Functional roles of conserved transmembrane prolines in the human VPAC(1) receptor.

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.

Kinetic stability of designed glycosylation mutants of Coprinus cinereus peroxidase.

The effect of glycans and surface mutations on protein unfolding induced by heat or urea has been studied. Removal of the only native high mannose type glycan in the N142P, N142T, and N142D CIP mutants reduced the lifetime to half of that of wtCIP at irreversible conditions of unfolding. The effect was moderate at reversible conditions. Five glycomutants designed to have 0, 1, 2, 4 and 6N glycans showed a correlation between increased carbohydrate mass and increased stability toward irreversible unfolding. The results are in agreement with a dampening effect of glycans on backbone fluctuation in both the native and the unfolded states. However, experiments in reversible conditions were less clear because of additional effects of an increasing number of amino acid substitutions and aggregation. Examples of strong effects from minor surface changes were also observed.

Characterization of a G protein coupling "YL" motif of the human VPAC1 receptor, equivalent to the first two amino acids in the "DRY" motif of the rhodopsin family.

The conserved residues Y239 and L240 of human VPAC1 receptor are predicted to be at the same location as the asparagine and arginine in the "DRY" motif in the Rhodopsin family of G protein-coupled receptors. By comparing vasoactive intestinal peptide (VIP) binding with or without the presence of GTP-gamma-S, it was found that the deltadelta G(o) for the endogenous G-protein coupling was 1.5 kJ/mol, 0.95 kJ/mol, and 3.4 kJ/mol for theY239A, L240A, and wild-type receptor, respectively. VIP-induced cAMP production in whole cells support the results of the binding studies, as Y239A had a moderate and L240A a pronounced impaired ability to produce cAMP. The mutants had a minor influence on the intrinsic "low affinity to high affinity equilibrium," suggesting that the dominating effect of these mutants is a perturbation of the G protein-binding site. Thus, the highly diverged chemical properties of the hydrophobic "YL" motif and charged "DR(Y)" motif could be a crucial difference between the Secretin Receptor Family and the Rhodopsin Family with respect to receptor activation and G-protein coupling.

Creation of a selective antagonist and agonist of the rat VPAC(1) receptor using a combinatorial approach with vasoactive intestinal peptide 6-23 as template.

We have used combinatorial chemistry with amino acid mixtures (X) at positions 6 to 23 in vasoactive intestinal peptide (VIP) to optimize binding affinity and selectivity to the rat VPAC(1) receptor. The most efficient amino acid replacement was a substitution of alanine at position 18 to diphenylalanine (Dip), increasing the displacement efficiency of (125)I-VIP by 370-fold. The [Dip(18)]VIP(6-23) was subsequently used to find a second replacement, employing the same approach. Tyrosine at position 9 was selected and the resulting [Tyr(9),Dip(18)]VIP(6-23) analog has a K(i) value of 90 nM. This analog was unable to stimulate cAMP production at 10(-6) M but was able to inhibit VIP-induced cAMP stimulation (K(b) = 79 nM). The K(i) values of [Tyr(9),Dip(18)]VIP(6-23) using the rat VPAC(2) and PAC(1) receptors were 3,000 nM and >10,000 nM, respectively. Thus, [Tyr(9),Dip(18)]VIP(6-23) is a selective VPAC(1) receptor antagonist. The C-terminally extended form, [Tyr(9),Dip(18)]VIP(6-28), displays improved antagonistic properties having a K(i) and K(b) values of 18 nM and 16 nM, respectively. On the contrary, the fully extended form, [Tyr(9),Dip(18)]VIP(1-28), was a potent agonist with improved binding affinity (K(i) = 0.11 nM) and ability to stimulate cAMP (EC(50) = 0.23 nM) compared with VIP (K(i) = 1.7 nM, EC(50) = 1.12 nM). Furthermore, the specificity of this agonist to the VPAC(1) receptor was high, the K(i) values for the VPAC(2) and PAC(1) receptors were 53 nM and 3,100 nM, respectively. Seven other analogs with the [Tyr(9),Dip(18)] replacement combined with previously published VIP modifications have been synthesized and described in this work.

Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC1R).

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.

Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was examined as host for heterologous expression of the G protein-coupled VPAC1 receptor. Rat VPAC1 receptor cDNA and two chimeric constructs encoding the yeast mating factor pre-pro alpha-leader peptide fused in-frame to rat VPAC1 receptor were expressed in yeast cells under control of a galactose inducible promoter. The rat VPAC1 receptor was fused to the HSV tag epitope to ensure proper immunological detection of the receptor. Crucial conditions for high-level expression of active rat VPAC1 receptor included growth in amino acid supplemented minimal medium, fusion to the yeast alpha-leader peptide and a temperature shift from 30 degrees C to 15 degrees C before promoter induction. Western blotting showed that the expressed receptor was highly glycosylated and a band of 47 kDa was obtained upon endoglycosidase H treatment. Binding with radioiodinated vasoactive intestinal polypeptide revealed a KD of 2.5 nM and an IC50 of 15 nM when displacing with unlabeled vasoactive intestinal polypeptide. VPAC1 receptor density quantified by Western blotting was 510 pmol/mg membrane protein of which only 66 pmol/mg were able to bind vasoactive intestinal polypeptide.

Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions.

Protein solubility is a fundamental parameter in biology and biotechnology. In the present study we have constructed and analyzed five mutants of Coprinus cinereus peroxidase (CIP) with 0, 1, 2, 4 and 6 N-glycosylation sites. All mutants contain Man(x)(GlcNAc)(2) glycans. The peroxidase activity was the same for wild-type CIP and all the glycosylation mutants when measured with the large substrate 2,2'-azino-bis(-3-ethylbenzthiazoline-6-sulfonic acid). The solubility of the five CIP mutants showed a linear dependence on the number of carbohydrate residues attached to the protein in buffered solution of both ammonium sulfate (AMS) and acetone, increasing in AMS and decreasing in acetone. Moreover, the change in free energy of solvation appears to be a constant, though with opposite signs in these solvents, giving DeltaDeltaG degrees (sol)=-0.32+/-0.05 kJ/mol per carbohydrate residue in 2.0 M AMS, a value previously obtained comparing ordinary and deglycosylated horseradish peroxidase, and 0. 37+/-0.10 kJ/mol in 60 v/v% acetone.

Identification of pituitary adenylate cyclase-activating polypeptide1-38-binding factor in human plasma, as ceruloplasmin.

125I-Pituitary adenylate cyclase-activating polypeptide (PACAP) 1-38 is able to bind a factor in human plasma, which can be displaced by unlabelled PACAP 1-38 and PACAP 28-38 but not by the other biologically active form, PACAP 1-27. Likewise, 125I-PACAP 28-38 binds this plasma factor, whereas 125I-PACAP 1-27 does not. Apparent Kd values were measured to be 12.0+/-1.3 and 3.4+/-1.5 nM for PACAP 1-38 and PACAP 28-38, respectively, using a competition assay with 125I-PACAP 28-38. Purification of the PACAP 1-38-binding factor from human blood was made by ethanol precipitation of serum followed by Ni2+-chelating and anion-exchange chromatography. A 120-kDa band on SDS/PAGE, as well as some proteolytic products, was blotted on to PVDF membrane and their N-terminal amino acid sequences determined. In combination with a mass-spectrometric fingerprinting of a tryptic digest of the 120-kDa band, this PACAP 1-38-binding factor was identified as ceruloplasmin. Purified commercial ceruloplasmin shows identical mobility on SDS/PAGE to the PACAP 1-38-binding factor and the same binding characteristics to PACAP 1-38, 1-27 and 28-38, using the same amount of ceruloplasmin as was expected to be found in the human plasma. Furthermore, the ability of plasma to bind 125I-PACAP 1-38 or 28-38 disappeared when ceruloplasmin was immunoprecipitated from plasma with rabbit anti-human ceruloplasmin Ig.

Proposed arrangement of the seven transmembrane helices in the secretin receptor family.

An arrangement of the seven transmembrane alpha-helices of the G protein-coupled Secretin receptor family is proposed. The helices of 27 homologous receptor sequences were plotted as helical wheels. The solvent inaccessible portion of each helix was used to assign relative orientations. They were arranged according to two criteria: 1) conserved, hydrophilic residues and aligned positions with restricted volume changes face the other helices and 2) aligned positions with low identity and large volume change face the lipid. The positive inside rule confirms the assumption that loops connecting transmembrane helices I-II, III-IV, V-VI and the C-terminal part of the receptors are intracellular. Our model approach was tested using the Bacteriorhodopsin family. The use of volume changes at each position in the transmembrane helix was crucial for the good correlation of the orientation of the helices using the model approach and the structure of bacteriorhodopsin solved by electron microscopy [Grigorieff N, Ceska TA, Downing KH, Baldwin JM, and Henderson R (1996) J Mol Biol 259 393-421]. The tests of our modelling approach showed that six helices were within a 15 degrees derivation in the orientation and five helices were within a horizontal derivation of two residues. The largest orientational derivations of a helix were 40 degrees and the largest horizontal displacement was four residues. A long stretch of side chains predicted to possess low resistance to movement in helix V of the Secretin receptor family suggests an involvement in receptor activation. Comparison of the Secretin receptor family and the larger G protein-coupled Rhodopsin family showed many similarities, despite the lack of obvious sequence identity.

Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase.

The influence of N-linked glycans on the stability of glycoproteins has been studied using horseradish peroxidase isoenzyme C (HRP), which contains eight asparagine-linked glycans. HRP was deglycosylated (d-HRP) with trifluoromethanesulfonic acid and purified to an enzymatically active homogeneous protein containing (GlcNAc)2 glycans. The thermal stability of HRP and d-HRP at pH 6.0, measured by residual activity, was indistinguishable and showed transition midpoints at 57 degrees C, whereas the unfolding in guanidinium chloride at pH 7.0, 23 degrees C was 2-3-fold faster for d-HRP than for HRP. The results are compatible with a glycan-induced decrease in the dynamic fluctuation of the polypeptide chain.

Importance of conserved cysteines in the extracellular loops of human PACAP/VIP1 receptor for ligand binding and stimulation of cAMP production.

The importance of two highly conserved cysteines in the human pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide 1 (VIP1) receptor was examined. Using site-directed mutagenesis, each Cys residue was converted into Ala or Ser. The mutant and wildtype genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215Ala/Ser and Cys285Ala/Ser showed at least a tenfold decrease in binding affinity and receptor potency when compared to the wildtype. In contradiction to the wildtype receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilizing the receptor in the correct conformation for ligand binding and activation.

A disulfide bond between conserved cysteines in the extracellular loops of the human VIP receptor is required for binding and activation.

The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.

Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on these processes.

The unfolding and refolding rates of the heme-and Ca2+ -containing Coprinus cinereus peroxidase (CIP) have been measured at pH 12.1, in 4 M urea, and at 61.2 degrees C. The change in peroxidase activity paralleled the change in the Soret band absorbance of the heme group. The unfolding rate constant (ku), was determined independently in thermolysin digestion and EDTA experiments at 59.4 degrees C. Both gave ku values of 1.5 ms-1, and also showed that the presence of 4 mM EDTA made CIP unfolding practically irreversible. Unfolding and refolding rates could therefore be determined under identical conditions of denaturation having either EDTA or Ca2+ in excess. The refolding rates at high pH and in 4 M urea were measured by adding Ca2+ to the unfolded CIP, whereas refolding at 61.2 degrees C was evaluated by comparing the unfolding carried out under reversible (excess of Ca2+) and irreversible conditions (excess EDTA). The activation energies for the unfolding at 61.2 degrees C are approximately delta G++(u) 100, T delta S++(u) 200, and delta H++(u) 300 kJ/mol. Five different additives, glycerol, EtOH, Na2SO4, guanidinium chloride (GdmCl), and NaCl, all at 100 mM, were used as probes to evaluate the mechanism of base, urea, and heat on unfolding and refolding. Salts destabilized CIP at high pH, primarily by enhancing the unfolding rate but also by decreasing the refolding rate. Glycerol had the reverse effects and thus stabilized CIP at high pH. The unfolding rate in urea was only slightly affected by the additives, with the exception of GdmCl which enhanced the unfolding rate. The refolding rate was decreased in urea by EtOH and GdmCl, in contrast to glycerol and Na2SO4 which increased the refolding rate. At high temperature the unfolding was affected only slightly by the additives, except for GdmCl, and to a lesser extent NaCl, which enhanced the unfolding rate. The refolding rates were greatly decreased by Na2SO4, EtOH, and GdmCl, whereas glycerol and Nacl enhanced the process. It appears that 100 mM NaCl functions as a catalyst for the temperature-induced process, enhancing both the unfolding and refolding rates. The results indicate that the mechanisms of CIP unfolding and refolding are similar in urea and at high temperature but different at high pH.

Mild chemical deglycosylation of horseradish peroxidase yields a fully active, homogeneous enzyme.

Horseradish peroxidase isoenzyme C (HRP) contains eight N-linked glycans composed of Man, Xyl, Fuc, and GlcNAc. These glycans were resistant to enzymatic hydrolysis by endoglycosidases peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, endo-beta-N-acetyl-glucosaminidase H, and endo-beta-N-acetylglucosaminidase F under conditions where ovalbumin was deglycosylated. However, using anhydrous trifluoromethanesulfonic acid (TFMS) in the presence of 90 mM phenol for 5 min at--10 degrees C, all carbohydrate except GlcNAc was removed. Sixty percent of deglycosylated HRP was active after this TFMS treatment. Benzhydroxamic acid affinity chromatography separated active and inactive deglycosylated HRP. TFMS treatment, however, introduced negative charges in all inactive HRP and in about 90% of the active deglycosylated HRP. The nature of this modification has not been identified. After ion-exchange chromatography, homogeneous and fully active deglycosylated HRP, showing the original pI of 9, electronic absorption spectrum, and enzyme kinetics, was obtained, In this purified product no amino acid modifications were detected by amino acid analysis, partial sequencing, and mass spectrometry of tryptic peptides. The deglycosylated product showed greatly reduced solubility in salt solution compared to that of authentic HRP.

Disulfide bonds and glycosylation in fungal peroxidases.

Four conserved disulfide bonds and N-linked and O-linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1-3, 2-7, 4-5 and 6-8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N-linked glycan composed only of GlcNAc and Man at residue Asn142, and two O-linked glycans near the C-terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N-linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man3 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C-terminal peptide of TvMP2 indicated the presence of five Man residues in O-linked glycans. No phosphate was found in these fungal peroxidases.

NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase.

Crystals suitable for an X-ray diffraction investigation have been obtained of recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae. The crystals were grown by the hanging drop method with polyethylene glycol 6000 as the precipitant. A pH range from 6.2 to 8.0 and CaCl2 or MgCl2 present at a concentration of 0.35 M were essential for the crystal growth. A metastable monoclinic modification can be obtained under certain conditions, and with variations in temperature they are transformed into a stable orthorhombic modification. With CaCl2 as the additive, the unit cell dimensions were a = 74.9 A, b = 76.8 A and c = 128.2 A. With two peroxidase molecules per asymmetric unit, the solvent content is 49% (v/v). In the diffraction pattern, the reflections Okl are systematically very weak for k = 2n + 1. Combined with an analysis of the Patterson function, this showed that the two independent molecules are related by the pseudotranslational symmetry 0.29a + 0.5b. The possible space groups are P2(1)2(1)2(1) or P2(1)22(1) because of this pseudosymmetry. The crystals diffract to a resolution of 2.9 A.