Proline Residue In Position Research Articles

Assignment of Ala, Ile, LeuproS, Met, and ValproS methyl groups of the protruding domain of murine norovirus capsid protein VP1 using methyl–methyl NOEs, site directed mutagenesis, and pseudocontact shifts

The protruding domain (P-domain) of the murine norovirus (MNV) capsid protein VP1 is essential for infection. It mediates receptor binding and attachment of neutralizing antibodies. Protein NMR studies into interactions of the P-domain with ligands will yield insights not easily available from other biophysical techniques and will extend our understanding of MNV attachment to host cells. Such studies require at least partial NMR assignments. Here, we describe the assignment of about 70% of the Ala, Ile, LeuproS, Met, and ValproS methyl groups. An unfavorable distribution of methyl group resonance signals prevents complete assignment based exclusively on 4D HMQC-NOESY-HMQC experiments, yielding assignment of only 55 out of 100 methyl groups. Therefore, we created point mutants and measured pseudo contact shifts, extending and validating assignments based on methyl-methyl NOEs. Of note, the P-domains are present in two different forms caused by an approximate equal distribution of trans- and cis-configured proline residues in position 361.

A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.

Identification of protein N-terminal methyltransferases in yeast and humans.

Protein modification by methylation is important in cellular function. We show here that the Saccharomyces cerevisiae YBR261C/TAE1 gene encodes an N-terminal protein methyltransferase catalyzing the modification of two ribosomal protein substrates, Rpl12ab and Rps25a/Rps25b. The YBR261C/Tae1 protein is conserved across eukaryotes; all of these proteins share sequence similarity with known seven beta-strand class I methyltransferases. Wild-type yeast cytosol and mouse heart cytosol catalyze the methylation of a synthetic peptide (PPKQQLSKY) that contains the first eight amino acids of the processed N-terminus of Rps25a/Rps25b. However, no methylation of this peptide is seen in yeast cytosol from a DeltaYBR261C/tae1 deletion strain. Yeast YBR261C/TAE1 and the human orthologue METTL11A genes were expressed as fusion proteins in Escherichia coli and were shown to be capable of stoichiometrically dimethylating the N-terminus of the synthetic peptide. Furthermore, the YBR261C/Tae1 and METTL11A recombinant proteins methylate variants of the synthetic peptide containing N-terminal alanine and serine residues. However, methyltransferase activity is largely abolished when the proline residue in position 2 or the lysine residue in position 3 is substituted. Thus, the methyltransferases described here specifically recognize the N-terminal X-Pro-Lys sequence motif, and we suggest designating the yeast enzyme Ntm1 and the human enzyme NTMT1. These enzymes may account for nearly all previously described eukaryotic protein N-terminal methylation reactions. A number of other yeast and human proteins also share the recognition motif and may be similarly modified. We conclude that protein X-Pro-Lys N-terminal methylation reactions catalyzed by the enzymes described here may be widespread in nature.

Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index

Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.

The Cytoplasmic Peptidase DPP9 Is Rate-limiting for Degradation of Proline-containing Peptides

Protein degradation is an essential process that continuously takes place in all living cells. Regulated degradation of most cellular proteins is initiated by proteasomes, which produce peptides of varying length. These peptides are rapidly cleaved to single amino acids by cytoplasmic peptidases. Proline-containing peptides pose a specific problem due to structural constrains imposed by the pyrrolidine ring that prevents most peptidases from cleavage. Here we show that DPP9, a poorly characterized cytoplasmic prolyl-peptidase, is rate-limiting for destruction of proline-containing substrates both in cell extracts and in intact cells. We identified the first natural substrate for DPP9, the RU1(34-42) antigenic peptide (VPYGSFKHV). RU1(34-42) is degraded in vitro by DPP9, and down-regulation of DPP9 in intact cells results in increased presentation of this antigen. Together our findings demonstrate an important role for DPP9 in peptide turnover and antigen presentation.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Governing the monomer-dimer ratio of human cystatin c by single amino acid substitution in the hinge region.

Three dimensional domain swapping is one of the mechanisms involved in formation of insoluble aggregates of some amyloidogenic proteins. It has been proposed that proteins able to swap domains may share some common structural elements like conformationally constrained flexible turns/loops. We studied the role of loop L1 in the dimerization of human cystatin C using mutational analysis. Introduction of turn-favoring residues such as Asp or Asn into the loop sequence (in position 57) leads to a significant reduction of the dimer fraction in comparison with the wild type protein. On the other hand, introduction of a proline residue in position 57 leads to efficient dimer formation. Our results confirm the important role of the loop L1 in the dimerization process of human cystatin C and show that this process can be to some extent governed by single amino acid substitution.

Purification, identification and characterisation of seprase from bovine serum

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC 50 of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC–MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S 1. This analysis revealed at least five subsites to be involved in enzyme–substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P 1 was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P 1′) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Structural Characterization of the HIV-1 Vpr N Terminus: EVIDENCE OF cis/trans-PROLINE ISOMERISM

The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major cellular isomerase cyclophilin A and Vpr, both of which are incorporated into HIV-1 virions.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Comparison of four variants of a major allergen in hazelnut ( Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97–99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Proline residue 280 in the second extracellular loop (EC2) of the VPAC 2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC 1 and the VPAC 2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC 1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (〈Pro〉294 VPAC 1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC 1 antagonist. Coupling of the 〈Pro〉294 VPAC 1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC 1 antagonist to partial agonist behavior. Deletion of the proline 280 (ΔPro280 VPAC 2) in the VPAC 2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC 2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.

Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen.

Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis. Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

Fluorescence study of neurohypophyseal hormones and their analogues. Distance distributions in a series of arginine-vasopressin analogues.

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or beta-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr2 and the disulphide bridge Cys1-Cys6. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field).

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel.

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.

Structural Study of the N‐Terminal Segment of Neuropeptide‐Tyrosine

A series of analogues of neuropeptide tyrosine (NPY) was synthesized by solid-phase peptide synthesis using BOP as a coupling reagent for the complete synthesis. A structure-activity study of the N-terminal portion of the molecule was performed with the analogues obtained by the successive replacement of the first 10 amino acids by the residue L-alanine. NPY and its analogues [Ala1-10]hNPY were tested for their potency on rat vas deferens and for their affinity to central nervous system receptors on a rat brain membrane preparation. The results suggest that the hypothetical polyproline type II helix structure of the N-terminal segment is involved in both potency and affinity. Indeed, the substitution by L-Ala of proline residues in position 2, 5, or 8 showed important losses of activity and affinity. The more important losses were observed with the replacement of Pro-5 or Pro-8. A critical loss of potency of hNPY was also observed after the substitution of the Tyr-1 residue by L-Ala, thus confirming the important role played by this residue for the full expression of the biological activity of NPY.

Rubredoxin from Clostridium thermosaccharolyticum. Amino acid sequence, mass-spectrometric and preliminary crystallographic data

Rubredoxin isolated from the thermophilic bacterium Clostridium thermosaccharolyticum has been sequenced and crystallized. The 52-residue sequence is similar to those of rubredoxins occurring in other anaerobic bacteria, but displays some unique features, including a tryptophan residue in position 4, two consecutive proline residues in positions 25 and 26, and an aspartic acid residue in position 41. The molecular mass (5988 Da) of the native rubredoxin has been measured by electrospray-ionization m.s., thus establishing the applicability of the technique to this type of iron-sulphur protein. C. thermosaccharolyticum rubredoxin crystallizes as dark-red elongated prisms with a flat diamond cross-section. The X-ray diffraction shows symmetry consistent with space group P2(1)2(1)2(1). Cell parameters are: a = 2.73 nm, b = 2.98 nm, c = 6.49 nm.

Structural study of the N-terminal segment of neuropeptide tyrosine

A series of analogues of neuropeptide tyrosine (NPY) was synthesized by solid-phase peptide synthesis using BOP as a coupling reagent for the complete synthesis. A structure-activity study of the N-terminal portion of the molecule was performed with the analogues obtained by the successive replacement of the first 10 amino acids by the residue L-alanine. NPY and its analogues [Ala1-10]hNPY were tested for their potency on rat vas deferens and for their affinity to central nervous system receptors on a rat brain membrane preparation. The results suggest that the hypothetical polyproline type II helix structure of the N-terminal segment is involved in both potency and affinity. Indeed, the substitution by L-Ala of proline residues in position 2, 5, or 8 showed important losses of activity and affinity. The more important losses were observed with the replacement of Pro-5 or Pro-8. A critical loss of potency of hNPY was also observed after the substitution of the Tyr-1 residue by L-Ala, thus confirming the important role played by this residue for the full expression of the biological activity of NPY.

Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

In N-glycosylated glycoproteins, carbohydrate is attached to Asn in the sequence Asn-X-Ser/Thr, where X denotes any amino acid. However, the presence of this consensus peptide does not always lead to glycosylation. We have compiled an extensive collection of glycosylated and non-glycosylated Asn-X-Thr/Ser sites and present a statistical study based on this data set. Our results indicate that non-glycosylated sites tend to be found more frequently towards the C termini of glycoproteins, and that proline residues in positions X and Y in the consensus Asn-X-Thr/Ser-Y strongly reduce the likelihood of N-linked glycosylation. Beyond this, there are no obvious local sequence features that seem to correlate with the absence or presence of N-linked glycosylation. These findings are discussed in terms of the prediction and engineering of glycosylation sites in secretory proteins.