Replacement Of Asn Research Articles

Proteolytic activation of MST/Krs, STE20-related protein kinase, by caspase during apoptosis.

The Fas system has been extensively investigated as a model of apoptosis and the caspase cascade has been shown to be a characteristic mechanism of signaling of apoptosis. We have identified and purified a kinase that was activated after the stimulation of Fas on human thymoma-derived HPB-ALL cells. Partial amino acid sequencing of the purified kinase revealed it to be MST/Krs, member of the yeast STE20 family of protein kinases. MST/Krs was activated by proteolytic cleavage and proteolytic activation was blocked by the caspase inhibitor, Z-VAD-FK. A mutant MST with Asp-->Asn replacement at a putative caspase cleavage site was resistant to either the proteolytic cleavage or the activation of the kinase activity. These findings suggest that proteolytic activation is one activation mechanism of MST and plays a role in apoptosis.

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon QB photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 → Asn replacement on QB−/QB FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 → Asp, Arg M233 → Cys, or Arg H177 → His suppressor mutation. The proton uptake by Glu L212 upon QB− formation, as reflected by the positive band at 1728 cm−1, is increased in the Asn M44 → Asp and Arg H177 → His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 → Asn mutant RCs. Only the Arg M233 → Cys suppressor mutation affected the 1728 cm−1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 → Asn lesion.

Polymorphisms in the coding exons of the human luteinizing hormone receptor gene

Four polymorphisms were identified in the coding exons of the human luteinizng hormone/chorionic gonadotropin receptor (hLHR) gene. A CTGCAG insertion occurred after nucleotide 54 in 8 of 34 independent chromosomes examined. The heterozygosity frequency was 0.353. This Leu-Gln dipeptide insertion in the first Leucine repeat of the hLHR extracellular domain did not affect the ligand binding affinity of the receptor. Among 54 chromosomes analyzed, 64.8% was A and 35.2% was G at nucleotide 872 in exon 10. The heterozygosity frequency was 0.115. The A/G substitution led to the replacement of Asn by Ser in the G alllele and the abolition of a potential N-glycosylation site. Another polymorphism occurred at nucleotide 935. Fifty nine percent of chromosomes examined were A and 41% were G at this site with the encoded amino acid being Ser in the former and Asn in the latter. The heterozygosity frequency was 0.192. This polymorphism did not have biological consequence. Both of the exon 10 polymorphisms showed ethnic prevalence with the 872 G allele and the 935 A allele predominantly in non-Caucasians. The fourth polymorphism was neutral and occurred at nucleotide 1065 in exon 11, with C in 60% and T in 40% of the 50 chromosomes examined. These polymorphisms are useful for tracking the inheritance of specific hLHR allele. Hum Mutat 11:333–334, 1998. © 1998 Wiley-Liss, Inc.

A novel missense mutation Ile538Val in the fibroblast growth factor receptor 3 in hypochondroplasia. Mutations in brief no. 122. Online.

Hypochondroplasia and achondroplasia are skeletal dysplasias, characterized by autosomal dominant inheritance and disproportionate short stature, which occurs mainly due to growth failure of the extremities. Both dysplasias have been mapped to fibroblast growth factor receptor 3 (FGFR3) gene. For hypochondroplasia, two point mutations, both responsible for the Asn540Lys substitution in the region coding the tyrosine kinase domain have been reported. Here we report an A to G transition at position 1651, predicting an Ile538Val substitution in the FGFR3, in hypochondroplasia. The substitution is found in a swedish family with three affected members. The criteria for hypochondroplasia were disproportionate short stature and radiological evidence of shortened long bones and decrease or absence of normal increase in interpedicular distances of the lumbar column. The mutation was detected by direct sequencing and restriction enzyme Tai I digestion. The base change was not found in the FGFR3 genes of unaffected members of the family nor in seventy-five unrelated unaffected individuals, suggesting that it was not a polymorphism. The Ile538Val substitution is a conservative amino acid change (a hydrophobic amino acid incorporated for another hydrophobic amino acid). Nevertheless, it is located in the stretch of nine amino acids, which is highly conserved among all the human fibroblast growth factor receptors. Considering the location of this substitution and the segregation with the phenotype in this family, we propose that it is a causative mutation of hypochondroplasia. It is difficult to establish whether the Ile538Val substitution is rare in hypochondroplasia patients or whether the individuals, who have a moderate degree of short stature, rarely seek medical help for the short stature and consequently are rarely diagnosed as affected by hypochondroplasia.

Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3.

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor. Herein prosaposin was found to increase sulfatide concentrations in primary and transformed Schwann cells (iSC) and oligodendrocytes (differentiated CG4 cells). Of the four mature saposins, only saposin C was found to increase sulfatide concentrations in these cell types. A similar result was obtained by using peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain. Dose-response curves demonstrated maximal enhancement by saposin C and prosaptides at low nanomolar concentrations (5-10 nM). The increase in sulfatide concentration by a 14-mer prosaptide, TX14(A), in CG4 oligodendrocytes was about 3-fold greater than in primary Schwann cells. A mutant prosaptide with a single amino acid replacement of Asn --> Asp was inactive. Prosaptides did not induce cell proliferation of primary Schwann cells, iSC cells, or CG4 oligodendrocytes but nanomolar concentrations of prosaptides prevented cell death of iSC cells and CG4 oligodendrocytes. Immunoblot analysis demonstrated that phosphorylation of both mitogen-activated protein kinase p-42 and p-44 isoforms were enhanced 3- to 5-fold after 5 min of treatment with prosaptides at concentrations of 1-5 nM. These findings suggest that prosaposin and prosaptides bind to a receptor that initiates signal transduction to promote myelin lipid synthesis and prolong cell survival in both Schwann cells and oligodendrocytes. Prosaposin may function as a myelinotrophic factor in vivo during development and repair of myelinated nerves explaining the deficiency of myelin observed in prosaposin-deficient mice and humans.

Identification and isolation of a bactericidal domain in chicken egg white lysozyme.

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fractionation of clostripain-digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I-V-S-D-G-N-G-M-N-A-W-V-A-W-R (amino acids 98-112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. The peptide I-V-S-D-G-N-G-M consisting of the eight amino acids of the N-terminal side had no bactericidal properties, whereas the peptide N-A-W-V-A-W-R of the C-terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R-A-W-V-A-W-R) increased the bactericidal activity considerably. The D enantiomer of R-A-W-V-A-W-R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

Amino-Acids of the Third Transmembrane Domain of the AT 1A Angiotensin-II Receptor Are Involved in the Differential Recognition of Peptide and Nonpeptide Ligands

The differential role of amino acids of the third transmembrane domain on peptide and nonpeptide recognition by the AT 1 angiotensin II receptor has been evidenced. The mutation of Ser 105 into alanine completely abolished peptide agonist and antagonist binding, while the binding of nonpeptide ligands, including the original radioligands [ 3H] LF 7-0156 and [ 3H] LF 8-0129, was more moderately affected. Reverse pharmacological changes, i.e., unchanged affinities for peptide agonists or antagonists and drastically reduced affinities for nonpeptide antagonists,were observed upon alanine replacement of Asn 111. These results confirm that the binding sites for peptide and nonpeptide molecules are not totally overlapping and delineate new amino acids as candidates for the selective receptor interaction with the two categories of ligands. Their integration in topographical studies is discussed.

Contribution of a single heavy chain residue to specificity of an anti‐digoxin monoclonal antibody

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene

Hemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC-->AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A-->T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP). At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

Novel missense mutation in cardiac β myosin heavy chain gene found in a japanese patient with hypertrophic cardiomyopathy

We have analyzed the exon 9, 13, 14, 15, and 16 of cardiac β myosin heavy chain gene in 96 Japanese patients with hypertrophic cardiomyopathy by using PCR-DNA conformation polymorphism analysis. The analysis revealed a sequence variation of the exon 16 in one patient. The sequence variation of a G to C transversion with replacement of Asn by Lys at the codon 615 was confirmed by sequencing and by dot-blot hybridization with an allele-specific oligonucleotide probe. Because the missense mutation was found at the residue conserved through birds to humans, this mutation was suggested to be a cause of hypertrophic cardiomyopathy in the patient. This is the first report of a mutant cardiac β myosin heavy chain gene in the Japanese population.

Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the C H2 domains

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the C H2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyi and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyi antibody is due to increased catabolism in the extravascular tissues.

Human growth hormone-releasing hormone analogues with much improved in vitro growth hormone-releasing potencies in rat pituitary cells

Enhancement of the amphiphilic α-helical properties of the central and C-terminal regions of growth hormone-releasing hormone (GRH) by substitution with helix-favouring amino acids, particularly Ala, can result in significant improvements in GH-releasing potencies using monolayer cultures of rat pituitary cells, a system which reflects analogue receptor affinity rather than effects of structural modifications on pharmacokinetic properties. For instance, previously reported, helix-enhanced [Ala 15]GRH-(1–29)NH 2 was presently 5 times more potent than [Gly 15]GRH-(1–29)NH 2] in this assay. The extent and importance of α-helical character further towards the N-terminus is less clear since Chou-Fasman probability calculations indicate also the possibility of β-bend formation in the 6–10 region. However, replacement of Asn[su8] with Ala resulted in a 4-fold improvement in potency and when this was combined with Ala 15 to give [Ala 8, 15]GRH-(1–29)NH 2 a 15-fold increase in potency was achieved and combination of D-Ala 2, Ala 8 and Ala 15 gave a 27-fold increase indicating that the effects of all of these modifications were additive. Computer analysis furthermore revealed that substitution of Ala for Ser in position 9 should also increase α-helix probability from 0.93 to 1.05. [D-Ala 2,Ala 8,9,15]GRH-(1-29)NH 2 was 49 times more potent than GRH itself making it by far the most potent analogue thus far reported in an in vitro assay system. The Ala 8 and Ala 9 substitutions were also effective in improving the inhibitory potency of a GRH receptor antagonist, [D-Arg 2,Leu 27]GRH-(1–29)NH 2. [D-Arg 2,Ala 8,15]GRH-(1-2 and [D-Arg 2,Ala 8,9,15]GRH-(1–29)NH 2 displayed IC 50 values of 5.9 × 10 −8 and 1.7 × 10 −8 M, respectively, against GRH-stimulated GH release compared to an IC 50 of 2.2 × 10 7 M for the unmodified control analogue and are thus commensurate with corresponding agonist analogue potency improvements.

Identification of hemoglobin G-Philadelphia (α68 Asn→Lys) and homoglonin matsue-oki (α75 Asp→Asn) in a black infant

Two α-chain variants, Hb G-Philadelphia and Hb Matsue-Oki, were present in members of a relatively large black family from South Carolina. The four Hb G-Philadelphia heterozygotes averaged 35.6% Hb G, suggesting the presence of an α-thalassemia-2 condition in cis to the Hb G mutation, which was confirmed by DNA structural analysis. The seven Hb Matsue-Oki heterozygotes averaged 22.2% Hb MO and likely have four active α-chain genes. One infant was a compound heterozygote for the two Hb variants which could not be separated from each other. The quantity of Hb G plus Hb MO was 58% by DEAE-cellulose chromatography and 69% by chain analyses. These results and the family data indicate that this child had three active α-chain genes, of which one regulated the synthesis of the normal a chain, one was mutated to give the α G chain, and one to give the α MO chain. The amino acid substitutions in Hb G-Philadelphia and Hb Matsue-Oki are located in the tryptic peptide αT-9, which is 29 amino acid residues long. Structural analyses of these abnormalities made use of high-pressure liquid chromatography for the separation of both tryptic and thermolytic peptides and of a highly sensitive ultra-micro sequencing procedure. Although the α68 Asn→Lys substitution is readily demonstrable in Hb G-Philadelphia the elucidation of the α75 Asp→Asn replacement in Hb Matsue-Oki was greatly facilitated by the use of these microprocedures.