Purification and characterization of active recombinant human napsin A.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to approximately 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ-Lucifer yellow (DS3). The Km values obtained were 1.7 microM and 6.2 microM, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 degrees C.

The influence of strongly acidic groups on the protonation of peptides in electrospray MS.

In order to obtain experimental data on the question of compensation of positive charges by anionic groups in multiply charged ions of polyfunctional molecules in electrospray MS, several pairs of peptides with the same basic structure but differentiated by one or two strongly acidic groups (phosphate or sulfonate) were investigated. It was found that depending on the density of basic centers and the strength of acidic groups present, the observed changes upon introduction of acidic groups ranged from complete elimination of the most highly charged state to absence of any difference.

Dioleoylmelittin as a novel serum-insensitive reagent for efficient transfection of mammalian cells.

Amphipathic peptides can be useful effectors to enhance gene delivery. However, peptide/DNA complexes usually require additional effectors, such as fusogenic lipids, to mediate efficient transfection. Due to weak and/or multiple interactions between the various components of the system, the transfecting complexes are often heterogeneous and unstable in biological fluids. Accordingly, a hybrid molecule resulting from the covalent coupling of an amphipathic, membrane-disturbing peptide to a lipid moiety might create a stable and efficient peptide-based gene transfer system. The present work describes such a novel hybrid molecule, dioleoylmelittin, resulting from the conjugation of dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)propionate] with [Cys1]melittin. Dioleoylmelittin had a lower hemolytic and membrane-disturbing activity than melittin. Size and zeta potential measurements, DNA gel electrophoresis, and electron microscopy showed that dioleoylmelittin, unlike melittin, was able to complex plasmid DNA to form spherical particles with a net positive charge and a diameter between 50 and 250 nm. These particles, prepared at an optimal 10/1 dioleoylmelittin/DNA ratio (w/w), mediated efficient transient transfection of reporter genes in cultured mammalian cells including primary cells. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line. Surprisingly, the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fold gene expression. Dioleoylmelittin represents a new class of efficient peptide-based transfection reagents, especially suited for serum-sensitive cells.

Labelling of CRF1 and CRF2 receptors using the novel radioligand, [3H]-urocortin.

The binding of the novel radioligand, [3H]-rat urocortin to homogenates of rat cerebellum and homogenates of cells stably transfected with the human CRF1, rat CRF2alpha and rat CRF2beta receptors was examined. In each case, specific reversible high affinity binding was observed (K[d]s between 0.18 and 0.31 nM). The density of sites was relatively low in the cerebellum (9 fmol/mg tissue) but high in the recombinant systems with expression levels of between 1.4 and 6.3 pmol/mg protein. Agents known to interact with CRF receptors potently competed for binding in each case. The pharmacological profile of binding to the recombinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF1 receptor, binding was inhibited with similar affinity by Urocortin, sauvagine, Urotensin 1 and CRF. The non-peptidic CRF antagonists (e.g. CP 154,526 and SC 241) also potently inhibited binding. The CRF2alpha and CRF2beta receptor recombinant systems had a very similar pharmacological profile with a clear rank order of potency for the peptide ligands (Urocortin > Sauvagine > Urotensin 1 > CRF), whereas the non-peptide CRF receptor antagonists had no measurable affinity. The pharmacological profile of specific [3H]-urocortin binding to homogentates of rat cerebellum was consistent with specific labelling of a CRF1 receptor. We conclude that [3H]-urocortin is a useful tool for the study of CRF receptors with the advantages that a filtration assay can be used, all CRF receptors can be labelled with the same ligand and the benefits associated with the low energy emittor, 3H.

Chemical synthesis of O-thiophosphotyrosyl peptides.

The synthon for O-thiophosphotyrosine, Fmoc-Tyr[PS(OBzl)2]-OH (1c), was prepared in 63% yield from Fmoc-Tyr-OH by first transient protection as the tBuMe2Si-ester and phosphinylation with (BzlO)2PNiPr2/tetrazole followed by oxidation of P(III) to P(V) with S8 in CS2. Building block 1c was incorporated in the Fmoc solid-phase synthesis of two O-thiophosphotyrosine-containing peptides H-Thr-Glu-Pro-Gln-Tyr(PS)-Gln-Pro-Gly-Glu-OH (2) and H-Thr-Arg-Asp-Ile-Tyr(PS)-Glu-Thr-Asp-Phe-Phe-Arg-Lys-OH (3), corresponding to sequences of the p60src (523-531) protein and an insulin receptor (IR) (1142-1153) analogue, respectively. An alternative approach of synthesis, the global phosphorylation of a resin-bound peptide, also proved useful. Thus, the free tyrosyl side-chain containing-peptide IR (1142-1153) on support was phosphinylated with the above phosphoramidite reagent followed by oxidation with either S8/CS2 or tetraethylthiuram disulfide/CH3CN solutions. Deprotection and peptide-resin cleavage was performed with a TFA/thiophenol (H2O) mixture. Crude peptides 2 and 3 were stable to the acidolytic deprotection. Preparative RP(C18)HPLC was initially performed using 0.1% TFA(aq)/EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosphopeptide in solution. Stability measurements both as a function of time and pH, further confirmed this initial finding. Purifications performed at intermediate pH using a triethylammonium acetate (pH 7.5)/CH3CN solvent system overcame this problem.

Chemical synthesis of an O-phosphorylated tyrosine analogue of human angiotensin II, [Tyr(P)4] angiotensin II, and its vasoconstrictor effect in intact sheep.

The derivative N alpha-fluorenylmethoxy-carbonyl-O-dimethylphosphono-L-tyrosi ne was utilized in continuous-flow solid-phase synthesis to prepare the O-phosphotyrosine analogue of human angiotensin II, [Tyr(P)4] angiotensin II. Side-chain deprotection, including the removal of the methyl phosphate groups, as well as cleavage of the peptide from the solid support was achieved with 1 M trimethylsilyl-bromide-thioanisole in trifluoroacetic acid. Overall yield of purified peptide was 46%. The pressor response in intact sheep to graded infusions of the synthetic [Tyr(P)4] angiotensin II showed it to have similar potency to the native angiotensin II. However, a prolonged, elevated mean arterial pressure was observed following cessation of the infusion.

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.

NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase.

An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142-1,153 of the insulin receptor was prepared and incubated with the 40-kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H-NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri-, di-, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.

Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system.

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.