, in the Hb F position (indicated as X1 in Fig. 4 ). It amounted to ~25% of the total Hb. A second difference was a distinct increase in the proportion of the Hb A1d peak, probably suggesting the presence of an additional Hb component that eluted in the same position (indicated as X2 in Fig. 4 ). This peak was ~9% of the total Hb, whereas in fresh hemolysates from healthy individuals it never exceeded 3%. The Hb A2 concentration was slightly reduced (1.8%), whereas Hb F was not detectable because it was overlapped by the variant peak. This elution pattern was confirmed in all Hb J Sardegna carriers examined. HPLC analysis of the hemolysate from one Hb J Sardegna carrier was periodically repeated during a 20-day storage at 4 °C. The elution profiles showed no evident differences in the relative proportion of the different Hb fractions. HPLC analysis of Hb was also performed in culture-grown erythroid cells obtained from an Hb J Sardegna carrier. The results (not reported in detail here) showed that Hb composition of BFU-e-derived erythroblasts was similar to that observed in the hemolysate prepared from peripheral blood.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Cation-exchange HPLC separation of whole blood hemolysates prepared from an Hb J Sardegna carrier (top) and from a healthy subject (bottom).

X1, Hb JAsp; X2, Hb JAsn.

The separation of globin chains from whole hemolysate obtained by analytical reverse-phase HPLC is shown in Fig. 5 . Three main peaks were present, corresponding to ß chains, native {alpha} chains, and more hydrophilic variant {alpha} chains, which accounted for 24% of the total {alpha} subunits. Globin chain biosynthesis was analyzed in BFU-e-derived erythroblasts and indicated an {alpha}-chain/non-{alpha}-chain ratio of 1.02, suggesting a balanced rate of synthesis of the globin chains. Furthermore, the HPLC elution pattern presented no different peaks with respect to those detected in the separation performed on peripheral blood.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Reverse-phase HPLC analysis of globin chains from an Hb J Sardegna carrier.

ßA, normal ß chain; {delta}, normal {delta} chain; {alpha}A, normal {alpha} chain; {alpha}X, variant {alpha} chain; NHP, non-hemoglobin proteins.

For structural studies, the variant {alpha} chains were separated by preparative ion-exchange chromatography and digested with trypsin. The separation of tryptic peptides from native and variant {alpha} chains by reverse-phase HPLC is shown in Fig. 6 . From the comparison of the tryptic digests, it was noted that one peptide peak was missing, whereas a new, more hydrophilic peak was present. N-terminal sequence analysis revealed this new peptide as {alpha}41–56, which corresponds to an {alpha}T-6 peptide in which the His at position 50 was replaced by Asp. During the N-terminal sequencing of tryptic peptide 6, the expected amount of L-aspartic acid was recovered. This finding should exclude the presence of isoaspartic acid, because it probably would not be released efficiently during the Edman degradation. Moreover, no other additional peaks, with the exception of a small quantity of Ala, were detected. The small amount of Ala was probably derived from cross-contamination of the chromatographic peak with ßT-9 peptide, which carry Ala in position 10.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Elution profile obtained by reverse-phase HPLC separation of tryptic digests of normal (top) and mutant (bottom) {alpha} chains.

The arrows indicate the corresponding T-6 peptides.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported the characterization of Hb J Sardegna by both DNA and protein analyses. The peculiar feature that emerged from our data was an evident disagreement between nucleotide sequence and protein analysis, the first showing a His->Asn mutation, the latter showing a His->Asp substitution in the same position. Moreover, we have proven that the mutation occurs in the {alpha}2 gene because the subjects heterozygous for Hb J Sardegna and {alpha}-thalassemia (deletion of one of the two {alpha}2 genes) possess an {alpha}2 gene sequence with no trace of the normal sequence of the {alpha}A-globin. Furthermore, the abnormal Hb seemed to be present as two fractions. One possible explanation for these findings is that a C->A mutation occurs in codon 50 of the {alpha}-globin gene, which causes the substitution of His for Asn, and that the new {alpha}50 Asn residue subsequently undergoes a posttranslational partial deamidation to Asp.

Indeed, two forms of Hb J Sardegna were found. Hb J Sardegna-Asp was easily seen in the ion-exchange HPLC chromatogram, as well as in the electrophoretic and isoelectrophoretic pattern, because it is more negatively charged than Hb A0. The presence of Asp at position {alpha}50 was confirmed by the Edman degradation. The Hb fraction that coeluted with Hb A1d in an intermediate position between Hb J Sardegna-Asp and Hb A0 is probably Hb J Sardegna-Asn, in agreement with a difference of charge attributable to the replacement of the slightly protonated His by a neutral amino acid. No evidence for two abnormal {alpha} chains was seen, however, in reverse-phase HPLC separation of globin chains where only variant {alpha}-Asp chains could be detected. The {alpha}-Asn chains have polar properties similar to normal {alpha} chains and would be expected to coelute with them. Therefore, we were not able to purify {alpha}-Asn chains, which are present in very small amounts, to prove the presence of Asn in position 50 by Edman degradation. However, the presence of {alpha}-Asn chains can be inferred from the results of the isoelectric focusing.

In previous reports on Hb J Sardegna, the fraction carrying Asn had never been observed, probably because its detection requires high resolution techniques. Manca and Masala (3), in their paper on the identification of Hb J Sardegna by means of isoelectric focusing, reported the presence of an additional band focusing closer to the anode than Hb A0 and accounting for 3.5–11.6% of the total Hb; however, they did not identify it. It is reasonable to suppose that this unknown band corresponds to Hb J Sardegna-Asn.

In an attempt to verify where the conversion of Hb J Sardegna-Asn to Hb J Sardegna-Asp occurs, we have analyzed the Hb composition of erythroid progenitor cells. The fraction containing Asp that was found in BFU-e-derived erythroblasts suggests that the conversion of Asn to Asp takes place in a very early phase of red cell maturation. Afterward, the proportions of the two Hb fractions do not seem to change in peripheral blood or in vitro during sample storage. On the other hand, in one study of Hb Redondo, deamidation reportedly occurred rapidly in vitro, and after a few days of storage at 4 °C the relative proportions of the two fractions changed (19).

In addition to Hb J Sardegna, six other rare Hb variants have been reported in which deamidation of an Asn residue to an Asp occurs as a spontaneous posttranslational modification. A list of these variants, including Hb J Sardegna, is given in Table 1 . The mutation, the amino acid sequence surrounding the Asn undergoing deamidation, and the relative percentage of the two protein forms containing, respectively, Asn or Asp detected in blood have been reported for each variant. In most of these variants, including Hb J Sardegna, the deamidation reaction involves the mutant Asn itself, whereas in Hb La Roche-sur-Yon (20) and Hb J Singapore (21) the variant amino acids, a Gly and a His, respectively, induce the deamidation of the adjacent Asn residue. To support the possibility of the existence of particular tridimensional structures and sequences favoring deamidation reaction, it may be observed that in three of the seven Hb variants—Hb J Singapore, Hb La Roche-sur-Yon, and Hb Providence (22)—the deamidation is localized within the EF corner, and in Hb Osler (23) it occurs in the C-terminal region, which exhibits a flexible local conformation. Moreover, the sequences flanking the Asn site often include amino acid residues known to be potentially catalytic in the deamination reaction (see below). In particular, in Hb J Sardegna the {alpha}50 Asn residue is located near a serine residue at position {alpha}49 and a Gly residue at position {alpha}51. In all reported cases, the Asn residue is only partially deamidated, as proven by the finding that both of the Hb forms, containing either Asn or Asp, are present in peripheral blood. Hb Redondo occurs in rather low proportion to Hb A0 because it is a highly unstable variant (19), whereas the unusually low percentage of Hb Wayne is probably the result of an increased rate of destruction because of a relevant structural abnormality of the 3' terminal region (24).


View this table:
[in this window]
[in a new window]
  		Table 1. Hb variants exhibiting posttranslational deamidation of an Asn residue.

Indeed, the molecular mechanism proposed for deamidation reactions of Asn residues involves the formation of a cyclic succinimidyl intermediate from whose opening aspartyl and isoaspartyl residues are formed (25). The reaction is influenced by different factors, including both primary and tertiary structural characteristics of the protein and general properties of the media surrounding the protein (26). From the comparison of amino acid sequences in model peptides, it has been noted that the Asn residue undergoing deamidation is frequently found nearby a residue with a small, nonbulky side chain, such as Gly, Ser, or Ala, which can be contiguous in sequence or brought close to the amide residue by the tertiary structure of the protein (27). Other studies point out that Asn is more susceptible to deamidation when it has a basic or acid residue on one side and a threonine, serine, or cysteine residue on the other side (28). In addition to the nature of neighboring residues, the polypeptide conformation seems to be equally important. It has been shown, in fact, that a high degree of flexibility of the polypeptide chain and the proximity of a His residue are required for the deamidation reaction (20)(29). Moreover, the Gln residues also may undergo posttranslational deamidation (27), and it has been postulated that these deamidations may serve as molecular timers that control protein turnover and aging (28). In addition, in the case of the deamidation of Gln residues, the polypeptide conformation and sequence, especially in the surroundings of Gln residues, has been proven to be important determinants for the occurrence of these spontaneous deamidations (27)(28)(30).

Apart from this proposed deamidation mechanism, there are other possible explanations, including errors in codon reading because of unusual mRNA structure or mischarging of tRNA (31). We did not test such hypotheses, but such alternative mechanisms do appear to take place rather rarely.

From a more general viewpoint, with regard to the eventual consequences of the deamidation of an Asn residue, it is important to remember that Asn residues are important determinants of posttranslational self-splicing (32), and that there is relatively extensive literature about deamidation of Asn residues, especially concerning well-characterized proteins of pharmaceutical interest (26)(33). Therefore, we strongly recommend that, in the case of mutant proteins, both DNA and protein structure analysis should be undertaken to fully characterize the mutation, especially when Asn or Gln residues have been introduced in the mutant protein.

In conclusion, in consideration of the widespread use of DNA techniques for the molecular characterization of mutant proteins, we would like to note that protein structural studies are sometimes not performed or are performed as a second step. We report here some cases in which, to our knowledge, only DNA analysis has been exploited to characterize the mutations: (a) factor V Leiden (1696G->A, 506Arg->Gln), a mutant of factor V associated with resistance to activated protein C known to be a genetic risk factor for venous thrombosis (34); (b) the first discovered apolipoprotein B-100 mutant (10708G->A, 3500Arg->Gln), which causes familial defective apolipoprotein B-100, an autosomal disorder associated with hypercholesterolemia (35); (c) a recently discovered polymorphism in human orosomucoid (ORM1F1 60CAA->CAG) (36); and (d) a variant of red cell pyruvate kinase (1529G->A, 509Arg->Gln), which causes severe nonspherocytic hemolytic anemia (37). We hope that this missing information (i.e., protein structure analysis) will soon be available to define these abnormal proteins completely.


   Acknowledgments
 
We thank Louise Benazzi (Instituto Technologie Biomediche Avanzare, Consiglio Nazionale Rierche, Milan, Italy), for performing the isoelectric focusing shown in Fig. 3Up and for help in preparing the manuscript, and Severino Ronchi (Università degli Studi, Milan, Italy) for helpful suggestions and discussion.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tangheroni W, Zorcolo G, Gallo E, Lehmann H. Haemoglobin J Sardegna: {alpha}50(CD8)Histidine->Aspartic Acid. Nature 1968;218:470-471. [Medline] [Order article via Infotrieve]
  2. Masala B. Hemoglobinopathies in Sardegna. Hemoglobin 1992;16:331-351. [Web of Science][Medline] [Order article via Infotrieve]
  3. Manca L, Masala B. Identification of Hb J-Sardegna [{alpha}50(CE8)His->Asp] by HPLC and its incidence in northern Sardinia. Hemoglobin 1989;13:33-44. [Web of Science][Medline] [Order article via Infotrieve]
  4. Gallo E, Pugliatti L, Ricco G, Pich PG, Pinna G, Mazza U. A case of haemoglobin J Sardegna-{alpha}-thalassemia double heterozygosis. Acta Haematol 1972;47:311-320. [Web of Science][Medline] [Order article via Infotrieve]
  5. Meloni T, Pilo G, Camardella L, Concedda F, Lania A, Pepe G, Luzzato L. Coexistence of three hemoglobins with different {alpha}-chains in two unrelated children (with family studies indicating polymorphism in the number of {alpha}-globin genes in the Sardinian population). Blood 1980;55:1025-1032. [Abstract/Free Full Text]
  6. Maccioni L, Galanello R, Melis MA, Cao A. Hemoglobin constitution of double heterozygotes for {alpha} or ß-thalassemia and Hb J Sardegna. Hemoglobin 1984;8:497-507. [Web of Science][Medline] [Order article via Infotrieve]
  7. Dode C, Krishnamoorthy R, Lamb J, Rochette J. Rapid analysis of -alpha 3.7 thalassaemia and alpha alpha alpha anti 3. 7 triplication by enzymatic amplification analysis. Br J Haematol 1993;83:105-111.
  8. Bowden DK, Vickers MA, Higgs DR. A PCR-based strategy to detect the common severe determinants of alpha thalassaemia. Br J Haematol 1992;81:104-108. [Web of Science][Medline] [Order article via Infotrieve]
  9. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-5467. [Abstract/Free Full Text]
  10. Chomeczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. [Web of Science][Medline] [Order article via Infotrieve]
  11. . US Department of Health and Human Services, Public Health Service. Laboratory methods for detecting hemoglobinopathies 1984:45-58 Centers for Disease Control Atlanta, GA. .
  12. Fairbanks VF, Klee GG. Biochemical aspects of hematology. Tietz NW eds. Fundamentals of clinical chemistry 3rd ed. 1987:803-806 WB Sauders Philadelphia, PA. .
  13. Mosca A, Paleari R, Rubino FM, Zecca L, De Bellis G, Debernardi S, et al. Hb Abruzzo [ß143(H21)His->Arg] identified by mass spectrometry and DNA analysis. Hemoglobin 1993;17:261-268. [Web of Science][Medline] [Order article via Infotrieve]
  14. Wimberley PD, Burnett RW, Covingon AK, Fogh-Andersen N, Maas AHJ, Muller-Plathe O, et al. Guidelines for routine measurement of blood hemoglobin oxygen affinity. J Int Fed Clin Chem 1991;3:81-86. [Medline] [Order article via Infotrieve]
  15. Clegg JB, Naughton MA, Weatherall JD. Abnormal human haemoglobins, separation and characterization of the {alpha} and ß chains by chromatography, and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok). J Mol Biol 1966;19:91-108. [Web of Science][Medline] [Order article via Infotrieve]
  16. Wilson JB, Lam H, Pravatmuang P, Huisman THJ. Separation of tryptic peptides of normal and abnormal {alpha}, ß, and {delta} hemoglobin chains by high-performance liquid chromatography. J Chromatogr 1979;179:271-290. [Web of Science][Medline] [Order article via Infotrieve]
  17. Fibach E, Manor D, Oppenheim A, Rachmilewitz EA. Proliferation and maturation of human erythroid progenitors in liquid culture. Blood 1989;73:100-103. [Abstract/Free Full Text]
  18. Leone L, Monteleone M, Gabutti V, Amione C. Reversed-phase high-performance liquid chromatography of human haemoglobin chains. J Chromatogr 1985;321:407-419. [Web of Science][Medline] [Order article via Infotrieve]
  19. Wajcman H, Vasseur C, Blouquit Y, Esperito Santo D, Peres MJ, Martins MC, et al. Hemoglobin Redondo [ß92(F8)His->Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms. Am J Hematol 1991;38:194-200. [Web of Science][Medline] [Order article via Infotrieve]
  20. Wajcman H, Kister J, Vasseur C, Blouquit Y, Trastour JC, Cottenceau D, Galacteros F. Structure of the EF corner favors deamidation of asparaginyl residues in hemoglobin: the example of Hb La Roche-sur-Yon [ß81(EF5)Leu->His]. Biochim Biophys Acta 1992;1138:127-132. [Medline] [Order article via Infotrieve]
  21. Blackwell RQ, Hock Boon W, Liu CS, Weng MI. Hemoglobin J Singapore: {alpha}78Asn->Asp; {alpha}79Ala->Gly. Biochim Biophys Acta 1972;278:482-490. [Medline] [Order article via Infotrieve]
  22. Moo-Penn WF, Jue DL, Bechtel KC, Johnson MH, Schmidt RM, McCurdy PR, et al. Hemoglobin Providence. A human hemoglobin variant occurring in two forms in vivo. J Biol Chem 1976;251:7557-7562. [Abstract/Free Full Text]
  23. Kattamis AC, Kelly KM, Ohene-Frempong K, Reilly MP, Keller M, Cubeddu R, et al. Hb Osler [ß145(HC2)Tyr->Asp] results from posttranslational modification. Hemoglobin 1997;21:109-120. [Web of Science][Medline] [Order article via Infotrieve]
  24. Seid-Akhavan M, Winter WP, Abramson RK, Rucknagel DL. Hemoglobin Wayne: a frameshift mutation detected in human hemoglobin alpha chains. Proc Natl Acad Sci U S A 1976;73:882-886. [Abstract/Free Full Text]
  25. Galletti P, Ingrosso D, Manna C, Clemente G, Zappia V. Protein damage and methylation-mediated repair in the erythrocyte. Biochem J 1995;306:313-325.
  26. Wright HT. Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Crit Rev Biochem Mol Biol 1991;26:1-52. [Web of Science][Medline] [Order article via Infotrieve]
  27. Wright HT. Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins. Protein Eng 1991;4:283-294. [Abstract/Free Full Text]
  28. Robinson AB. Evolution and the distribution of glutamyl and asparaginyl residues in proteins. Proc Natl Acad Sci U S A 1974;71:885-888. [Abstract/Free Full Text]
  29. Clarke S. Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int J Peptide Protein Res 1987;:808-821. [Abstract/Free Full Text]
  30. Ingrosso D, Clarke S. Human erythrocyte D-aspartyl/L-isoaspartyl methyltransferases: enzymes that recognize age-damaged proteins. Adv Exp Med Biol 1991;307:263-275. [Medline] [Order article via Infotrieve]
  31. Momand JA, Clarke S. The fidelity of protein synthesis: can mischarging by aspartyl-tRNA (Asp) synthetase lead to the formation of isoaspartyl residues in proteins?. Biochim Biophys Acta 1990;1040:153-158. [Medline] [Order article via Infotrieve]
  32. Clarke ND. A proposed mechanism for the self-splicing of proteins. Proc Natl Acad Sci U S A 1994;91:11084-11088.
  33. Bischoff R, Kolbe HV. Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology. J Chromatogr B 1994;662:261-278. [Web of Science][Medline] [Order article via Infotrieve]
  34. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-67. [Medline] [Order article via Infotrieve]
  35. Soria LF, Ludwig EH, Clarke HRG, Vega GL, Grundy SM, McCarthy BJ. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B-100. Proc Natl Acad Sci U S A 1989;86:587-591. [Abstract/Free Full Text]
  36. Yuasa I, Umetsu K, Vogt U, Nakamura H, Nanba E, Tamaki N, Irizawa Y. Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S. Hum Genet 1997;99:393-398. [Web of Science][Medline] [Order article via Infotrieve]
  37. Rouger H, Valentin C, Craescu CT, Galacteros F, Cohen-Solal M. Five unknown mutations in the LR pyruvate kinase gene associated with severe hereditary nonspherocytic haemolytic anemia in France. Br J Haematol 1996;92:825-830. [Web of Science][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
D. Tchernitchko, M. Goossens, and H. Wajcman
In Silico Prediction of the Deleterious Effect of a Mutation: Proceed with Caution in Clinical Genetics
Clin. Chem., November 1, 2004; 50(11): 1974 - 1978.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. E. Robinson and A. B. Robinson
Deamidation of human proteins
PNAS, October 12, 2001; (2001) 221463198.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. E. Robinson and A. B. Robinson
Prediction of protein deamidation rates from primary and three-dimensional structure
PNAS, April 10, 2001; 98(8): 4367 - 4372.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Qin, D.-S. Yang, Y. Yang, M. A. Chishti, L.-J. Meng, H. A. Kretzschmar, C. M. Yip, P. E. Fraser, and D. Westaway
Copper(II)-induced Conformational Changes and Protease Resistance in Recombinant and Cellular PrP. EFFECT OF PROTEIN AGE AND DEAMIDATION
J. Biol. Chem., June 16, 2000; 275(25): 19121 - 19131.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. E. Robinson and A. B. Robinson
Deamidation of human proteins
PNAS, October 23, 2001; 98(22): 12409 - 12413.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paleari, R.
Right arrow Articles by Galanello, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paleari, R.
Right arrow Articles by Galanello, R.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Proteomics and Protein Markers
Right arrow Hematology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS