Site‐specific fluorescein labeling of human insulin

Three fluorescein derivatives of human insulin (HI, 1 ) labeled at positions N^αA1, N^αB1 and N^εB29 respectively, were synthesized using an N‐trifluoroacetyl‐based protecting group scheme. The Tfa protecting group introduced by reaction with ethyl trifluoroacetate was found to be stable in aqueous and organic media and efficiently removed under mild basic conditions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Characterizing the release of bioactive N‐glycans from dairy products by a novel endo‐β‐N‐acetylglucosaminidase

Endo‐β‐N‐acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI‐1) is a novel enzyme that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐glycan core of high mannose, hybrid, and complex N‐glycans. These conjugated N‐glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N‐glycans from bovine colostrum whey by EndoBI‐1. A central composite design was used, including a two‐level factorial design (2⁴) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N‐glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan‐free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI‐1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1331–1339, 2015     

Site‐specific conjugation of the quencher on peptide's N‐terminal for the synthesis of a targeted non‐spreading activatable optical probe

Optical imaging offers high sensitivity and portability at low cost. The design of ‘smart’ or ‘activatable’ probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its ‘off ’ state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α‐amino group of the peptide's N‐terminus, (ii) conjugate the dye on the ε‐amino group of a lysine in C‐terminus, and (iii) conjugate the carboxyl group of the peptide's C‐terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site‐specific conjugation, presents several drawbacks including ‘on beads labeling’, additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α‐amino N‐terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo‐bilabeling, (ii) increase the yield of the N‐terminal labeling by two folds, and (iii) decrease the cost by 44‐fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Structural studies of the tethered N‐terminus of the Alzheimer's disease amyloid‐β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ_1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.     

A general method for preparation of N‐Boc‐protected or N‐Fmoc‐protected α,β‐didehydropeptide building blocks and their use in the solid‐phase peptide synthesis

N‐(tert‐butyloxycarbonyl) or N‐(9‐fluorenylmethoxycarbonyl) dipeptides with C‐terminal (Z)‐α,β‐didehydrophenylalanine (?^ZPhe), (Z)‐α,β‐didehydrotyrosine (?^ZTyr), (Z)‐α,β‐didehydrotryptophan (?^ZTrp), (Z)‐α,β‐didehydromethionine (?^ZMet), (Z)‐α,β‐didehydroleucine (?^ZLeu), and (Z/E)‐α,β‐didehydroisoleucine (?^Z/EIle) were synthesised from their saturated analogues via oxidation of intermediate 2,5‐disubstituted‐oxazol‐5‐(4H)‐ones (also known as azlactones) with pyridinium tribromide followed by opening of the produced unsaturated oxazol‐5‐(4H)‐one derivatives in organic‐aqueous solution with a catalytic amount of trifluoroacetic acid or by a basic hydrolysis. In all cases, a very strong preference for Z isomers of α,β‐didehydro‐α‐amino acid residues was observed except of the ΔIle, which was obtained as the equimolar mixture of Z and E isomers. Reasons for the (Z)‐stereoselectivity and the increased stability of the aromatic α,β‐didehydro‐α‐amino acid residue oxazol‐5‐(4H)‐ones over the corresponding aliphatic ones are also discussed. It is the first use of such a procedure to synthesise peptides with the C‐terminal unsaturated residues and a peptide with 2 consecutive ΔPhe residues. This approach is very effective especially in the synthesis of peptides with aliphatic α,β‐didehydro‐α‐amino acid residues that are difficult to obtain by other methods. It allowed the first synthesis of the ?Met residue. It is also more cost‐effective and less laborious than other synthesis protocols. The dipeptide building blocks obtained were used in the solid‐phase synthesis of model peptides on a polystyrene‐based solid support. Peptides containing aromatic α,β‐didehydro‐α‐amino acid residues were obtained with PyBOP or TBTU as a coupling agent with good yields and purities. In the case of aliphatic α,β‐didehydro‐α‐amino acid residues, a good efficiency was achieved only with DPPA as a coupling agent.     

Identification of genes encoding N‐glycan processing β‐N‐acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems

Glycoproteins produced by non‐engineered insects or insect cell lines characteristically bear truncated, paucimannose N‐glycans in place of the complex N‐glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N‐glycan processing β‐N‐acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N‐glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing β‐N‐acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing β‐N‐acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing β‐N‐acetylglucosaminidases than degradative or chitinolytic β‐N‐acetylglucosaminidases. In addition, over‐expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing β‐N‐acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus‐insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Peptide purification using the chemoselective reaction between N‐(methoxy)glycine and isothiocyanato‐functionalized resin

An efficient peptide purification strategy is established, comprising the selective reaction of an N‐terminal N‐(methoxy)glycine residue of the peptide and isothiocyanato‐functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation‐prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240–272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

The β‐N‐acetylhexosaminidase FDL specifically removes the β‐1,2‐GlcNAc residue conjugated to the α‐1,3‐mannose residue of the core structure of insect N‐glycans, playing significant physiological roles in post‐translational modification in the Golgi apparatus. Little is known about its enzymatic properties. We obtained the OfFDL gene from the insect Ostrinia furnacalis by RT‐PCR. The full length cDNA of FDL is 2241 bp carrying an opening reading frame of 1923 bp encoding 640 amino acids. The recombinant protein OfFDL in a soluble and active form was obtained with high purity through a two‐step purification strategy. The recombinant OfFDL exclusively hydrolyzes the terminal β‐1,2‐GlcNAc residue from the α‐1,3 branch instead of the α‐1,6 branch of the substrate GnGn‐PA. Several kinetic parameters including k_cat/K_m values toward four artificial substrates and K_i values of three representative hexosaminidase inhibitors were obtained.     

Antimicrobial activity of human α‐defensin 6 analogs: insights into the physico‐chemical reasons behind weak bactericidal activity of HD6 in vitro

Human α‐defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α‐defensins in the crystalline state. However, the physico‐chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α‐defensins, shows broad‐spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N‐terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N‐terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full‐length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of marmoset and human FSH using synthetic peptides of the β‐subunit L2 loop region and anti‐peptide antibodies

Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH–FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti‐peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β‐subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32–50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone–receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross‐species peptide and FSH in ELISA. The anti‐peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH–FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

N‐cadherin regulates the proliferation and differentiation of ventral midbrain dopaminergic progenitors

Adherens junction (AJ) between dopaminergic (DA) progenitors maintains the structure of ventricular zone and polarity of radial glia cells in the ventral midbrain (vMB) during embryonic development. However, it is unclear how loss of N‐cadherin might influence the integrity of the AJ and the process of DA neurogenesis. Here, we used conditional gene targeting approaches to perform the region‐specific removal of N‐cadherin in the neurogenic niche of DA neurons in the vMB. Removal of N‐cadherin in the vMB using Shh‐Cre disrupts the AJs of DA progenitors and radial glia processes in the vMB. Surprisingly, loss of N‐cadherin in the vMB leads to a significant expansion of DA progenitors, including those expressing Sox2, Ngn2, and Otx2. Cell cycle analyses reveal that the cell cycle exit in the progenitor cells is decreased in the mutants from E11.5 to E12.5. In addition, the efficiency of DA progenitors in differentiating into DA neurons is decreased from E10.5 to E12.5, leading to a marked reduction in the number of DA neurons at E11.5, E12.5, and E17.5. Loss of N‐cadherin leads to the diffuse distribution of β‐catenin proteins, which are a critical component of AJ and Wnt signaling, from the AJ throughout the entire cytoplasm in neuroepithelial cells, suggesting that canonical Wnt signaling might be activated in the DA progenitors in vMB. Taken together, these results support the notion that N‐cadherin regulates the proliferation of DA progenitors and the differentiation of DA neurons through canonical Wnt‐β‐catenin signaling in the vMB. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 518–529, 2013     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

Evaluation of the double‐disk synergy test for New Delhi metallo‐β‐lactamase‐1 and other metallo‐β‐lactamase producing gram‐negative bacteria by using metal‐ethylenediaminetetraacetic acid complexes

New Delhi metallo‐β‐lactamase‐1 (NDM‐1), one of the metallo‐β‐lactamases (MBLs), has been identified from clinical isolates worldwide. Rapid detection of NDM‐1 producers is necessary to prevent their dissemination. Seven types of EDTA complexes were evaluated as MBL inhibitors in double‐disk synergy tests (DDSTs), resulting in detection of the first isolate of NDM‐1‐producing Escherichia coli (NDM‐1 Dok01) in Japan. NDM‐1 Dok01 was detected when EDTA magnesium disodium salt tetrahydrate (Mg‐EDTA), EDTA calcium disodium salt dihydrate, EDTA cobalt disodium salt tetrahydrate and EDTA copper disodium salt tetrahydrate were used as MBL inhibitors. The sensitivity and specificity of DDSTs using Mg‐EDTA for 75 MBL producers and 25 non‐MBL producers were 96.0% and 100%, respectively. These findings indicate that the DDST method using Mg‐EDTA can detect MBL‐producing strains, including NDM‐1 producers.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.