Biomedical subjects

J Hofsteenge

Publications and source records attributed to J Hofsteenge.

At least 37 records · Page 2Linked to original sources

PubMed1998

Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp.

C2-alpha-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1-13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

Amino Acid Sequence↗

PubMed1997

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.

Animals↗

PubMed1997

C-Mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells.

C2-alpha-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) is C-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was not C-mannosylated. These results not only form the basis for further studies on the biochemical aspects of C-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins.

Animals↗

PubMed1996

Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms.

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

Amino Acid Sequence↗

PubMed1996

Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation.

Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.

Animals↗

PubMed1996

Structural determinants of the uridine-preferring specificity of RNase PL3.

RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.

Animals↗

PubMed1995

The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is alpha-mannopyranose.

Recently, the novel C-glycosidic linkage of a hexopyranosyl residue to the indole ring of tryptophan residue 7 of human RNase U(s) was reported [Hofsteenge, J., Müller, D. R., de Beer, T., Löffler A., Richter, W. J., & Vliegenthart, J. F. G. (1994) Biochemistry 33, 13524-13530]. Identification of this monosaccharide is a prerequisite for studies of its biosynthesis and its biological relevance. Using vicinal proton-proton coupling constants and rotating-frame nuclear Overhauser enhancements, ewe demonstrate that the C-linked substituent is alpha-mannopyranose. Furthermore, the nuclear magnetic resonance (NMR) data indicate that the mannopyranose moiety in a glycopeptide derived from RNase U(s) adopts several conformations on the NMR time scale.

Amino Acid Sequence↗

PubMed1995

AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity.

AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. By using an AUUUA matrix, we have affinity-purified a 32-kDa protein, microsequenced it, and cloned the corresponding cDNA. In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. Thus, this gene, designated AUH, encodes an RNA binding protein with intrinsic enzymatic activity. Protein immobilized on an AUUUA matrix was enzymatically active, suggesting that hydratase and AU-binding functions are located on distinct domains within a single polypeptide.

Amino Acid Sequence↗

PubMed1994

New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.

We report a new type of linkage between a carbohydrate and a protein, involving the rarely modified side chain of a tryptophan residue. An aldohexopyranosyl residue was found to be linked via a C-C bond to the indole ring of the tryptophan residue at position 7 of human RNase Us. Mass spectrometric analysis of peptides containing this residue showed a molecular mass 162 Da higher than that expected for tryptophan. The fragmentation pattern of the modified amino acid side chain was reminiscent of that of aromatic C-glycosides, suggesting a direct attachment of a hexose residue to a C-position of the tryptophan indole moiety. 1H and 13C NMR spectroscopic data confirmed this inference and unequivocally demonstrated the substituent to be an aldohexopyranosyl residue, C-glycosidically linked to the C2 atom of the indole. This mode of attachment differs from the ones known so far, in which carbohydrates are linked to an amino acid side chain by N- or O-glycosidic bonds.

Amino Acid Sequence↗

PubMed1994

Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences.

The primary structure of pseudo-hevein, a minor hevein component from the latex of the rubber tree, Hevea brasiliensis, was determined. Six differences with the sequence of the major hevein component were found, one of which is a replacement of tryptophan by tyrosine in the carbohydrate binding region of the molecule. Analysis by ion-spray mass spectrometry showed that pseudo-hevein has a heterogeneous C-terminal extension of several glycine residues and that hevein itself also contains minor components with additional C-terminal amino-acid residues. A seventh difference between the two sequences occurs in these extensions.

Amino Acid Sequence↗

PubMed1994

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.

Aminobenzoates↗

PubMed1994

The amino acid sequence of iguana (Iguana iguana) pancreatic ribonuclease.

The pyrimidine-specific ribonuclease superfamily constitutes a group of homologous proteins so far found only in higher vertebrates. Four separate families are found in mammals, which have resulted from gene duplications in mammalian ancestors. To learn more about the evolutionary history of this superfamily, the primary structure and other characteristics of the pancreatic enzyme from iguana (Iguana iguana), a herbivorous lizard species belonging to the reptiles, have been determined. The polypeptide chain consists of 119 amino acid residues. The positions of insertions and deletions in the sequence are identical to those in the enzyme from snapping turtle. However, the two enzymes differ at 54% of the amino acid positions. Iguana ribonuclease contains no carbohydrate, although the enzyme possesses three recognition sites for carbohydrate attachment, and has a high number of acidic residues in a localized part of the sequence.

Amino Acid Sequence↗

PubMed1994

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor.

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.

Animals↗

PubMed1994

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.

Amino Acid Sequence↗

PubMed1994

'Holy' proteins. I: Ribonuclease inhibitor.

The X-ray structure of the ribonuclease inhibitor from porcine pancreas shows a remarkable non-globular fold. It possesses a large central hole that forms part of the RNase A binding site.

Amino Acid Sequence↗

PubMed1993

Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding; a reply.

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.

Amino Acid Sequence↗

PubMed1993

Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing.

The trimeric form of protein phosphatase 2A consisting of 36-, 65-, and 72-kDa subunits (previously termed polycation-stimulated protein phosphatase M) was purified from rabbit skeletal muscle. Amino acid sequence data of the 72-kDa regulatory subunit (termed PR72) were used to isolate cDNAs from human heart and fetal brain libraries and libraries derived from WI-38 and MCF-7 cells. The clones isolated from the heart cDNA library revealed an open reading frame encoding a protein with a predicted molecular mass of 62 kDa. All the peptides sequenced from the protein matched with the sequence predicted from the cDNA. However, in vitro transcription and translation from this cDNA yielded a protein with an apparent molecular mass of 72 kDa on sodium dodecyl sulfate-polyacrylamide gels. From brain we isolated cDNA clones spanning an open reading frame encoding a 130-kDa protein (termed PR130). The apparent molecular mass of the protein produced by in vitro transcription and translation was 130 kDa. This protein has exactly the same deduced C-terminal protein sequence as the PR72 subunit from amino acids 45 to 527 but has an N-terminal extension of 665 amino acids. It is likely, therefore, that these two proteins arise from the same gene by alternative splicing. In human tissues several transcripts were detected by Northern analysis generated probably by the use of different polyadenylation signals and alternative splicing. High levels of the PR72 mRNAs were detected in heart and muscle, while lower levels of PR130 transcripts were found in heart, brain, placenta, lung, muscle, and kidney.

Alternative Splicing↗

PubMed1993

Crystallization of porcine liver ribonuclease inhibitor a member of the family of proteins containing leucine-rich repeats.

Single crystals of the RNase inhibitor from porcine liver have been obtained from 30 to 34% saturated ammonium sulphate solutions at pH 6.0 to 7.2, containing 20 mM dithiothreitol, at room temperature over a period of two to three weeks. Because the inhibitor contains 30 1/2-cystinyl residues, all of which occur in the free thiol form, crystallization experiments were carried out in a desiccator under a nitrogen atmosphere. The crystals belong to the tetragonal space group I4, with cell dimensions a = b = 134.76 A and c = 83.65 A. The asymmetric part of the unit cell contains two molecules with a molecular mass of 49,093 Da, as could be shown with a self-rotation function calculated in the resolution range 10.0 to 3.2 A. The crystals diffract to at least 3.2 A resolution and are suitable for an X-ray structure determination.

Amino Acid Sequence↗