Soluble Lactobacillus delbrueckii subsp. bulgaricus 92059 PrtB proteinase derivatives for production of bioactive peptide hydrolysates from casein.

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities.

Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (< or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.

Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase.

A number of isoenzymes of the catalytic subunit of cAMP-dependent protein kinase arise through posttranslational modifications of the enzyme outside the catalytic domain; the biological significance of these is not yet fully clear. A clustering of sites for such modification exists at the N-terminus of the protein, where myristoylation (of Gly1), phosphorylation (at Ser10), and deamidation of Asn2 have been observed. As the first two are known to govern membrane binding and thus subcellular compartmentalization in some proteins, it was of interest to see whether the local structure of the N-terminus was being influenced by one or more of these modifications. A series of synthetic peptides mimicing the 16 N-terminal residues of the catalytic subunit Calpha was produced covering the full range of possible modifications, singly and in combination, and tested for possible effects on local structure by measuring the circular dichroism under varying polarity. It was found that myristoylation and phosphorylation modify the structure in this region in opposite ways and in a manner designed to amplify the action of a potential myristoyl/electrostatic switch. To what extent deamidation of Asn2 may oppose a potential membrane binding is unknown. Deamidation, however, had no effect on the structure of the peptide either alone or in combination with acylation and/or phosphorylation, suggesting that the change of the nuclear/cytoplasmic disribution in cells caused by deamidation [Pepperkok et al. (2000) J. Cell Biol. 148, 715-726] is due to a more complex signaling mechanism. The structural implications of the data are discussed.

Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Relative peak-height ratios of products to substrates determined by MALDI-TOFMS allow the quantitative analysis of enzyme catalyzed reactions for screening purposes. Two examples were investigated: the first one was a lipase catalyzed reaction which produces 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) using rac-alpha-phenylethylamine (PEA) as substrate. The second one was the pyruvate decarboxylase catalyzed formation of (1R)-1-hydroxy-1-phenyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corresponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r2 = 0.985 for MET, r2 = 0.991 for PAC) were linear over two orders of magnitude for MET and PAC concentrations. After optimization of the sample preparation an average relative standard deviation of 12.5% was obtained in both cases.

Direct effects of phosphorylation on the preferred backbone conformation of peptides: a nuclear magnetic resonance study.

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Halpha coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues.

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collision offset and of the charge state of the molecular ion on phosphate-specific fragmentation processes was investigated in detail. H--D exchange experiments and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C-beta hydrogen of homoserine is transferred to the protonated phosphate group. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these phosphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the negative ion product spectra, which is consistently elevated in tyrosine phosphopeptides as compared with serine, threonine and homoserine phosphopeptides. The fragment ions of methylphosphono- and H-phosphonopeptides can be explained by the same mechanisms as are applicable to phosphopeptides.

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry.

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.

Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides.

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible posttranslational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce sold-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh3)4. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of 1H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by 1H-tetrazole and followed by oxidation led to the methyphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide.

Synthesis and NMR spectroscopy of peptides containing either phosphorylated or phosphonylated cis- or trans-4-hydroxy-L-proline.

Many proteins are regulated by reversible O-glycosylation and O-phosphorylation. Whereas O-glycosylation of hydroxy-L-proline is common and well investigated, phosphorylation has not been proved so far in vivo, but this post-translational modification is entirely possible. As a first step to identify this phosphoamino acid, we describe both the syntheses of peptides phosphorylated at 4-hydroxy-L-proline and the 1H and 31P NMR parameters of these phosphopeptides. The model peptides were synthesized on solid-phase using Fmoc-strategy. Both natural isomers of 4-hydroxy-L-proline (containing the hydroxyl group in either the cis or trans position) were introduced without side-chain protection. All peptides were globally phosphorylated with O,O'-tert-butyl-N,N-diethylphosphoramidite on the solid phase and cleaved with trifluoroacetic acid. Additionally, we synthesized two classes of phosphonopeptides that mimic phosphopeptides, namely H- and methylphosphonopeptides. The NMR data were based on the model peptide Gly-Gly-Hyp-Ala, which is regarded as a typical random-coil sequence. The NMR parameters showed a significant influence of the phosphate group on the cis-trans isomerization of the Gly-Hyp bond, which may reflect a possible regulation of proteins by changing their local conformations. The 1H and 31P NMR parameters differed for each isomer, and were distinct from the parameters of phosphorylated serine, threonine and tyrosine. These known shifts can be used to identify both cis- and trans-O-phospho-4-hydroxy-L-proline in vivo.

Solid-phase synthesis of H- and methylphosphonopeptides.

We introduce solid-phase syntheses of H- and methylphosphonopeptides, giving access for the first time to a new class of mimics for o-phosphoamino acids. The model peptides H-GlyGlyXaaAla-OH (Xaa = Ser, Thr) were synthesized on a solid-phase using Fmoc/tBu strategy and HBTU/HOBt activation by incorporation of hydroxyl-protected serine and threonine. As selectively cleavable hydroxyl-protecting groups we used triphenylmethyl and tert-butyldimethylsilyl for both amino acids, as described in the literature. All peptides were phosphitilated with O, O-di-tert-butyl-N,N-diethylphosphoramidite and yielded H-phosphonopeptides after trifluoroacetic acid cleavage. Alternatively we phosphonylated the peptides with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite, which was synthesized by a two-step one-pot procedure starting from commercially available chemicals. All H- and methylphosphonopeptides were obtained in high purities and yields, as shown by reversed-phase high-performance liquid chromatography and anion-exchange chromatography. The phosphonopeptides were characterized by 1H and 31P NMR. We confirmed their molecular masses by electrospray mass spectrometry and analyzed their fragmentation schemes, which seemed to be characteristic for each class of analogues. The H-phosphonopeptides lost phosphonic acid (H3PO3, 82 mass units) and the methylphosphonopeptides lost methylphosphonic acid (MeH2PO3, 96 mass units). Both H- and methylphosphonopeptides represent a new and simply accessible class of mimics for phosphopeptides. Compared with the corresponding phosphopeptides all phosphonopeptides were synthesized in higher yields and purities (> 80%).