Terutoshi Kimura

A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta.

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase ζ (PTPζ) with high affinity. In this study, we examined the binding of midkine to PTPζ by solid-phase binding assay. Midkine and pleiotrophin binding to PTPζ were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nm) and high (0.58 nm) affinity binding sites. These results suggested that PTPζ is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPζ binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPζ exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPζ by interacting with the chondroitin sulfate portion of this receptor.

Nocistatin, a peptide that blocks nociceptin action in pain transmission

Prolonged tissue damage or injury often leads to chronic pain states such that noxious stimuli evoke hyperalgesia and innocuous tactile stimuli evoke pain (allodynia),. The neuropeptide nociceptin,, also known as orphanin FQ (ref. 5), is an endogenous ligand for the orphan opioid-like receptor which induces both hyperalgesia and allodynia when administered by injection through the theca of the spinal cord into the subarachnoid space (that is, intrathecally),. Here we show that the nociceptin precursor, contains another biologically active peptide which we call nocistatin. Nocistatin blocks nociceptin-induced allodynia and hyperalgesia, and attenuates pain evoked by prostaglandin E2. It is the carboxy-terminal hexapeptide of nocistatin (Glu-Gln-Lys-Gln-Leu-Gln), which is conserved in bovine, human and murine species, that possesses allodynia-blocking activity. We have also isolated endogenous nocistatin from bovine brain. Furthermore, intrathecal pretreatment with anti-nocistatin antibody decreases the threshold for nociceptin-induced allodynia. Although nocistatin does not bind to the nociceptin receptor, it binds to the membrane of mouse brain and of spinal cord with high affinity. Our results show that nocistatin is a new biologically active peptide produced from the same precursor as nociceptin and indicate that these two peptides may play opposite roles in pain transmission.

SOLUTION STRUCTURE OF MIDKINE, A NEW HEPARIN-BINDING GROWTH FACTOR

Midkine (MK) is a 13 kDa heparin‐binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C‐terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel β‐strands, but the C‐terminal domain has a long flexible hairpin loop where a heparin‐binding consensus sequence is located. Basic residues on the β‐sheet of the C‐terminal domain form another heparin‐binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head‐to‐head dimer model of MK. The model was supported by the results of cross‐linking experiments using transglutaminase. The dimer has a fused heparin‐binding site at the dimer interface of the C‐terminal domain, and the heparin‐binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin‐binding activity and biological activity of the dimer.

Structure-activity relationship of endothelin: Importance of charged groups

Endothelin (ET)-related peptides including ET-1 (1-39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at positions 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.

Synthesis of endothelin-1 analogues, endothelin-3, and sarafotoxin S6b: Structure - activity relationships

Summary Two disulfide analogues (types A and B) of endothelin-3 (ET-3; formerly, rat ET), sarafotoxin S6b, and apamin, were synthesized to determine their disulfide structures as in the case of endothelin-1 (ET-1; formerly human and porcine ET). The disulfide structures of ET-3 and sarafotoxin S6b were found to be identical with that of ET-1 (type A) but distinct from that of apamin (type B). The vasoconstricting activities of ET-3 and sarafotoxin S6b were about one-60th and one-third that of ET-1, respectively. Such different biological potencies between endothelins and sarafotoxin S6b could be largely attributed to the sequence heterogeneity at the N-terminal portion. ET-1 analogues were also synthesized to clarify the structure-activity relationships. The opening of any disulfide bond in the ET-1 molecule extremely decreased the activity, while oxidation of the Met residue did not alter it. Amidation of the terminal COOH group and extension of the Lys-Arg sequence to the N-terminus led to 16-and 540-fold decreases in activity, respectively. Removal of the C-terminal Trp residue resulted in complete loss of the activity. The other disulfide analogues (type B and C) of ET-1 showed markedly lower activity than the parent molecule (type A). These results indicated the importance of the whole molecule with the proper double cyclic structure for determining its active conformation.

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.

Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds

Human midkine (hMK), a novel heparin‐binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121‐residue peptide was assembled from two large fully protected intermediates, Boc‐(1–5 9)‐OH and H‐(60–121)‐OBzl, in CHL/TFE (3:1, v/v) using water‐soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N‐terminal and C‐terminal half domains [(1–59) and (60–121)] were also synthesized by the same procedure used for the hMK synthesis. The C‐half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N‐half one showed much less activity in general.

Anti-nociceptive responses produced by human putative counterpart of nocistatin

b‐nocistatin is a heptadecapeptide produced from bovine prepronociceptin and blocks the induction of hyperalgesia and touch‐evoked pain (allodynia) by intrathecal administration of nociceptin or prostaglandin E2 (PGE2). Human prepronociceptin may generate a 30‐amino acid peptide different in length from b‐nocistatin. Here, we examine whether the human putative counterpart of nocistatin (h‐nocistatin) possessed the same biological activities as b‐nocistatin. Simultaneous intrathecal injection of h‐nocistatin in mice blocked the induction of allodynia by nociceptin and PGE2 in a dose‐dependent manner with ID50 values of 329 pg kg−1 and 16.6 ng kg−1, respectively. h‐nocistatin was about 10 times less potent than b‐nocistatin. h‐nocistatin also attenuated the nociceptin‐ and PGE2‐induced hyperalgesia. These results demonstrate that h‐nocistatin is biologically active and may be involved in the processing of pain at the spinal level in humans.

Oxidative folding of ω-conotoxin MVIIC: Effects of temperature and salt

Oxidative folding of o-conotoxin MVIIC, a highly basic 26-amino acid peptide with three disulfide bonds, predominantly gave two products with mismatched disulfide bonds in 0.1M NH4OAc buffer (pH 7.7) at 21°C both in the presence and absence of redox reagents such as reduced and oxidized glutathione. A low reaction temperature (5°C) and a high salt concentration in buffer such as 2M (NH4)2SO4 were necessary to obtain the correctly folded biologically active product. The folding reaction was found to proceed via a two-stage pathway of (I) the formation and (II) the rearrangement of the mismatched disulfide bonds. Both the reaction temperature and the salt strongly affected the equilibrium between mismatched and correctly formed disulfide bonds in the second stage. Such an effect of salts on the rearrangement reaction could be explained by anion binding at a low concentration and the salting out effect at a high concentration by analyzing the rank order of their effectiveness. The anion-binding effect was also confirmed by examining the folding of the tetra-acetylated peptide at the Lys side chains. CD study suggested that the yield of the biologically active product was correlated with its conformational change as functions of temperature and salt concentration.

Structure-activity relationships of α-human atrial natriuretic peptide

Abstract The spasmolytic activity of synthetic α-human atrial natriuretic peptide (α-hANP) and its related peptides was determined in vitro using the chick rectum and the rat aorta. Natriuretic activity was also measured in the anesthetized rat. α-hANP-(7–28), with the NH 2 -terminal hexapeptide truncated, had grater spasmolytic and natriuretic effect than did α-hANP-(1–28). These responses were reduced by truncation of the COOH-terminal residues. α-hANP-(7–23), the cyclic structure of α-hANP-(1–28), exhibited weak aortic relaxation and natriuretic activities. However, α-hANP-(7–23) produced a greater relaxation than did α-hANP-(1–28) in the chick rectum. Elimination of Gly at position 9 reduced the spasmolytic and natriuretic activity. Substitution of amino acid residues at position 8, 12 and 13 changed these activities. Analogues containing the ethylene linkage instead of the disulphide bond had weak biological activity. These results indicate that the size of the 17-amino acid ring and the COOH-terminal residues of α-hANP are important for the expression of spasmolytic and natriuretic activity.