Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.     

Development of novel lipid-peptide hybrid compounds with antibacterial activity from natural cationic antibacterial peptides

Seven depsipeptides were synthesized by appending seven amino acids (Lys, Leu, Val, Phe, Ser, Gln, and Pro) at the N-terminus of the active fragment [TE-(33-43)], respectively corresponding to the C-terminal beta sheet domain of tenecin 1, an antibacterial protein and their activities were measured against Staphylococcus aureus. Considering the relationship between the activity and the characteristic of amino acid at the N-terminal of the peptide, novel derivatives were designed and synthesized from TE-(33-43) by introduction of fatty acids at the N-terminal. In this process, we synthesized novel lipid-peptide hybrid compounds with a potent antibacterial activity and more improved bioavailabilities. We characterized the important structural parameters of the lipid-peptide hybrid compounds for the antibacterial activities.     

Role of the C-terminus in the biological activity of human interleukin 5

Alterations of the C-terminal amino acid sequence of recombinant human interleukin 5 (rhIL-5) caused significant changes in its biological activity. Removal of eight amino acids from the C-terminus of rhIL-5 by CNBr treatment led to a complete loss of biological activity as determined by the BCL1 cell IgM-inducing assay. Oxidation of Met residue located at C-terminus also resulted in a loss of activity. These results suggest that the C-terminal amino acids of rhIL-5 are crucial for its biological actions.     

Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein.

In a previous report (Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments. The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity. We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein. We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product. The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment. We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced. The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523). The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein. Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction. Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523). A model is proposed for the domain organization of the gyrase A protein.     

Interrelationships between the imprinting potential and biological activity of insulin derivatives: studies on Tetrahymena and Chang liver cells

Insulin dimers deprived of biological activity by linking with suberic acid symmetrically in position B29 or B1 were not able to induce imprinting. Lack of N-terminal phenylalanine or even of five C-terminal amino acids did not interfere with imprinting, regardless of whether or not it was associated with an activity loss. It appears that while hormonal imprinting is closely associated with the hormone's ability to bind to the receptor, it may be related as well as unrelated to the hormone's biological activity. The imprinted Tetrahymena and Chang cells bound the insulin and its derivatives in a similar manner.     

Effect of synthetic pban and derived peptides on sex pheromone biosynthesis in Heliothis peltigera (Lepidoptera: Noctuidae)

The activity of synthetic Heliothis zea PBAN (Hez-PBAN) and four shorter peptides on the sex pheromone biosynthesis in Heliothis peltigera was investigated in order to characterize their biological potency, and to determine the structure-activity relationship. Hez-PBAN (PBAN 1–33) is very potent and stimulates sex pheromone biosynthesis at the picomolar range both in photophase and scotophase. Removal of eight amino acids from the N-terminal region of the peptide Hez-PBAN had only a minor effect on the biological activity. A shorter fragment of Hez-PBAN, lacking 18 amino acids from the N-terminus, was less active. Two short peptides, consisting of eight and six amino acids, derived from the C-terminal region of Hez-PBAN had very little biological activity. In addition, it was found that PBAN 1–33 undergoes oxidation during storage. The oxidation of the peptide resulted in a loss of its biological activity, which could be restored by reduction with N-methylmercaptoacetamide. Unlike PBAN 1–33, PBAN 9–33 did not lose activity as a function of time, and its activity was fully preserved after prolonged storage. The results indicate that PBAN 1–33 and PBAN 9–33 have similar activities, and that the sequence containing the eight N-terminal amino acids is not essential for the biological activity of Hez-PBAN on the biosynthesis of H. peltigera sex pheromone.     

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion

Phytochromes are widely distributed photochromic biliprotein photoreceptors. Typical bacterial phytochromes such as Agrobacterium Agp1 have a C-terminal histidine kinase module; the N-terminal chromophore module induces conformational changes in the protein that lead to modulation of kinase activity. We show by protein cross-linking that the C-terminal histidine kinase module of Agp1 mediates stable dimerization. The fragment Agp1-M15, which comprises the chromophore module but lacks the histidine kinase module, can also form dimers. In this fragment, dimer formation was stronger for the far-red-absorbing form Pfr than for the red-absorbing form Pr. The same or similar behavior was found for Agp1-M15Delta9N and Agp1-M15Delta18N, which lack 9 and 18 amino acids of the N-terminus, respectively. The fragment Agp1-M20, which is derived from Agp1-M15 by truncation of the C-terminal "PHY domain" (191 amino acids), can also form dimers, but dimerization is independent of irradiation conditions. The cross-linking data also showed that the PHY domain is in tight contact with Lys 16 of the protein and that the nine N-terminal amino acids mediate oligomer formation. Limited proteolysis shows that the hinge region between the chromophore module and the histidine kinase and a part of the PHY domain become exposed upon Pr to Pfr photoconversion.     

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.     

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA.

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein-RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA.     

Novel chloroenyne-modified amino acid derivatives

Three groups of chloroenyne-modified amino acids were synthesized. Chloroenyne moiety was attached at the N- or C-terminal amino acid (Tyr, Phe, Val, Gly, Lys) position carrying different protecting groups. Prepared derivatives will be used as building blocks in the synthesis of enediyne-peptide conjugates. Furthermore, reactivity of modified amino acids in the peptide bond formation reaction was tested.     

Investigations on possible roles of C-terminal propeptide of a Ca-independent α-amylase from bacillus

Previously, an extracellular α-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [BKAΔ(N44) and BKAΔ(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The Km and V(max) values for BKAΔ(N44) were lower than BKAΔ(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.     

Human interferon gamma: significance of the C-terminal flexible domain for its biological activity

The significance of the C-terminal part of human interferon gamma (hIFNgamma) for its biological activity was studied by 3(')-end gene mutagenesis. A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus. To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNgamma proteins was measured in clear cell lysates containing the soluble fractions only. The deletion of the C terminus had a two-step effect on both hIFNgamma antiviral and antiproliferative activities. Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNgamma, the deletion of more than 9 amino acids had an opposite effect. The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNgamma. The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the alpha-helical domain F.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

Interaction with DNA and aggregation properties of C-terminal fragment of RecA protein

C-terminal fragment of Escherichia coli RecA protein 150 amino acids residues in length--the product of RecA protein BrCN-hydrolysis--was isolated by single stranded DNA-cellulose chromatography. In low salt buffer this fragment tightly bounds with single and double stranded DNAs. Aggregational properties of the fragment are similar to such of native protein: the fragment oligomerises in low salt buffer and precipitates in the presence of Mg2+. Double stranded DNA in the last case coprecipitates with the fragment, forming a complex which is stable at higher salt concentration, like the complexes with a native RecA protein. These results indicate that the C-terminal half of RecA protein participates in DNA binding and intersubunits interaction of RecA protein.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

N-terminal portion of motilin determines its biological activity.

This study aimed to identify the portion of the 22 amino acid sequence of motilin responsible for the biological activity of the peptide. The contraction of rabbit duodenal muscle in vitro was measured when exposed to synthetic fragments of motilin corresponding to various sequences of the C- or N-terminal portions of the molecule. Fragments 2-22 or 3-22 (where the initial amino acids of the N-terminal ending were removed) were more than 1000 times less potent than the native molecule 1-22. Fragment 1-9 (where the last 13 amino acids located at the C-terminal side of motilin were removed) was devoid of any contractile capacity, while synthetic fragments whose C-terminal structure extended beyond the 1-9 motilin sequence maintained almost complete biological activity. N-terminal amino acid sequence 1-9 is therefore an essential determinant of the contractile activity of motilin.