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PubMed2006

Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

Animals↗

PubMed2004

Peptides derived from the histidine-proline domain of the histidine-proline-rich glycoprotein bind to tropomyosin and have antiangiogenic and antitumor activities.

The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline-rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in fibroblast growth factor-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor growth in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 approximately 600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a 16-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor growth, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.

Angiogenesis Inhibitors↗

PubMed2002

Histidine-proline-rich glycoprotein has potent antiangiogenic activity mediated through the histidine-proline-rich domain.

Histidine-proline-rich glycoprotein (HPRG) is an abundant multidomain plasma protein evolutionarily related to high-molecular-weight kininogen. The cleaved form of high-molecular-weight kininogen has recently been demonstrated to exhibit antiangiogenic activities in vitro (J. C. Zhang et al., FASEB J., 14: 2589-2600, 2000), mediated primarily through domain 5. HPRG contains a histidine-proline-rich (H/P) domain with sequence and functional similarities to HKa-D5. We hypothesized that HPRG may also have antiangiogenic properties, localized within its H/P domain. The H/P domain is highly conserved among species, and because rabbit H/P domain is more resistant to internal proteolytic cleavage than the human domain, the rabbit HPRG (rbHPRG) was primarily used to assess the antiangiogenic activity of HPRG. Rabbit HPRG inhibited human umbilical vein endothelial cell (HUVEC) tube formation stimulated by fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor on a Matrigel surface as well as cell proliferation of FGF-2 stimulated HUVECs. The antiangiogenic activity of rbHPRG was localized to the H/P domain by use of proteolytic fragments of rbHPRG and was further confirmed and characterized in two in vivo models of angiogenesis: the chorioallantoic membrane of the chick assay and the mouse Matrigel plug assay. Caspase-3 activation was observed in HUVECs stimulated with FGF-2 in the presence of rbHPRG, suggesting that apoptosis of activated endothelial cells may be one of the mechanisms underlying its antiangiogenic activity. Finally, the H/P domain of rbHPRG reduced tumor cell number when tumor cells were co-inoculated in the Matrigel plug assay. In conclusion, the H/P domain within HPRG induces the apoptosis of activated endothelial cells leading to potent antiangiogenic effects.

Angiogenesis Inhibitors↗

PubMed1982

Fractionation and characterization of basic proline-rich peptides of human parotid saliva and the amino acid sequence of proline-rich peptide P-E.

Basic proline-rich peptides of human parotid saliva were fractionated and characterized. The amino acid sequence of one of the purified peptides, P-E, was determined to be (formula: see text). The results demonstrate the repetitiveness of the partial sequence within the molecule and the occurrence of structures common to those of other salivary polypeptides such as P-C and Protein C.

Adult↗

PubMed2003

Toxicity of free proline revealed in an arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase.

The toxicity of proline (Pro) to plant growth has raised questions despite its protective functions in response to environmental stresses. To evaluate Pro toxicity, we isolated an Arabidopsis T-DNA-tagged mutant, pdh, that had a defect in Pro dehydrogenase (AtProDH), which catalyzes the first step of Pro catabolism. The pdh mutant showed hypersensitivity to exogenous application of < or =10 mM L-Pro, at which wild-type plants grew normally. A dose-dependent increase in internal free Pro accumulation was observed in pdh plants during external Pro supply. These results do not just prove the toxicity of Pro, but also suggest that AtProDH is the only enzyme acting as a functional ProDH in Arabidopsis: To further analyze the targets of Pro toxicity, we compared the expression of thousands of genes by pdh plants with that by wild-type plants by cDNA microarray analysis. Most genes were unaffected. Here we demonstrate Pro toxicity by using the pdh mutant and discuss a cause-and-effect action between an excess of free Pro and growth inhibition in Arabidopsis.

Arabidopsis↗

PubMed2002

ACTCAT, a novel cis-acting element for proline- and hypoosmolarity-responsive expression of the ProDH gene encoding proline dehydrogenase in Arabidopsis.

Proline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration. ProDH is also induced by L-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5' to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in L-Pro-induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to L-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary for L-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 L-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the L-Pro-responsive gene expression.

3' Flanking Region↗

PubMed1998

A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis.

The cDNA clone ERD5 (early responsive to dehydration), isolated from 1-h-dehydrated Arabidopsis, encodes a precursor of proline (Pro) dehydrogenase (ProDH), which is a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The transcript of the erd5 (ProDH) gene was undetectable when plants were dehydrated, but large amounts of transcript accumulated when plants were subsequently rehydrated. Accumulation of the transcript was also observed in plants that had been incubated under hypoosmotic conditions in media that contained L- or D-Pro. We isolated a 1.4-kb DNA fragment of the putative promoter region of the ProDH gene. The beta-glucuronidase (GUS) reporter gene driven by the 1.4-kb ProDH promoter was induced not only by rehydration but also by hypoosmolarity and L- and D-Pro at significant levels in transgenic Arabidopsis plants. The promoter of the ProDH gene directs strong GUS activity in reproductive organs such as pollen and pistils and in the seeds of the transgenic plants. GUS activity was detected in vegetative tissues such as veins of leaves and root tips when the transgenic plants were exposed to hypoosmolarity and Pro solutions. GUS activity increased during germination of the transgenic plants under hypoosmolarity. The relationship between Pro metabolism and the physiological aspects of stress response and development are discussed.

Arabidopsis↗

PubMed1991

Effects of proline ring conformation on theoretical pi-pi* absorption and CD spectra of helical poly(L-proline) forms I and II.

Absorption and CD spectra of the pi-pi* transition near 200 nm are calculated for helical (Pro)10 forms I and II with a variable proline ring conformation characterized by torsion angle chi 2 in the range -60 degrees to 60 degrees. The spectra for poly(Pro) I are not sufficiently sensitive to chi 2 to suggest a preferred ring conformation. The spectra for poly(Pro) II are more sensitive to chi 2, and suggest preferred ring conformations near either or both of the chi 2 regions -50 +/- 10 degrees and 50 +/- 10 degrees.

Circular Dichroism↗

PubMed1989

Analysis by fast atom bombardment mass spectrometry of 4,4-dicarboxy-5-(pyridoxyl-5'-phosphate)-proline, of 4-carboxy-5-(pyridoxyl-5'-phosphate)-proline and 4,4-dicarboxy-5-pyridoxylproline.

gamma-Carboxyglutamic acid (Gla) reacts easily and quantitatively with piridoxal-5'-phosphate in H2O at room temperature, to give the 4,4-dicarboxy-5-(pyridoxyl-5'-phosphate)-proline (1). This product has been characterized by fast atom bombardment (FAB) mass spectrometry, including the B/E linked scan technique, ultraviolet and 1H nuclear magnetic resonance (NMR) analysis. In order to support further on the structure of 1, the 4-monodecarboxyderivative (2), obtained by heating compound 1 at 100 degrees C for 30 min and the 5'-dephosphoderivative (3), obtained by enzymatic hydrolysis of 1 with alkaline phosphatase, were also analysed by FAB and 1H NMR techniques. FAB was shown to be the only adequate ionization technique to deal with such thermolabile and non-volatile compounds and could provide an elegant and fast method to detect Gla in biological matrices.

Magnetic Resonance Spectroscopy↗

PubMed2005

Improved proton affinity measurements for proline and modified prolines using triple quadrupole and ion trap mass spectrometers.

Proton affinity (PA) of compounds such as proline, cis-3-methylproline, cis-3-ethylproline, cis-3-isopropylproline and cis-3-isopentanylproline was determined by kinetic method with amines as the reference bases. The effective temperatures determined using ion trap and triple quadrupole mass spectrometers were found to be significantly different. In the case of the triple quadrupole instruments, the effective temperature depends significantly on the collision energy. The influence of the apparent basicity (GBapp) on the effective temperature may be used to estimate the difference in protonation entropy (DeltaDeltaS degrees) between the sample and reference compounds. In case of the ion trap mass spectrometer, the variation of the effective temperature as a function of the excitation amplitude is small, so it is difficult to account for the contribution of the entropy effects to the proton affinity value. A better estimation of the PA and DeltaDeltaS degrees values for the investigated molecules is obtained by combining the GBapp and Teff data pairs that are obtained from both the mass spectrometers.

Kinetics↗

PubMed1982

A new synaptosomal biosynthetic pathway of proline from ornithine and its negative feedback inhibition by proline.

The biosynthesis of L-proline (Pro) was studied in sonicates of mouse brain synaptosomes (P2). Incubation with L-[14C]glutamic acid (Glu) resulted in formation of [14C]gamma-aminobutyric acid (GABA) and L-[14C]aspartic acid (Asp), but not [14C]Pro. On the other hand, L-ornithine (Orn) was found to be an effective precursor of Pro. Incubation of [3H]Orn with the sonicated mouse brain P2 fraction not only resulted in the extensive formation of [3H]Pro, but also yielded [3H]Glu and [3H]GABA. Almost 50% of [3H]Orn consumed was converted into [3H]Pro. In vitro addition of Pro (0.1-1 mM) inhibited the formation of [3H]Pro from [3H]Orn to the extent of 40-75%, while increasing the accumulations of [3H]Glu and [3H]GABA without affecting the consumption of [3H]Orn. These results clearly demonstrate that a potential site exists for negative feedback inhibition in Pro biosynthesis from Orn in the mammalian central nervous system.

Animals↗

PubMed1984

The transneuronal transport of proline within the mouse visual system: some characteristics of the [3H]-proline containing material.

Following the intraocular injection of tritiated proline in the mouse, the progressive transport of radioactivity in the brain and the nature of the cortical material(s) to which the label is bound was examined. About 35-40% of the radioactivity that was present in the cerebral cortex at four weeks post-injection was extractable with either distilled water or various buffers. By using 0.1% SDS this value can be increased up to 94%. Polyacrylamide gel electrophoresis of the extracted proteins showed that a major part of the radioactivity is accumulated in one band of proteins. This heavily labeled band could not be identified in the homolateral parietooccipital cortex or in the frontal cortex. Similarly, SDS electrophoresis of the SDS-extracted proteins also demonstrated the presence of a major band of proteins whose molecular weight was estimated at approximately 68,000 daltons. The protein was purified by ammonium sulfate precipitation and DEAE-Sephadex separation.

Animals↗

PubMed2006

The special five-membered ring of proline: An experimental and theoretical investigation of alkali metal cation interactions with proline and its four- and six-membered ring analogues.

The interaction of the alkali metal cations, Li+, Na+, and K+, with the amino acid proline (Pro) and its four- and six-membered ring analogues, azetidine-2-carboxylic acid (Aze) and pipecolic acid (Pip), are examined in detail. Experimentally, threshold collision-induced dissociation of the M+(L) complexes, where M = Li, Na, and K and L = Pro, Aze, and Pip, with Xe are studied using a guided ion beam tandem mass spectrometer. From analysis of the kinetic energy dependent cross sections, M(+)-L bond dissociation energies are measured. These analyses account for unimolecular decay rates, internal energy of reactant ions, and multiple ion-molecule collisions. Ab initio calculations for a number of geometric conformations of the M+(L) complexes were determined at the B3LYP/6-311G(d,p) level with single-point energies calculated at MP2(full), B3LYP, and B3P86 levels using a 6-311+G(2d,2p) basis set. Theoretical bond energies show good agreement with the experimental bond energies, which establishes that the zwitterionic form of the alkali metal cation/amino acid, the lowest energy conformation, is formed in all cases. Despite the increased conformational mobility in the Pip systems, the Li+, Na+, and K+ complexes of Pro show higher binding energies. A meticulous examination of the zwitterionic structures of these complexes provides an explanation for the stability of the five-membered ring complexes.

Cations↗

PubMed1975

13C-nuclear magnetic resonance study of [85% 13C-enriched proline]thyrotropin releasing factor: 13C-13C vicinal coupling constants and conformation of the proline residue.

To understand fully interactions between peptides and cellular receptors, peptide side chain conformation must be defined. In many cases the complexity of proton nuclear magnetic resonance (NMR) prevents this but the present work demonstrates this problem can be solved by using 13C enrichment. Selective 13C enrichment of a natural peptide hormone has been achieved by preparing [85% 13C-enriched proline]thyrotropin releasing factor which was examined by 13C NMR spectroscopy at various pH values. Because of the 13C enrichment, one-bonded and three-bonded (vicinal) 13C-13C coupling constants have been determined. The latter vary from 0 to 5 Hz and show bond angle dependence. These data indicate that in this hormone the pyrrolidine ring is not free but fixed in the Cgamma-endo puckered conformation. It has also been possible to assign chemical shift values for a second order 13C NMR spectrum.

Carbon Isotopes↗

PubMed1993

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

Amino Acid Isomerases↗

PubMed1979

Preparation of racemic 2r-carboxy-trans, trans-3,4-dideutero-pyrrolidine (DL-trans, trans-3,4-dideutero-proline) and unambiguous assignment of protons in the 1H n.m.r. spectrum of proline.

Racemic trans-3,4-dideutero proline has been prepared by catalytic deuteration of 3-pyrroline-2-carboxylic acid. The stereospecificity of the reduction (i.e. trans to the carboxylic group) was ascertained after transformation of the dideutero compound into the diketopiperazide c/Pro, Aib/, for which unambiguous proton assignments in the 1H n.m.r. spectrum had been obtained previously. The identification of the configuration of the dideuteroproline allows for the affirmation of the correctness of proton assignments as previously proposed in literature.

Chemical Phenomena↗

PubMed1992

cDNA clones for mouse parotid proline-rich proteins. mRNA regulation by isoprenaline and the nucleotide sequence of proline-rich protein cDNA MP5.

cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline-rich protein (PRP) oligonucleotide. They were divided into two cross-hybridisation groups, A and B, which were shown by hybrid-selected translations to encode acidic PRP and basic PRP, respectively. The A-type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B-type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B-type clones contained a novel PRP cDNA (MP5) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260-residue-long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGPPPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3'-untranslated region to rat salivary glutamate/glutamine-rich protein (GRP) cDNA. Two MP5 clones contained a 273-bp intron-like insertion in the 3' untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts. Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B-type PRP mRNA was present in normal parotid glands. RNA dot-blots probed with PRP-gene-specific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.

Amino Acid Sequence↗

PubMed2006

Proline betaine uptake in Sinorhizobium meliloti: Characterization of Prb, an opp-like ABC transporter regulated by both proline betaine and salinity stress.

Sinorhizobium meliloti uses proline betaine (PB) as an osmoprotectant when osmotically stressed and as an energy source in low-osmolarity environments. To fulfill this dual function, two separate PB transporters, BetS and Hut, that contribute to PB uptake at high and low osmolarity, respectively, have been previously identified. Here, we characterized a novel transport system that mediates the uptake of PB at both high and low osmolarities. Sequence analysis of Tn5-luxAB chromosomal insertions from several PB-inducible mutants has revealed the presence of a four-gene locus encoding the components of an ABC transporter, Prb, which belongs to the oligopeptide permease (Opp) family. Surprisingly, prb mutants were impaired in their ability to transport PB, and oligopeptides were not shown to be competitors for PB uptake. Further analysis of Prb specificity has shown its ability to take up other quaternary ammonium compounds such as choline and, to a lesser extent, glycine betaine. Interestingly, salt stress and PB were found to control prb expression in a positive and synergistic way and to increase Prb transport activity. At low osmolarity, Prb is largely implicated in PB uptake by stationary-phase cells, likely to provide PB as a source of carbon and nitrogen. Furthermore, at high osmolarity, the analysis of prb and betS single and double mutants demonstrated that Prb, together with BetS, is a key system for protection by PB.

ATP-Binding Cassette Transporters↗