scholarly journals Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination center

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Carina Jägers ◽  
Henk Roelink
Keyword(s):  
Extracellular Matrix ◽  
Sonic Hedgehog ◽  
Calcium Ions ◽  
Calcium Binding ◽  
Peptidase Activity ◽  
Coordination Center ◽  
Zinc Coordination ◽  
Bacterial Peptidoglycan ◽  
Catalytic Cleft ◽  
Sequence Similarities

Abstract Background Sonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh. Results We found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 μM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh. Conclusions Our results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.

scholarly journals Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

2019 ◽  
Author(s):  
Carina Jägers ◽  
Henk Roelink
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Sonic Hedgehog ◽  
Calcium Ions ◽  
Heparan Sulfate Proteoglycans ◽  
Peptidase Activity ◽  
Zinc Coordination ◽  
Bacterial Peptidoglycan ◽  
Catalytic Cleft ◽  
Sequence Similarities

AbstractSonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination domain, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and thus potentially involved in the extracellular distribution of Shh. We found that the zinc-coordination fold of Shh is required for its association with ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 μM zinc and is prevented by mutations affecting critical conserved catalytical residues as well as extracellular calcium. Our results demonstrate that the zinc-coordination fold is required for ECM-association and suggest that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling.

Computational insights into the calcium ions role in protein-glycosaminoglycan systems

2021 ◽  
Author(s):  
Małgorzata M Kogut ◽  
Martyna Maszota-Zieleniak ◽  
Mateusz Marcisz ◽  
Sergey Samsonov
Keyword(s):  
Extracellular Matrix ◽  
Calcium Ions ◽  
Vital Role ◽  
Biological Processes ◽  
Periodic Linear

Glycosaminoglycans (GAGs) are anionic periodic, linear polysaccharides which are composed of periodic disaccharide units. They play a vital role in many biological processes ongoing in the extracellular matrix. In terms...

Fibrinogen Bern I: A Hereditary Fibrinogen Variant With Defective Conformational Stabilization By Calcium Ions

1981 ◽  
Author(s):  
C Rupp ◽  
C Kuyas ◽  
A Häberli ◽  
M Furlan ◽  
E A Beck
Keyword(s):  
Calcium Ions ◽  
Gel Electrophoresis ◽  
Calcium Binding ◽  
Negative Charge ◽  
Fibrinopeptide A ◽  
Isoelectric Focussing ◽  
High Calcium ◽  
Two Generations ◽  
Polypeptide Chains ◽  
Conformational Stabilization

Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.

Sonic hedgehog synergizes with the extracellular matrix protein vitronectin to induce spinal motor neuron differentiation

Development ◽  
2000 ◽  
Vol 127 (2) ◽  
pp. 333-342 ◽  
Author(s):  
S. Pons ◽  
E. Marti
Keyword(s):  
Extracellular Matrix ◽  
Motor Neuron ◽  
Sonic Hedgehog ◽  
Motor Neurons ◽  
Neural Tube ◽  
Matrix Protein ◽  
Floor Plate ◽  
Binding Domain ◽  
Extracellular Matrix Protein ◽  
Neuron Differentiation

Patterning of the vertebrate neural tube depends on intercellular signals emanating from sources such as the notochord and the floor plate. The secreted protein Sonic hedgehog and the extracellular matrix protein Vitronectin are both expressed in these signalling centres and have both been implicated in the generation of ventral neurons. The proteolytic processing of Sonic hedgehog is fundamental for its signalling properties. This processing generates two secreted peptides with all the inducing activity of Shh residing in the highly conserved 19 kDa amino-terminal peptide (N-Shh). Here we show that Vitronectin is also proteolitically processed in the embryonic chick notochord, floor plate and ventral neural tube and that this processing is spatiotemporally correlated with the generation of motor neurons. The processing of Vitronectin produces two fragments of 54 kDa and 45 kDa, as previously described for Vitronectin isolated from chick yolk. The 45 kDa fragment lacks the heparin-binding domain and the integrin-binding domain, RGD, present in the non-processed Vitronectin glycoprotein. Here we show that N-Shh binds to the three forms of Vitronectin (70, 54 and 45 kDa) isolated from embryonic tissue, although is preferentially associated with the 45 kDa form. Furthermore, in cultures of dissociated neuroepithelial cells, the combined addition of N-Shh and Vitronectin significantly increases the extent of motor neuron differentiation, as compared to the low or absent inducing capabilities of either N-Shh or Vitronectin alone. Thus, we conclude that the differentiation of motor neurons is enhanced by the synergistic action of N-Shh and Vitronectin, and that Vitronectin may be necessary for the proper presentation of the morphogen N-Shh to one of its target cells, the differentiating motor neurons.

HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR

1987 ◽  
Author(s):  
A KÖhlin ◽  
J Stenflo
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Plasma Proteins ◽  
Metal Ion ◽  
Protein C ◽  
Protein S ◽  
Protein Z ◽  
High Affinity ◽  
Epidermal Growth

In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.

scholarly journals The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors

1996 ◽  
Vol 320 (2) ◽  
pp. 687-691 ◽  
Author(s):  
Armelle MÉNARD ◽  
Emanuele PAPINI ◽  
Michèle MOCK ◽  
Cesare MONTECUCCO
Keyword(s):  
Bacillus Anthracis ◽  
Mechanism Of Action ◽  
Lethal Factor ◽  
Bifunctional Enzyme ◽  
Peptidase Activity ◽  
Macrophage Cell ◽  
Anthrax Lethal Factor ◽  
Leukotriene A4 Hydrolase ◽  
Leukotriene A4 ◽  
Sequence Similarities

The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase.

Binding of calcium ions to bovine αsl-casein and precipitability of the protein–calcium ion complexes

1980 ◽  
Vol 47 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Douglas G. Dalgleish ◽  
Thomas G. Parker
Keyword(s):  
Ionic Strength ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Binding Constants ◽  
Substitution Effects ◽  
Binding Isotherms ◽  
Decreasing Ph ◽  
Increasing Temperature

SummaryBinding isotherms for the calcium ion–αsl-casein system have been measured, as functions of ionic strength, temperature, and pH, and the isotherms have been analysed in terms of binding constants modified by substitution effects. The results demonstrate that the strength of binding is increased with increasing temperature and decreased by increasing ionic strength or decreasing pH, all of which may be explained semi-quantitatively. Parallel studies on the precipitability of the αsl-casein–Ca2+ complexes showed that there is considerable variation in the extent of calcium binding required to initiate precipitation of the protein, and in the calcium concentration necessary to achieve the required extent of ligand binding.

scholarly journals The Caenorhabditis elegans homologue of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility.

1993 ◽  
Vol 4 (9) ◽  
pp. 941-952 ◽  
Author(s):  
J E Schwarzbauer ◽  
C S Spencer
Keyword(s):  
Extracellular Matrix ◽  
Caenorhabditis Elegans ◽  
Calcium Binding ◽  
Fusion Gene ◽  
Muscle Cells ◽  
Gene Organization ◽  
The Body ◽  
Matrix Assembly ◽  
Developmentally Regulated ◽  
C Elegans

The extracellular matrix-associated protein, SPARC (osteonectin [Secreted Protein Acidic and Rich in Cysteine]), modulates cell adhesion and induces a change in cell morphology. SPARC expression in mammals is developmentally regulated and is highest at sites of extracellular matrix assembly and remodeling such as parietal endoderm and bone. We have isolated cDNA and genomic DNA clones encoding the Caenorhabditis elegans homologue of SPARC. The gene organization is highly conserved, and the proteins encoded by mouse, human, and nematode genes are about 38% identical. SPARC consists of four domains (I-IV) based on predicted secondary structure. Using bacterial fusion proteins containing nematode domain I or the domain IV EF-hand motif, we show that, like the mammalian proteins, both domains bind calcium. In transgenic nematodes expressing a SPARC-lacZ fusion gene, beta-galactosidase staining accumulated in a striated pattern in the more heavily stained muscle cells along the body. Comparison of the pattern of transgene expression to unc-54-lacZ animals demonstrated that SPARC is expressed by body wall and sex muscle cells. Appropriate levels of SPARC are essential for normal C. elegans development and muscle function. Transgenic nematodes overexpressing the wild-type SPARC gene were abnormal. Embryos were deformed, and adult hermaphrodites had vulval protrusions and an uncoordinated (Unc) phenotype with reduced mobility and paralysis.

scholarly journals A mysterious family of calcium-binding proteins from parasitic worms

2016 ◽  
Vol 44 (4) ◽  
pp. 1005-1010 ◽  
Author(s):  
Charlotte M. Thomas ◽  
David J. Timson
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Cell Biology ◽  
Fasciola Hepatica ◽  
Family Members ◽  
Ion Concentration ◽  
Pharmacological Significance ◽  
Ef Hand ◽  
Parasitic Worms

There is a family of proteins from parasitic worms which combine N-terminal EF-hand domains with C-terminal dynein light chain-like domains. Data are accumulating on the biochemistry and cell biology of these proteins. However, little is known about their functions in vivo. Schistosoma mansoni expresses 13 family members (SmTAL1–SmTAL13). Three of these (SmTAL1, SmTAL2 and SmTAL3) have been subjected to biochemical analysis which demonstrated that they have different molecular properties. Although their overall folds are predicted to be similar, small changes in the EF-hand domains result in differences in their ion binding properties. Whereas SmTAL1 and SmTAL2 are able to bind calcium (and some other) ions, SmTAL3 appears to be unable to bind any divalent cations. Similar biochemical diversity has been seen in the CaBP proteins from Fasciola hepatica. Four family members are known (FhCaBP1–4). All of these bind to calcium ions. However, FhCaBP4 dimerizes in the presence of calcium ions, FhCaBP3 dimerizes in the absence of calcium ions and FhCaBP2 dimerizes regardless of the prevailing calcium ion concentration. In both the SmTAL and FhCaBP families, the proteins also differ in their ability to bind calmodulin antagonists and related drugs. Interestingly, SmTAL1 interacts with praziquantel (the drug of choice for treating schistosomiasis). The pharmacological significance (if any) of this finding is unknown.

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