Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways.

Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

A novel anti-inflammatory compound, artonkin-4'-O-glucoside, from the leaves of Artocarpus tonkinensis suppresses experimentally induced arthritis.

Artocarpus tonkinenesis (Moraceae) has been used in Vietnamese traditional medicine for the treatment of backache and joint diseases since many 100 years. We have previously shown that a crude extract of A. tonkinensis elicited anti-inflammatory effects in rat collagen-induced arthritis (CIA), with significant improvement of disease symptoms. However, the pharmacological basis of the bioactivity of A. tonkinensis extract is not known. In the present study, we have isolated four individual active components from A. tonkinensis extract by reverse phase high-pressure liquid chromatography. The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and their biological effects investigated. A novel biologically active flavonoid glucoside (5-hydroxy-8-hydroxymethyl-8-methyl-2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenyl]-8H-pyrano[3,2-g]chromen-4-one) with an average molecular mass of 514.49 Da was isolated.We have named the compound artonkin-4'-O-glucoside. The name 'artonkin' for the novel flavonoid part of the compound was coined from the Latin name of its source Artocarpus tonkinensis. The three other active flavonoid glucosides isolated and characterized were alphitonin-4-O-beta-D-glucoside, maesopsin-4-O-beta-D-glucoside and kaempherol-3-O-beta-D-glucoside. All four compounds were found to cause anti-inflammatory effect with different potencies. The anti-inflammatory effects demonstrated in the rat model of arthritis correlate well with the inhibition of mitogen-induced T-cell proliferation. Furthermore, the compounds inhibit production of cytokines, such as tumour necrosis factor-a and interferon-c, in mitogen-stimulated T cells in a concentration-dependent manner. We postulate that the isolated flavonoids suppress T-cell proliferation as well as cytokine expression and thereby contribute to an amelioration of arthritis severity in CIA.

Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.

Identification of the bioactive peptide PEC-60 in brain.

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

Isolation of peptides from porcine intestinal tissue that induce extracellular acidification in CHO cells: identification of the active peptide as IGF-I and characterization of a fragment of calponin H1 processed at a dibasic site.

Chinese hamster ovary (CHO) cells are widely used as hosts for receptor expression and pharmacological studies. However, several endogenous receptor populations are present on these cells. Intestinal tissue extracts were found to induce strong extracellular acidification responses (ECAR) in CHO cells, yet several pure hormonal peptides, such as VIP, secretin, CCK, GIP, and galanin were ineffective. It is not known, which are the active compounds in the extracts that can stimulate the extracellular acidification in CHO cells. These active substances may be ligands for yet unknown receptors that are present natively in this cell type. We therefore decided to identify the active compound(s) by isolation from intestinal extract and structural characterization. Using chromatographic separations in combination with microphysiometry we have purified and characterized one such bioactive ligand. Structural analysis indicated that the isolated peptide was identical to insulin-like growth factor I (IGF-I). In the intestine, IGF-I is present in low amounts and has previously been detected only with radioimmunoassays. The results indicate that CHO cells express functional receptors for IGF-I. Among the peptides extracted from the intestine IGF-I is probably the strongest stimulator of ECAR in CHO cells. Moreover, IGF-I acts synergistically with other factors present in the crude tissue extract. Additionally, a fragment of calponin H1 (residues 1-43), previously not described at the protein level, was identified in the IGF-I containing fractions. The fragment was characterized by mass spectrometry and found to be N-terminally modified by acetylation suggesting that the whole protein bears the same posttranslational modification.

Ser(13)-phosphorylated PYY from porcine intestine with a potent biological activity.

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI-TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser(13). Phospho-PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC(50) values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho-PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho-PYY potently inhibits forskolin-stimulated cAMP accumulation in SK-N-MC cells with an IC(50) value of 0.5 nM compared to 0.15 nM for non-phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.

Large-scale HPLC purification of calbindin D9k from porcine intestine.

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.

A C-terminally elongated form of PHI from porcine intestine.

A C-terminally elongated form of peptide histidine isoleucine amide (PHI) was isolated from porcine intestine based on its effect on cAMP production in IMR-32 cells. The structure was determined by amino acid sequence analysis of tryptic fragments and by mass spectrometry. The peptide has 42 amino acid residues like those described from human, rat and mouse, but the amino acid sequence of the C-terminal extension of pig PHI is unique. Unlike the other peptides, it has a C-terminal Ala and it differs at five positions from the human form and at six positions from the rat form, while the human and the rat forms differ by only two substitutions. To avoid confusion arising from different C-terminal residues, a unifying nomenclature is proposed: PHI-27 for the hormone and PHI-42 for the elongated product.

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin.

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.

The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.

In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.

C-terminal sequence determination of modified peptides by MALDI MS.

Peptides, cleaved by a mixture of carboxypeptidases CPP and CPY, can be detected by MALDI MS and the amino acid sequence thereby determined by calculation of the differences between consecutive peaks. In the present study we have used derivatizations of Lys and Cys to facilitate identification of these residues. Since the mass values do not readily distinguish Lys from Gln, we have converted Lys to homoarginine by guanidination, allowing simple detection of Lys. To identify the Cys positions in peptides that contain cystine, cysteic acid, or carboxymethylcysteine is not possible using CPY and CPP because of the lack of proteolytic cleavage. Instead we find that identification of Cys residues within the sequence can be achieved after conversion to a basic derivative, 4-thialaminine (Thi), by trimethylaminoethylation.

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues.

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Solution structure of a naturally-occurring zinc-peptide complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM domain is an independent folding unit.

The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by 1H NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 A for the backbone atoms of residues 3-30. Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1-zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat CRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N delta in contrast to the more usual coordination by N epsilon observed for other zinc-finger domains. The present structure determination of the ZF-1-zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.

The secretory trypsin inhibitor like-peptide, PEC-60 increases dopamine D2 receptor agonist induced inhibition of GABA release in the dorsolateral neostriatum of the awake freely moving rat. An in vivo microdialysis study.

The effects of local perfusion with the secretory trypsin inhibitor like-peptide, PEC-60 on dopamine and gamma-aminobutyric acid (GABA) release in the dorsolateral neostriatum and GABA release in the globus pallidus were studied using in vivo microdialysis in the awake freely moving rat. Local perfusion with PEC-60 (500 nM and 1 microM) increased dopamine release in the dorsolateral neostriatum while the highest (1 microM) concentration of PEC-60 decreased striatal but not pallidal GABA release. An inactive form of the peptide, S-carboxyamidomethylated PEC-60 (1 microM) failed to influence either striatal dopamine and GABA or pallidal GABA release. In addition, when PEC-60, at a dose which did not affect striatal and pallidal GABA release (100 nM), was co-perfused together with the dopamine D2 receptor agonist pergolide (500 nM), a potentiation in the ability of pergolide to reduce GABA release in the dorsolateral neostriatum was observed and this effect was counteracted by co-perfusion with the selective dopamine D2 receptor antagonist raclopride (1 microM). In contrast, the pergolide induced inhibition of striatal dopamine release was unaffected by PEC-60 (100 nM). These data indicate that PEC-60 differentially regulates dopamine and GABA release in the dorsolateral neostriatum by a selective and facilitory interaction with the postsynaptic dopamine D2 receptor possibly involving high-affinity PEC-60 like-peptide binding sites located on local axon collaterals of a discrete subpopulation of efferent GABA neurons and/or on GABA interneurons.

Endosulfine, endogenous ligand for the sulphonylurea receptor: isolation from porcine brain and partial structural determination of the alpha form.

Anti-diabetic sulphonylureas act via high affinity binding sites coupled to K-ATP channels. Endosulfine, an endogenous ligand for these binding sites, was shown to exist in two molecular forms, alpha and beta, in both the pancreas and the central nervous system. We describe here the isolation, and partial structural characterization of alpha endosulfine derived from porcine brains by means of a series of chromatography runs and gel electrophoresis. Porcine alpha endosulfine is a protein with a molecular mass of 13,196 daltons as determined by mass spectrometry and which is N-terminally blocked. Tryptic digestion followed by separation of the fragments by HPLC and automated Edman degradation yielded a total of 72 amino acids in four partial sequences. Comparison of these sequences with that present in the National Biomedical Research Foundation protein data bank indicated a 82% identity with a 112-amino acid protein with a molecular mass of 12,353 daltons called "cyclic AMP-regulated phosphoprotein-19', isolated from the bovine brain as a substrate for protein kinase A.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.