Basic Amino Acid Residues Research Articles

High temperature induced structural changes of apo-lactoferrin and interactions with β-lactoglobulin and α-lactalbumin for potential encapsulation strategies

There has been increasing interest in lactoferrin as a bioactive compound from milk, as it functions as an antimicrobial and also in delivering iron. Despite the potential as a food ingredient, the low bioavailability of lactoferrin has limited opportunities for commercialisation. Therefore, protective encapsulation of lactoferrin has been explored as a means of enhancing its bioavailability. In this study, a novel in silico approach was adopted to explore the possibility of employing whey protein components to encapsulate lactoferrin. The most unstable form of the protein, apo-lactoferrin has been studied, particularly interactions with the whey proteins β-lactoglobulin and α-lactalbumin. Temperature conditions that might be encountered during spray drying (95 and 180 °C) have been modelled in order to provide an in-depth understanding of the interactions at the molecular level. The findings indicate that β-lactoglobulin and α-lactalbumin are able to preserve the two antibacterial regions within the apo-lactoferrin structure (lactoferricin and lactoferampin). Intra-molecular residue contact analyses within apo-lactoferrin illustrate the tendency for agglomeration under these temperature conditions, indicating that it is most likely to be readily dispersible during subsequent rehydration. Furthermore, free energy calculations found electrostatic attractions to be the primary mode of interaction between apo-lactoferrin, β-lactoglobulin, and α-lactalbumin. This data is confirmed by a network analysis that demonstrates how interactions amongst apo-lactoferrin, β-lactoglobulin, and α-lactalbumin are predominantly a result of the acidic and basic amino acid residues of the proteins. Overall, this study provides molecular insights in understanding the interactions between lactoferrin with β-lactoglobulin and α-lactalbumin for potential encapsulation strategies via spray drying.

The membrane binding and deformation property of vaccinia virus K1 ankyrin repeat domain protein.

Membrane lipids are essential participants in cellular events, but only a small number of lipid-interacting proteins have been characterized. Taking advantage of the small genome (~270 genes) of the vaccinia virus, we screened for soluble lipid-binding proteins and found 27 proteins to be soluble after expression in Escherichia coli. Among them, 4 proteins were found to strongly bind to the total bovine brain lipid extract (Folch I fraction) that contained large amounts of phosphatidylserine in vitro. Out of the 4 proteins, 3 were unique proteins to viruses. Another protein, K1, solely contained an ankyrin repeat domain (ARD). ARD is conserved in large numbers of proteins in bacteria, archaea, eukaryotes and viruses, suggesting the possibilities of the membrane binding of ARDs in varieties of proteins. Furthermore, K1 deformed the lipid membrane dependently on the charged lipids. The tubulation and membrane binding was enhanced with increased negative membrane charge from phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ). The basic amino acid residues in the ARD were essential for membrane deformation, suggesting electrostatic interactions between K1 and the membrane for membrane deformation.

Ultrashort Cationic Lipopeptides-Effect of N-Terminal Amino Acid and Fatty Acid Type on Antimicrobial Activity and Hemolysis.

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that hypothetically may be alternatively used to combat pathogens such as bacteria and fungi. In general, USCLs consist of fatty acid chains and a few basic amino acid residues. The main shortcoming of USCLs is their relatively high cytotoxicity and hemolytic activity. This study focuses on the impact of the hydrophobic fatty acid chain, on both antimicrobial and hemolytic activities. To learn more about this region, a series of USCLs with different straight-chain fatty acids (C8, C10, C12, C14) attached to the tripeptide with two arginine residues were synthesized. The amino acid at the N-terminal position was exchanged for proteinogenic and non-proteinogenic amino acid residues (24 in total). Moreover, the branched fatty acid residues were conjugated to N-terminus of a dipeptide with two arginine residues. All USCLs had C-terminal amides. USCLs were tested against reference bacterial strains (including ESKAPE group) and Candida albicans. The hemolytic potential was tested on human erythrocytes. Hydrophobicity of the compounds was evaluated by RP-HPLC. Shortening of the fatty acid chain and simultaneous addition of amino acid residue at N-terminus were expected to result in more selective and active compounds than those of the reference lipopeptides with similar lipophilicity. Hypothetically, this approach would also be beneficial to other antimicrobial peptides where N-lipidation strategy was used to improve their biological characteristics.

PtdIns4P-mediated electrostatic forces influence S-acylation of peripheral proteins at the Golgi complex.

Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.

CD40 ligand dictated signaling bias

T cells are pivotal for innate and adaptive immune response. On, T cell a co-stimulatory molecule CD40 ligand is present that binds to the CD40 receptor. CD40L a type II membrane protein, consists of 261 amino acids belong to a member of the TNF-family. There are 193 amino acids present at the extracellular domain of CD40 ligand requires basic amino acid residue K143, R203, R207 for receptor binding, however, the CD40 molecule requires acidic amino acid residue D84, E114, E117 for activation of the different signaling cascade. Binding of CD40L with CD40 receptors activate JAK-STAT signaling, inflammatory cytokines, chemokines along with various downstream transcription factor molecules. As it is well studied, deficiency in CD40 ligand leads to an autoimmune disorder called Hyper X-linked IgM syndrome. So studies on HXIM syndrome revealed that an extracellular domain of CD40 ligand is cardinal for receptor binding. Depending on binding strength, signaling skew will be decided. Stronger will activate p38 MAPK (anti-parasitic in nature) by inducing IL-12 however, weaker will activate ERK-1/2 (pro-parasitic in nature) by inducing IL-10. The specific binding of the receptor is necessary to decode the message encoded by binding stimulation. Therefore, CD40 ligand wild type residues bind with CD40 site-directed mutagenized with different amino acids. These mutant CD40 ligands upon binding with CD40 reveal a better insight into the nature of host-pathogen interaction, inflammatory cytokines, chemokines and signaling molecules. Therefore, we can assume CD40 ligand mutants have a role in anti-leishmanial function.

Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) contains two major open reading frames (ORFs) and the ORF2 gene encodes the major structural capsid protein. In this study, nuclear localization of ORF2 was demonstrated by fluorescence observation and subcellular fractionation assays in ORF2-transfected PK-15 cells. The subcellular localization of truncated ORF2 indicated that the 38 N-terminal amino acids were responsible for the nuclear localization of ORF2. The truncated and site-directed mutagenesis of this domain were constructed, and the results demonstrated that the basic amino acid residues at positions 8–32 were essential for the strict nuclear localization. The basic motifs 8RRR-R-RRR16 and 16RRRHRRR22 were further shown to be the key functional nucleolar localization signals that guide PCV3 ORF2 into nucleoli. Furthermore, sequence analysis showed that the amino acids of PCV3 nuclear localization signals were highly conserved. Overall, this study provides insight into the biological and functional characteristics of the PCV3 ORF2 protein.

Penetration of phospholipid membranes by poly-l-lysine depends on cholesterol and phospholipid composition

Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio.

Discovery & Characterization of Therapeutically Targetable Ntrk Point Mutations in Leukemia

Discovery & Characterization of Therapeutically Targetable Ntrk Point Mutations in Leukemia

Zinc-Dependent Homomultimerization of Mutant Calreticulin Is Required for MPN Pathogenesis

Recurrent mutations in the ER chaperone calreticulin (CALR) are found in ~30% of MPNs. All known CALR mutations result in a +1-frameshift of the CALR reading frame that retains the N-domain and P-arm of wild-type CALR and generates a novel mutant-specific C-terminal domain encoded by an alternative reading frame. Multiple groups have previously demonstrated that mutant CALR (CALRMUT) physically binds to and activates the thrombopoietin receptor MPL and causes inappropriate receptor activation and downstream JAK-STAT signaling in a ligand-independent manner. These oncogenic activities of CALRMUT have been shown to be dependent on a threshold of basic amino acid residues in the mutant-specific C-terminus and on the lectin motif within the N-domain. In this study, we extended the analysis of the role of the N-domain in CALRMUT activity by performing a comprehensive alanine mutagenesis screen of all 177 non-alanine residues within the N-domain and assessed the ability of alanine-mutants to confer cytokine independent growth in Ba/F3-MPL cells. We identified two functional groups of residues as being important for CALRMUT activity: (i) Residues associated with CALR lectin motif. Consistent with previous work which had shown that Asp135 and Asp317 was essential for CALRMUT activity, we observed that loss of any of 4 residues within the lectin motif (Cys105, Lys111, Gly133 and Asp135) led to total impairment in the ability of CALRMUT to confer cytokine independence, bind MPL or elicit JAK-STAT signaling. Surprisingly, other residues implicated in lectin activity (Tyr109, Tyr128, Met131) had either only a partial effect or no effect on CALRMUT oncogenic activity, suggesting that different regions within the lectin motif of CALR have differential effects in facilitating MPL binding. (ii) Residues associated with zinc binding. We also identified a group of zinc-binding histidine residues (His99, His145 and His170) as being important for modulating CALRMUT activity. We observed that loss of any single histidine residue (1xHis) led to partial abrogation of cytokine independence in Ba/F3-MPL cells. Strikingly, combined loss of two (or more) histidines (2xHis) led to complete impairment in the capacity to confer cytokine independent growth or elicit JAK-STAT signaling, in conjunction with impaired ability to physically associate with Zn2+ by affinity chromatography. In addition, 2xHis-CALRMUT failed to bind MPL in pulldown assays or colocalize with MPL in FRET-based immunocytochemical analysis. We next investigated in greater detail how the zinc binding capacity of CALRMUT regulates its oncogenic activity. Previous studies have shown that CALRMUT forms homomultimers (Araki, Leukemia, 33:122), and that zinc is able to increase homomultimerization of recombinant wild-type CALR in vitro (Li, Biochemistry, 40:11193). We therefore tested the multimerization capacity of lectin-deficient or zinc binding-deficient CALRMUT by assessing the ability of heterologously-expressed FLAG-tagged CALRMUT species to complex and co-immunoprecipitate with a V5-tagged CALRMUT species in 293T cells. We observed that lectin-deficient CALRMUT and 1xHis-CALRMUT retained the capacity to form homomultimers, but the 2xHis- CALRMUT could not, suggesting that zinc binding was required for homomultimer formation. Treatment with ZnCl2 which increases free intracellular Zn2+ resulted in increased CALRMUT homomultimerization in a dose-dependent manner, as well as increased binding to MPL within heteromeric complexes. Finally, treatment with the zinc chelator TPEN led to decreased CALRMUT homomultimer formation, decreased CALRMUT-MPL heteromultimeric complexes and attenuated JAK-STAT signaling. Cumulatively, our data suggest that zinc is required for homomultimerization of CALRMUT, and is a necessary prerequisite event to facilitate binding to and activation of MPL in CALR-mutated MPNs. Disclosures Mullally: Janssen: Research Funding.

Influence of Short Cationic Lipopeptides with Fatty Acids of Different Chain Lengths on Bacterial Biofilms Formed on Polystyrene and Hydrogel Surfaces.

Nowadays, biomaterials are applied in many different branches of medicine. They significantly improve the patients’ comfort and quality of life, but also constitute a significant risk factor for biofilm-associated infections. Currently, intensive research on the development of novel materials resistant to microbial colonization as well as new compounds that are active against biofilms is being carried out. Within this research, antimicrobial peptides (AMPs) and their analogues are being intensively investigated due to their promising antimicrobial activities. The main goal of this study was to synthesize and evaluate the antimicrobial efficacy of short cationic lipopeptides that were designed to imitate the features of AMPs responsible for antimicrobial activities: positive net charge and amphipacity. The positive charge of the molecules results from the presence of basic amino acid residues: arginine and lysine. Amphipacity is provided by the introduction of decanoic, dodecanoic, tetradecanoic, and hexadecanoic acid chains to the molecules. Lipopeptides (C16-KR-NH2, C16-KKK-NH2, C16-KKC-NH2, C16-KGK-NH2, C14-KR-NH2, C14-KKC-NH2, C12-KR-NH2, C12-KKC-NH2, and (C10)2-KKKK-NH2) were synthesized using a novel solid-phase temperature-assisted methodology. The minimum inhibitory concentrations (MICs), minimum biofilm eradication concentrations (MBECs), and minimum biofilm formation inhibitory concentrations (MBFICs) were determined for the following bacterial strains: Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Proteus mirabilis PCM 543. The biofilms were cultured on two types of surfaces: polystyrene plates (PS) and contact lenses (CL). The lipopeptides exhibited the ability to inhibit the growth of bacteria in a liquid medium as well as on the PS and CL. The compounds also eliminated the bacterial biofilm from the surface of both materials. In general, the activity against gram-positive bacteria was stronger in comparison to that against gram-negative strains. There were certain discrepancies between the activity of compounds against the biofilm cultured on PS and CL. This was especially noticeable for staphylococci—the lipopeptides presented much higher activity against biofilm formed on the PS surface. It is worth noting that the obtained MBEC values for lipopeptides were usually only a few times higher than the MICs. The results of the performed experiments suggest that further studies on lipopeptides and their potential application in the treatment and prophylaxis of biofilm-associated infections should be conducted.

Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

TRPM6 N-Terminal CaM- and S100A1-Binding Domains.

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member—the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

Protein import into chloroplasts via the Tic40-dependent and -independent pathways depends on the amino acid composition of the transit peptide

Preprotein import into chloroplasts is mediated by the coordinated actions of translocons at the outer and inner envelopes of chloroplasts (Toc and Tic, respectively). The cleavable N-terminal transit peptide (TP) of preproteins plays an essential role in the import of preproteins into chloroplasts. The Tic40 protein, a component of the Tic complex, is believed to mediate the import of preproteins through the inner envelope. In this study, we aimed to obtain in vivo evidence supporting the role of Tic40 in preprotein import into chloroplasts. Contrary to previous findings, the import of various preproteins with wild-type TPs showed no difference between tic40 and wild-type protoplasts of Arabidopsis thaliana. However, the import of N-terminal mutants of the RbcS protein (RbcS-nt), in which basic amino acid residues (arginine and lysine) in the central region of the TP were substituted with neutral (alanine) or acidic (glutamic acid) amino acid residues, was dependent on Tic40. In addition, in tic40 protoplasts, the inner envelope protein Tic40 tagged with HA (hemagglutinin) showed more intermediate form present in the stroma. Based on these results, we propose that protein can be imported into chloroplast by either Tic40-independent or Tic40-dependent pathways depending on the types of TP.

Identification of functionally important amino acid sequences in cobra venom factor using human C3/Cobra venom factor hybrid proteins

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF,Bb is an enzyme that cleaves C3 and C5. However, CVF,Bb exhibits significantly different functional properties from C3b,Bb. The CVF,Bb convertase is physico-chemically very stable, and completely resistant to an activation by Factors H and I. These two properties, in contrast to C3b,Bb, allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the functional differences between CVF and C3, we have created several hybrid proteins of CVF and human C3. Here we report that replacing the C-terminal 168 amino acid residues of human C3 with the corresponding residues from CVF results in a hybrid protein (HC3-1496) which is essentially a human C3 derivative exhibiting the functional properties of CVF. This result demonstrates that the important structures for the CVF-specific functions reside within the C-terminal 168 amino acid residues of CVF. We further demonstrate that reverting the 46 C-terminal CVF residues of HC3-1496 to human C3 sequence results in a hybrid protein (HC3-1496/1617) that exhibits a physico-chemically unstable convertase with only residual complement depleting activity. This result demonstrates that most, but not all, structural requirements for CVF activity reside within the 46 C-terminal amino acid residues. We also investigated the potential role of position 1633, which is an acidic residue in human C3 (glutamic acid) but a basic amino acid residue (histidine) in CVF. However, the charge at position 1633 appears to be of no functional relevance. Exchanging the neutral amino acids present in CVF at positions 1499 and 1501 with the two charged amino acids at these positions in human C3 (aspartic acid and lysine) resulted in a hybrid protein that exhibited significantly slower convertase formation although both binding to Factor B and C3 cleavage was not affected, demonstrating that the charged amino acid residues at these two positions interfere with the formation of the convertase. In conclusion, our work demonstrates that hybrid proteins of human C3 and CVF present valuable tools to identify functionally important amino acid residues in CVF.

Three Structural Features of Functional Food Components and Herbal Medicine with Amyloid β42 Anti-Aggregation Properties.

Aggregation of amyloid β42 (Aβ42) is one of the hallmarks of Alzheimer’s disease (AD). There are numerous naturally occurring products that suppress the aggregation of Aβ42, but the underlying mechanisms remain to be elucidated. Based on NMR and MS spectroscopic analysis, we propose three structural characteristics found in natural products required for the suppressive activity against Aβ42 aggregation (i.e., oligomerization by targeting specific amino acid residues on this protein). These characteristics include (1) catechol-type flavonoids that can form Michael adducts with the side chains of Lys16 and 28 in monomeric Aβ42 through flavonoid autoxidation; (2) non-catechol-type flavonoids with planarity due to α,β-unsaturated carbonyl groups that can interact with the intermolecular β-sheet region in Aβ42 aggregates, especially aromatic rings such as those of Phe19 and 20; and (3) carboxy acid derivatives with triterpenoid or anthraquinoid that can generate a salt bridge with basic amino acid residues such as Lys16 and 28 in the Aβ42 dimer or trimer. Here, we summarize the recent body of knowledge concerning amyloidogenic inhibitors, particularly in functional food components and Kampo medicine, and discuss their application in the treatment and prevention of AD.

Enkephalin related peptides are released from jejunum wall by orally ingested bromelain

Stem bromelain [EC 3.4.22.32] is a thiol-endopeptidase and orally recommended in traditional medicine due to its analgesic activity, but the mechanisms are not known. Proenkephalin is expressed in the nervous system, but also in the gastrointestinal tract, where it can be assessed by ingested stem bromelain. Here we demonstrated that stem bromelain hydrolyses synthetic proenkephalin fragments after basic amino acid residues flanking the enkephalin sequences. We also observed with in vivo studies that oral administration of bromelain reduced jejunum proenkephalin levels and increased the serum enkephalin in mice. Effective anti-nociceptive effects in mice were observed 3 h after oral administration of 3 mg/kg stem bromelain by the acetic acid-induced writhing test. However, with higher doses this effect is reduced due to hydrolysis of enkephalin that possibly occurs by the presence of ananain in commercial pineapple stem bromelain preparations, that is also a thiol-protease with broad specificity. The analgesic effects were also evaluated by hot-plate and formalin tests and the obtained results indicated that enkephalin generated in intestine acts in periphery where it also can have anti-inflammatory activity.

ASB20123: A novel C-type natriuretic peptide derivative for treatment of growth failure and dwarfism

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1–22) and human ghrelin(12–28, E17D). Compared to CNP(1–22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1–22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Role of heparan sulfate in entry and exit of Ross River virus glycoprotein-pseudotyped retroviral vectors

Variants of Ross River virus (RRV) that bind to heparan sulfate (HS) were previously selected by serial passaging in cell culture. To explore the effects of mutations that convey HS utilization, we pseudotyped Moloney murine leukemia virus (MoMLV), with the RRV envelope. We substituted amino-acid residues 216 and 218 on RRV-E2-envelope glycoprotein with basic amino-acid residues, because these mutations confer affinity for HS upon RRV. However, T216R-RRV- and N218R-RRV-pseudotyped viruses possessed lower transduction titers, and we demonstrated that HS-affinity impeded release of pseudotyped virus from producer cells. Addition of heparinase to HS-expressing target cells reduces the transduction efficiency of the T216R-RRV- and N218R-RRV-pseudotyped viruses, whereas no such effect is seen in cells lacking HS. Under appropriate conditions, these T216R-RRV- and N218R-RRV-pseudotyped viruses have enhanced capacities for transducing HS-expressing cells. General principles concerning viral adaptation to the use of attachment factors and design of pseudotyped viral vectors are discussed.

Site-directed mutagenesis of cysteine to serine residues affects heparin binding and mitogenicity in fibroblast growth factor 4 produced in Escherichia coli

The binding activity to heparan sulphate is crucial for the mitogenic activity of fibroblast growth factor 4 (FGF4) in developing mammalian embryos. There are two conserved cysteine residues in FGF family proteins, Cys-84 and Cys-151 in mouse FGF4, and these constitute all of the cysteine residues in FGF4. However, the relationships among the heparin binding activity, growth promoting activity, and the two conserved cysteine residues in FGF4 are still unclear. Consequently, we generated in Escherichia coli three kinds of point-mutated mouse FGF4, namely C84S, C151S, and C84S;C151S, by converting the cysteine residues to serine residues. In heparin column chromatography, the heparin binding activities of these mutants were attenuated. In particular, the activity of the double-mutated C84S;C151S was weakened considerably. The growth promoting activities of these mutants correlated well with their heparin binding activities. We also demonstrated that an octapeptide, the Leu-76 to Tyr-83 region, which contained four basic amino acid residues and flanked Cys-84 in mouse FGF4, enhanced the heparin binding activity when fused to glutathione-S-transferase as a recombinant protein. Overall, our findings imply that the two conserved cysteine residues in FGF4 are both involved in the heparin binding activity and mitogenicity likely by affecting the configuration of heparin binding sites in FGF4.

Determination of conjugated protein on nanoparticles by an adaptation of the Coomassie blue dye method

A new accurate spectrophotometric method for protein determination on nanoparticles is described. The method is based on the Coomassie blue dye that binds to the basic and aromatic amino acid residues of proteins, especially arginine and lysine. A known amount of reagent dye was mixed with a variety of protein-loaded nanoparticles. Thereafter the unconjugated reagent was mixed with excess protein (bovine serum albumin) and titrated. In this method, the reacted dye on the protein coating of nanoparticle is directly determined, in opposite to the conventional method, in which the conjugated protein is determined as the difference between the non-conjugated protein found in the supernatant after centrifugation, and the total amount of protein originally used. This method is able to measure amounts of coated protein lower than 1 ppm.•Simple and accurate method especially adapted for protein-coated nanoparticles.•The amino acid residues of protein in the nanoparticle surface react with Coomassie brilliant blue dye.•The unreacted dye is titrated with an excess of a standard protein.