Structure determination of two conotoxins fromConus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods

Kalume, Dário Eluan; Stenflo, Johan; Czerwiec, Eva; Hambe, Björn; Furie, Barbara C.; Furie, Bruce; Roepstorff, Peter

doi:10.1002/(sici)1096-9888(200002)35:2<145::aid-jms922>3.0.co;2-i

Cited by 45 publications

(17 citation statements)

References 44 publications

(57 reference statements)

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“…The immunoassay results and microscopic data together support a concept for the release of immunodetectable osteocalcin during bone resorption. In agreement with our results, Kurihara et al (46) have provided evidence for osteocalcin immunoreactivity in the supernatant of human bone particle-derived osteoclasts cultured on human bone slices a Because ␥-carboxylation is destroyed in MALDI-MS and not included in observed ions (51,52), all calculations of theoretical molecular masses were performed using the sequence of bovine osteocalcin (average molecular masses) with glutamic acid at positions 17, 21, and 24 instead of ␥-carboxyglutamic acid.…”

Section: Discussionsupporting

confidence: 89%

Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Ivaska

Hentunen

Vääräniemi

et al. 2004

Journal of Biological Chemistry

178

114

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Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation. Osteocalcin (OC)1 is a 6-kDa noncollagenous protein produced by osteoblasts (1), osteocytes (2), and odontoblasts (3).Osteocalcin messenger RNA has also been detected in tissues other than bone, but it appears to be processed properly only in the bone microenvironment (4, 5). The structure of osteocalcin is characterized by three glutamic acid residues, which undergo a vitamin K-dependent carboxylation. The ␥-carboxyglutamic acid residues (Gla) provide osteocalcin with the ability to bind bone hydroxyapatite with a high affinity (6, 7). Osteocalcin is the second most abundant protein in the bone matrix, and it is highly conserved among all vertebrate species (8). The biological function of osteocalcin is probably related to the regulation of bone turnover and/or mineralization (9, 10).The expression of osteocalcin is a marker of late osteoblast differentiation and is induced only after the expression of other osteoblastic markers such as alkaline phosphatase and type I collagen (11,12). Newly synthesized osteocalcin is mostly (60 -90%) adsorbed to the bone hydroxyapatite via the Gla residues, but a part of it leaks into the circulation where it can be detected (13,14). Although osteoblasts synthesize only intact osteocalcin (15), osteocalcin may further undergo intracellular processing or ...

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Section: Discussionsupporting

confidence: 89%

Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Ivaska

Hentunen

Vääräniemi

et al. 2004

Journal of Biological Chemistry

178

114

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“…Other than this example, to our knowledge, no posttranslationally brominated polypeptides have been reported in terrestrial animals. Some peptides from the venom of carnivorous marine cone snails, such as -conotoxin, are shown to have brominated tryptophan(s) (22)(23)(24)(25). Like NPB, brominations on these peptides of conus venom were found at the C-6-position of the indole moiety.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

Tanaka

Yoshida

Miyamoto

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

181

217

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GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC 50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and͞or GPR8.T here are a large number of orphan G protein-coupled receptors (GPCR) whose cognate ligands have yet to be identified (1, 2). The search for endogenous ligands of orphan GPCRs is important because GPCRs are in general excellent drug targets (3). GPR7 and GPR8 are two orphan GPCRs (4). They were originally cloned from human genomic DNA by degenerative PCR using primers based on the sequences of the ␦-opioid receptor and somatostatin receptor. Indeed, GPR7 and GPR8 each share Ϸ40% overall amino acid identities with opioid and somatostatin receptors. Human GPR7 and GPR8 are 70% identical to each other at the nucleotide level and 64% identical at the amino acid level. Interestingly, no orthologue exists for the GPR8 gene in rodents, indicating that GPR8 may have originated as a replicate of GPR7 after divergence of the rodent from other species in mammalian evolution (5).The observation that GPR7 and GPR8 mRNAs are expressed in distinct areas in the central nervous system (CNS) (4, 5), together with the similarity of GPR7 and GPR8 with opioid and somatostatin receptors, strongly argue for the existence of endogenous peptide ligand(s) in CNS. In this study, we have purified a neuropeptide ligand for GPR7 from the bovine hypothalamus. This peptide, termed neuropeptide B (NPB), consists of 29 aa with a unique brominated N-terminal tryptophan. Through EST database searches, we also identified another isopeptide of the same family, termed neuropeptide W (NPW). While this manuscript was being prepared, three papers were published reporting two endogenous ligands for GPR7 and GPR8 (6-8). Sequ...

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“…A differentially expressed ϳ60-kDa protein that was evident in TGF-␤-CDM was excised from the gel and subjected to trypsin digestion as described previously (12). The resulting peptides were sprayed from nanoelectrospray needles supplied by Protana A/S (Odense, Denmark) and analyzed on a Micromass q-Tof mass spectrometer (Manchester, UK) essentially as described earlier (13). Data acquisition and analysis were performed on a Mass Lynx Windows NT PC data system.…”

Section: Methodsmentioning

confidence: 99%

Context-specific Effects of Fibulin-5 (DANCE/EVEC) on Cell Proliferation, Motility, and Invasion

Schiemann¹,

Blobe²,

Kalume³

et al. 2002

Journal of Biological Chemistry

144

100

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The Fibulin gene family comprises five distinct genes that encode more than eight protein products via alternative splicing. Fibulins are widely expressed secretory proteins found in the blood and in the basement membranes and stroma of most tissues, where they self-associate (e.g. Fibulins 1 and 2) (1, 2) and/or interact with a variety of extracellular matrix components, including fibronectin, laminin, nidogen, aggrecan, versican, endostatin, and elastin (3-5). Thus, Fibulins likely participate in the assembly and stabilization of extracellular matrix structures; they also have been implicated in regulating organogenesis, vasculogenesis, fibrogenesis, and tumorigenesis (6 -8). Although the molecular mechanisms underlying the various biological activities of Fibulins remain to be elucidated, recent work suggests that Fibulins may interact directly with cell surface receptors, raising the possibility that these secretory proteins also function in mediating cell-cell and cell-matrix communication.The newest member of the Fibulin family is Fibulin-5 (FBLN-5; also known as EVEC (9) or DANCE (10)), 1 a 448-amino acid glycoprotein with interesting structural features; it contains an integrin-binding RGD motif, six calcium-binding epidermal growth factor-like repeats, a Pro-rich insert in the first calcium-binding epidermal growth factor-like repeat, and a globular C-terminal domain (9, 10). Functionally, FBLN-5 binds ␣ v ␤ 3 , ␣ v ␤ 5 , and ␣ 9 ␤ 1 integrins (4) and mediates endothelial cell adhesion via its RGD motif (10). In response to mechanical injury, FBLN-5 expression is induced dramatically in vascular endothelial and smooth muscle cells (9, 10), suggesting that FBLN-5 regulates vasculogenesis and endothelial cell function. Inactivation of the FBLN-5 gene in mice produces profound elastinopathy in the skin, lung, and vasculature (4, 5), demonstrating its importance in scaffolding cells to elastic fibers.Despite these recent advances, many questions regarding the role of FBLN-5 in mammalian biology remain to be answered, particularly (i) what are the signaling systems/molecules that regulate (ii) what are the effects of FBLN-5 on cell proliferation, migration, and invasion, and (iii) what are the signaling systems/molecules stimulated by FBLN-5. Through the use of expression proteomics, we show here that FBLN-5 is a novel TGF-␤-inducible protein that

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Structure determination of two conotoxins fromConus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods

Cited by 45 publications

References 44 publications

Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

Context-specific Effects of Fibulin-5 (DANCE/EVEC) on Cell Proliferation, Motility, and Invasion

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