Biomedical subjects

A Schierhorn

Publications and source records attributed to A Schierhorn.

At least 19 recordsLinked to original sources

PubMed1999

Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3.

Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.

Amino Acid Sequence↗

PubMed1999

Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein.

Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed by o-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit. L-[14C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.

Amino Acid Oxidoreductases↗

PubMed1999

Using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer for combined in-source decay/post-source decay experiments.

Mass spectrometric experiments with fragment ions have not yet been possible with a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer because intense signals of fragment ions were rarely observed during continuous extraction and the mass resolution of in-source formed fragment ions has been low. This paper describes a combination of MALDI in-source decay and post-source decay experiments on a MALDI-TOF mass spectrometer equipped with delayed extraction. Fragment ions initially formed in the ion source were selected by the precursor ion gate and investigated by post-source decay. The in-source formed fragment ions were sufficiently excited to undergo further metastable decay. The new method was applied to linear peptides, the cyclic peptide gramicidin S and a pentasaccharide, leading to unambiguous structural information.

Carbohydrate Sequence↗

PubMed1999

A strategy for rapid and efficient sequencing of Lys-C peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry post-source decay.

A modification procedure for Lys-C peptides is described which simplifies the correct assignment of the amino acid sequence. Release of the C-terminal lysine from Lys-C peptides by carboxypeptidase B and subsequent N-terminal acetylation of the resulting peptides leads to predictable shifts of the C- and N-terminal fragment ions in Matrix-assisted laser desorption/ionisation time-of-flight post-source decay mass spectra and facilitates the correct assignment of mostly complete amino acid sequences for oligopeptides. The derived sequences of peptides from unknown proteins were used to search in databases for homologous protein sequences. Our method was applied to an unknown protein isolated from eggs of Drosophila melanogaster, resulting in the identification of a peptidyl prolyl cis-trans-isomerase.

Acetylation↗

PubMed1999

Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation.

Ribonuclease (RNase) A and the more stable glycosylated RNase B differ by a carbohydrate moiety (GlcNAc2Man5-9) attached to Asn34. As previously shown, the first proteolytic cleavage sites to appear on thermal denaturation of both enzymes are in the structural region around Asn34. To discriminate the contribution of the modifying moiety to the stabilization toward thermal unfolding, on the one hand, and proteolytic fragmentation, on the other hand, the carbohydrate chain of RNase B was shortened by treatment with glycosidases to obtain GlcNAc-RNase and (GlcNAc)2Man3 -RNase and extended by binding to concanavalin A or concanavalin A-agarose. The results show a saltatory increase of the thermal unfolding constants and transition temperatures of GlcNAc-RNase in comparison to RNase A, whereas the extension of the modification at Asn34 in the other RNase species does not further increase thermal stability. Therefore, the stability difference between RNase A and RNase B derivatives is attributed to the first carbohydrate unit. In contrast, the rate of proteolysis decreases gradually with increasing volume of the modifying moiety. As concluded from the analysis of the primary cleavage fragments, the main degradation pathway is shifted from the Asn34-Leu35 to the Thr45-Phe46 peptide bond due to increasing shielding effects.

Animals↗

PubMed1998

Influence of the carbohydrate moiety on the proteolytic cleavage sites in ribonuclease B.

The influence of glycosylation on proteolytic degradation was studied by comparing cleavage sites in ribonuclease A (RNase A) and ribonuclease B (RNase B), which only differ by a carbohydrate chain attached to Asn34 in RNase B. Primary cleavage sites in RNase B were determined by identifying complementary fragments using matrix-assisted laser desorption/ionization mass spectrometry and compared with those in RNase A [Arnold et al. (1996), Eur. J. Biochem. 237, 862-869]. RNase B was cleaved by subtilisin even at 25 degrees C at Ala2-Ser21 as known for RNase A. Under thermal unfolding, the peptide bonds Asn34-Leu35 and Thr45-Phe46 were identified as primary cleavage sites for thermolysin and Lys31-Ser32 for trypsin. These sites are widely identical with those in RNase A. Treatment of reduced and carbamidomethylated RNase A and RNase B with trypsin led to a fast degradation and revealed new primary cleavage sites. Therefore, the state of unfolding seems to determine the sequence of degradation steps more than steric hindrance by the carbohydrate moiety does.

Carbohydrate Metabolism↗

PubMed1997

Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12.

A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cis/trans isomerase (PPIase) domain of E. coli trigger factor (TF) that was thought to be significant with regard to the enzymatic properties of the bacterial enzyme. We examined whether the alteration of a negatively charged side-chain at position 37 (FKBP numbering) and a phenylalanine at position 99, both highly conserved through both types of enzymes, leads to parallel effects on the catalytic activity of both FKBP12 and TF-PPIase domain in a series of tetrapeptide substrates with different P1 subsites. For the latter enzyme, substitution of Glu178 by Val or Lys, which aligns to Asp37 in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp37Leu and Asp37Val variants of FKBP12. Regardless of the P1 subsite of the substrate used for the assay, mutation of Phe233Tyr generated a protein variant of the TF-PPIase domain with about 1% of the wild type PPIase activity. Dependent on the substrate nature, a moderate decrease as well as a 4.8-fold increase in k(cat)/K(M) could be determined for the corresponding Phe99Tyr FKBP12 variant. Neither of the mutations of the TF-PPIase domain was able to implant FK506 inhibition found as a major characteristic of the FKBP family of PPIases.

Amino Acid Isomerases↗

PubMed1996

Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.

Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.

Amino Acid Isomerases↗

PubMed1996

Isolation and amino acid sequence of a new 22-kDa FKBP-like peptidyl-prolyl cis/trans-isomerase of Escherichia coli. Similarity to Mip-like proteins of pathogenic bacteria.

We identified a periplasmic peptidyl-prolyl cis/trans-isomerase (PPIase) of the (FK506-binding protein (FKBP) type in Escherichia coli (FK506 represents a natural peptidomacrolide containing an acylated pipecolic acid residue). After purification to homogeneity, its complete amino acid sequence was determined by a combination of Edman degradation and electrospray mass spectrometry of the authentic protein and peptides generated by proteolysis. The molecular mass calculated from the amino acid sequence of the protein was 22,085.53 Da, which corresponded perfectly with the value of 22,084 +/- 1.47 Da as determined by mass spectrometry. The corresponding gene was cloned and analyzed, and Southern blot experiments revealed the existence of similar genes in various Gram-negative bacteria. The amino acid sequence of the novel FKBP22 shows similarity to Mip (macrophage infectivity potentiator)-like proteins produced by a number of pathogenic bacteria. However, FKBP22 is inhibited more strongly by FK506 than are other Mip-homologues, as indicated by the Ki value of 25 nM. The subsite specificity regarding the P1 position of the substrate resembles that for Mip-FKBP25 from Legionella pneumophila. The mature FKBP22 enzyme of 205 amino acids exists as a dimer in solution.

Amino Acid Isomerases↗

PubMed1996

N-peptidyl, O-acyl hydroxamates: comparison of the selective inhibition of serine and cysteine proteinases.

Two series of N-aminoacyl, O-benzoyl hydroxamates were designed to investigate the influence of the substituted benzoyl residue on the hydrolytic stability and the reactivity of these potential inhibitors towards selected cysteine and serine proteinases. The inactivators react more rapidly with cysteine proteinases than with the serine enzymes tested. While Z-Phe-Gly-NHO-Nbz is the most reactive inhibitor of cathepsin L, inhibiting the target protein by a second order rate constant of 932.000 M-1 s-1, the bacterial serine proteinase thermitase is inhibited best by Z-Gly-Phe-NHO-Nbz, exhibiting a second-order rate constant of 1.170 M-1 s-1. Thiolsubtilisin, having the thiol-group as the reactive nucleophile instead of serine, exhibits specificity constants of the inactivation two orders of magnitude smaller than subtilisin. The degree of selectivity of the inhibitors relative to cathepsin B, cathepsin L, cathepsin S and papain varies up to two orders of magnitude with respect to their second order rate constant of inactivation. The inhibitory reactivity of these compounds varies only up to sixfold depending on the benzoyl substituent. Similarly, the rate constants for the hydrolytic decomposition of the compounds vary by a factor of about 6, suggesting that the structural and mechanistic features of the compounds which are responsible for decomposition as well as for the enzyme inhibition are the same. Comparing both reactions, the data allow the calculation of an acceleration factor of 2.4 x 10(10) for the inhibition of cathepsin L by its most effective inhibitor, clearly characterizing this enzyme inhibition reaction as enzyme-activated.

Cathepsins↗

PubMed1996

Thermal unfolding and proteolytic susceptibility of ribonuclease A.

With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20-65 degrees C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electrophoretic bands, partial N-terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix-assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31-Ser32 and Arg33-Asn34 for trypsin and Asn34-Leu35 and Thr45-Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent beta-structure containing Thr45-Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.

Amino Acid Sequence↗

PubMed1996

Substrates containing phosphorylated residues adjacent to proline decrease the cleavage by proline-specific peptidases.

Thirteen dipeptide rho-nitroanilides of the common structure H-Xaa-Pro-4-NA (Xaa = serine, threonine and tyrosine) and seven tripeptide rho-nitroanilides of the common structure H-Gly-Xaa-Pro-4-NA (Xaa = serine or threonine) were prepared and analyzed as substrates of the proline-specific peptidases dipeptidyl peptidase IV and prolyl endopeptidase, respectively. The side chains of the hydroxy amino acids were synthetically modified by various acyl-, benzyl- and phosphate residues. The presence of aliphatic or aromatic residues attached to the side chain of the P2-hydroxy amino acids resulted in no significant change of the specificity constants of the enzyme-catalyzed substrate hydrolysis. In some cases, however, substrate inhibition was observed. In contrast, the reactivity of dipeptidyl peptidase IV and prolyl endopeptidase decreases more than two orders of magnitude towards the phosphorylated di- and tripeptide substrates compared to the hydrolysis of unmodified substrates. The kinetic data obtained with the model compounds suggest that side-chain modification of proline-containing peptide substrates may influence their resistance towards the hydrolytic activity of proline-specific hydrolases. Additionally, the results support that structural changes of the substrate during enzyme-hydrolysis may be involved in the mechanism of action of proline-specific serine peptidases. From this result we speculate that posttranslational phosphorylation of peptide sequences found in protein kinase recognition motifs such as -Xaa-Ser/Thr-Pro-Yaa- and -Xaa-Pro-Ser/Thr-Yaa- may serve as structural determinants that modulate their proteolytic stability.

Amino Acid Sequence↗

PubMed1996

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

Amino Acid Isomerases↗

PubMed1996

High-resolution separation of amyloid beta-peptides: structural variants present in Alzheimer's disease amyloid.

In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid beta-peptide (Abeta). Structural variants of AD-associated Abeta peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Abeta variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Abeta peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Abeta variants terminating at either residue 40 or 42. Abeta variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate the structural isomers of Abeta1-40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Abeta variants, because each variant can now be studied individually.

Alzheimer Disease↗

PubMed1996

Solid-phase synthesis of peptide-4-nitroanilides.

A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides and other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing the side-chain carboxyl groups for transient anchoring to the resin. Suitable synthons for this method, Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptides of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Glu/Asp, Gln) were synthesized and were shown to be substrates for the protease subtilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans-isomerases (PPIases E.C. 5.2.1.8.). The method was extended to amino acids possessing a side chain missing an anchor for binding to the matrix. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptide derivative Fmoc-Glu-Phe-NH-Np with the carboxyl group to Rink amide resin using standard SPPS procedures. Additionally this procedure allowed us the preparation of peptidyl-arylamides, utilizing the commercial available Fmoc-Glu-OAll as building block. A mixture of pentapeptide-4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np was synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the protease subtilisin Carlsberg. It could be shown that peptides with the hydrophobic amino acids Phe, Tyr, Leu and Val in the varied P3-position were most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed.

Anilides↗

PubMed1994

A homodimer represents an active species of the peptidyl-prolyl cis/trans isomerase FKBP25mem from Legionella pneumophila.

The molecular mass of the native FK506-binding peptidyl-prolyl cis/trans isomerase (PPIase) FKBP25mem from Legionella pneumophila (Mip (macrophage infectivity potentiator) protein) was determined by two methods. By gel-permeation chromatography we found no indication of the presence of the monomeric enzyme. However, an oligomeric state with a molecular mass of about 62 kDa was detected. By cross-linking with dimethyl pimelimidate and subsequent SDS-PAGE of either the surface proteins of intact L. pneumophila cells or the purified recombinant FKBP25mem in solution, we observed an immunoreactive band indicative of a mass in the dimer range. In contrast to human recombinant FKBP12, the enzymatic activity of Legionella FKBP25men was strongly dependent on the protein concentration, pointing to a dimer as the most active species. However, the inhibition by FK506 yielded a nearly constant value of Ki of about 250 nM when measured in the same range of FKBP25mem concentration. These results may be explained by the fact that monomeric FKBP25mem has little, if any, influence on enzymatic activity when compared with the homodimer.

Amino Acid Isomerases↗

PubMed1994

Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging.

The Alzheimer A beta amyloid peptide (A beta) is the principal proteinaceous component of amyloid associated with Alzheimer disease (AD). We have determined the relative abundance of A beta structural variants present in amyloid from brains of 10 individuals with sporadic AD, 2 individuals with familial AD carrying specific mutations in the Alzheimer amyloid precursor protein gene, and 5 nondemented elderly controls. A procedure of isolation based on the extreme insolubility of A beta amyloid was used. The purified, nondigested A beta was analyzed by N-terminal sequencing and electrospray-ionization mass spectrometry. Three principal A beta variants were detected--A beta-(1-40), A beta-(1-42), and A beta-(11-42)--in all brains analyzed. The predominant variant in sporadic AD was A beta-(1-40), whereas the principal A beta variant in nondemented elderly controls was A beta-(1-42). The ratio A beta-(1-40)/A beta-(1-42) differed by 10-fold between brains from nondemented controls and those with sporadic AD.

Aged↗

PubMed1994

A novel peptidyl-prolyl cis/trans isomerase from Escherichia coli.

A novel peptidyl-prolyl cis/trans isomerase was isolated from Escherichia coli cell extract and characterized partially. Determination of the molecular mass by electrospray mass spectrometry indicated a protein of 10102 +/- 2 Da, smaller than cyclophilins or FK 506 binding proteins currently known. The specificity constant kcat/Km determined with Succinyl-Ala-Xaa-Pro-Phe-4-nitroanilide (Xaa = Leu) had a value comparable to those from cyclophilins from the same organism. However, the pattern of subsite specificity (Xaa = Gly, Ala, Val, Ile, Leu, Phe, Trp, His, Lys and Glu) was reminiscent of FK506 binding peptidyl-prolyl cis/trans isomerases. The enzyme activity was not inhibited by cyclosporin A or FK506 at inhibitor concentrations of < 5 microM, concentrations that affect most bacterial peptidyl-prolyl cis/trans isomerases. Computer-assisted analysis of 21 amino acid residues of the N-terminus determined by Edman degradation revealed no homology to known peptidyl-prolyl cis/trans isomerases.

Amino Acid Isomerases↗