Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.     

The N-terminal sequence of the heavy chain of rabbit immunoglobulin IgG

The absence of an N-terminal amino acid with a free alpha-amino group from the heavy chain of rabbit immunoglobulin IgG has been confirmed and no evidence could be found of a blocking formyl, acetyl or propionyl group. The N-terminal amino acid appears to be pyrrolid-2-one-5-carboxylic acid (PCA) in all molecules. A mixed amino acid sequence follows in the approximate proportions: PCA-Ser-Val-Glu-Glu-Ser-Gly-Gly-Arg, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu(NH(2)), 20%. The heavy chains of a purified antibody, namely anti-(human serum albumin), and of immunoglobulin IgG from a rabbit homozygous at the allotypic loci both showed a similar mixed N-terminal sequence.     

Proteinase Inhibitors in Plant Seeds

The proteinase inhibitors I (R-I) and III (R-III) isolated from Japanese radish seed were characterized in terms of their N-terminal amino acids, amino acid composition and reacting groups. The amino acid composition of two proteins differed from each other, while histidine, methionine and tryptophan contents were all low. N-Terminal amino acids of these inhibitors determined by Edman degradation were the same; valine.

By modifying free amino groups in the inhibitors with trinitrobenzenesulfonic acid, R-III was greatly inactivated in proportion to the modification of amino groups, but the activity of R-I was not affected.

However, modification of arginyl residues of R-I by cyclohexanedione reduced its activity. These results indicate that R-I is an arginine-type and R-III is a lysine-type inhibitor.     

Changes in the Bohr effect due to pyridoxylation of the alpha-chain terminal amino groups of hemoglobin

Deoxyhemoglobins substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at the N-terminal amino groups of the alpha-chains were investigated by 31P-NMR spectroscopy. Titration curves of the 5'-side-chains show a substantial increase in acid strength in alpha-pyridoxylated deoxyhemoglobins when compared to the corresponding CO-liganded hemoglobins. These derivatives therefore contain a new oxygenation-linked acid group which opposes the normal Bohr effect. The loss in stabilization of the monoanion of the phosphate or phosphonate group derived from the three-dimensional structure can account for the lower pK of this ionization in deoxyhemoglobin as compared to CO-liganded hemoglobin. The reduction in the Bohr effect caused by modification of the alpha-chains with pyridoxal 5'-deoxymethyl-enephosphonate is quantitatively equal to the expected contribution of alpha-chain N-terminal amino groups.     

Glutathionylation of lens proteins through the formation of thioether bond

Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.     

Functionalization of aminomodified probes for atomic force microscopy

Probes for atomic force microscopy functionalized by bovine serum albumin were obtained, which may be used for molecular recognition studies. The procedure of the modification and functionalization of probes includes three stages. First, amino probes are obtained by modification in vapors of amino silane derivative. Then a homobifunctional amino reactive cross-linker is covalently linked to surface amino groups of the amino probe. And finally, the probe with the covalently attached cross-linker is functionalized by bovine serum albumin molecules. The probes obtained were characterized at different stages of the modification by atomic force microscopy: the adhesion force and the work of adhesion force were determined from histograms. The modification of probe surface was confirmed by visualization of bovine serum albumin and supercoiled pGEMEX DNA molecules immobilized on the amino mica and amino mica modified by cross-linker.     

A modification of neurotoxin RTX-III from Radianthus macrodactylus actinin with acetic anhydride

Upon modification of neurotoxin RTX-III at amino groups with [3H]acetic anhydride, four monoacetyl and four diacetyl derivatives have been obtained. Acetylation of the N-terminal amino group led to 12-fold decrease of toxicity in mice. Monoderivatives of the toxin with either Lys or two out of three C-terminal Lys residues modified showed a 2-fold drop in toxicity as compared with the native RTX-III. Diacetylation caused a 30 to 35-fold decrease in toxicity, the N-terminal amino group being modified in all the derivatives. As assessed by circular dichroism method, the modification of amino groups, except for N-terminal one, affected secondary structure of the toxin. The data suggest the N-terminal amino group to be essential for toxicity of RTX-III.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Isolation and properties of bovine kidney aminopeptidase A]

Aminopeptidase A, which specifically hydrolyses N-terminal dicarbonic amino acid residues containing free alpha-amino groups, is isolated from bovine kidney. The enzyme is 500-fold purified and is homogenous under electrophoresis and ultracentrifugation. Aminopeptidase A has pH optimum of 7.5, it is activated with Ca2+ and inactivated with EDTA. Its molecular weight is 53000. The enzyme hydrolyses alpha-L-aspartyl-beta-naphtylamide and splits peptides having N-terminal glycine, lysine, arginine and alanine are hydrolyzed by the enzyme much slower. Aminopeptidase A does not attack alpha-L-alanyl-beta-naphtylamide, leucineamide, insulin, peptides with blocked N-terminal amino acid and peptides which have proline to be the second N-terminal amino acid.     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Amino acid sequences of fragments I and II obtained by cyanogen bromide cleavage of rat serum albumin

The amino acid sequences of fragment I, the N-terminal cyanogen bromide fragment, and fragment II, a fragment between the first and second methionine residues, of rat serum albumin were determined by conventional methods in consideration of the sequences of human and bovine serum albumin. These sequences were compared with those of human and bovine serum albumin.     

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.     

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.     

Molecular characterization of a new lectin from the marine alga Ulva pertusa

A new lectin, named UPL1, was purified from a green alga Ulvapertusa by an affinitychromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectinwas about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinatingactivity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. Thelectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and hadhigher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined(P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned byrapid amplification ofcDNA ends (RACE) method (AY433960). Sequence analysis of upll indicated it was! 084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the matureUPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 aminoacids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not showamino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigmof a novel lectin family.     

Preparation and characterization of antibodies with specificity for the amino-terminal tetrapeptide sequence of the platelet-derived connective tissue activating peptide-III

Antisera selectively reactive with the N-terminal tetrapeptide sequence of the platelet-derived connective tissue activating peptide-III mitogen were prepared and characterized. Solid phase synthesized Z-Asn-Leu-Ala-Lys(Z)-OH tetrapeptide representing the N-terminus of the mitogen was used as an immunogen after carbodiimide mediated coupling to methylated bovine serum albumin carrier and subsequent removal of Z groups. Anti-tetrapeptide sera demonstrated cross-reactivity to the mitogen but not beta-thromboglobulin, fibroblast growth factor, or epidermal growth factor, and a limited cross-reactivity to parathyroid hormone. The studies indicate that the N-terminal sequence of the mitogen is accessible for binding with antibody and the antitetrapeptide sera provide a reagent for the selective measurement of biologically active mitogen in the presence of structurally similar beta-thrombo-globulin. In addition, computer analysis of amino acid sequences revealed that few proteins contain the Asn-Leu-Ala-Lys sequence and of those that do, many are retroviral proteins or transforming polyproteins.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase

A new post-translational modification site in the growth factor domain of urinary type plasminogen activator has been identified. A glycopeptide containing the monosaccharide, fucose, covalently linked directly to the peptide backbone has been isolated from the tryptic digest of pro-urokinase expressed in a mouse hybridoma cell line Sp 2/0 Ag 14. The glycopeptide was isolated by semi-preparative reversed phase high performance liquid chromatography. The identity of a fucose containing peptide was confirmed by carbohydrate analysis, amino acid analysis and plasma desorption mass spectrometry (PDMS). A combination of these methodologies showed an equimolar ratio of peptide and fucose in the glycopeptide. This modification is not detected without mass spectrometry because the fucose residue is hydrolyzed under standard acidic conditions of amino acid composition and N-terminal sequence analysis. The site of attachment of fucose to the peptide has been localized towards the N-terminus (within first 23 amino acids) of the protein. Also, the carbohydrate composition of recombinant pro-urokinase is reported.     

The effect of chemical modification on the immunochemical activity of Rm-III neurotoxin from the anemone Radiantus macrodactylus]

A chemical modification was used for studying the organization of antigenic determinants of neurotoxin Rm-III from the sea anemone Radianthus macrodactylus. Immunochemical experiments were performed using competitive enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies to Rm-III. The modification affected N-terminal amino group of Gly1, Lys4, Arg13, Trp30 residues, a residue in the Lys46-Lys47-Lys48 sequence, two different residues in the Asp6-Asp7-Glu8 sequence in two samples of the toxin, and two disulphide bonds. Only the modification of the disulphide bonds led to a considerable change in the toxin's affinity to antibodies. The modification of Trp30 resulted in two-fold decrease of the toxin concentration necessary for 50% inhibition of the test-system, whereas upon modification of any other amino acid residue this concentration increased but not more than by 2.2 times. It is suggested that Rm-III sequence lacks individual residues which are of great importance for the toxin's antigenic activity, its conformation being of vital importance for the formation of the toxin's antigenic determinants.     

Effect on brain monoamines in the rat of substituted and protected analogues of the oxytocin fragment, prolyl-leucyl-glycinamide following N-terminal substitution by D- and L-pipecolic acid

The effect of modified and substituted analogues of prolyl-leucyl-glycinamide (PLG, MIF-I) was investigated on the steady-state level of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in various brain regions. Proline was replaced by D- or L-pipecolic acid (D- or L-Pip), which analogues in turn were protected by benzoxy-carbonyl (Z) group. Substitution by D- or L-pipecolic acid caused opposite changes in the DA level of the dorsal hippocampus. These effects were absent it the N-terminal of either analogues was protected by Z-group. Following the above mentioned N-terminal modification, the amino group of the C-terminal glycine was also substituted by methyl-esther (Gly-OMe), Z-D-Pip-Leu-Gly-OMe decreased the mesencephalic DA level, while Z-L-Pip-Leu-Gly-OMe increased the 5-HT content of the mesencephalon and striatum. In general, N-terminal substitution by D-pipecolic acid decreased, whereas that by L-pipecolic acid increased the monoamine level in the brain.     

Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.