Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.

To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.     

Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

BACKGROUND: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice. AIM: In an attempt to broaden the concept that neutrophils and MRP-14 controls inflammatory pain induced by different type of irritants, in the present study, after demonstrating that carrageenan (Cg) also induces atinociception in mice, we investigated the participation of both neutrophils and MRP-14 in the phenomenon. METHODS: Male Swiss mice were injected intraperitoneally with Cg and after different time intervals, the pattern of cell migration of the peritoneal exudate and the nociceptive response of animals submitted to the writhing test were evaluated. The participation of neutrophils and of the MRP-14 on the Cg effect was evaluated by systemic inoculation of monoclonal antibodies anti-granulocyte and anti-MRP-14. RESULTS: Our results demonstrate that the acute neutrophilic peritonitis evoked by Cg induced antinociception 2, 4 and 8 h after inoculation of the irritant. Monoclonal antibodies anti-granulocyte or anti-MRP-14 reverts the antinociceptive response only 2 and 8 h after Cg injection. The antibody anti-MRP-14 partially reverts the antinociception observed after 4 h of Cg injection while the anti-granulocyte antibody enhances this effect. This effect is reverted by simultaneous treatment of the animals with both antibodies. After 4 h of Cg injection in neutrophil-depleted mice a significant expression of the calcium-binding protein MRP-14 was detected in the cytoplasm of peritoneal macrophages. This suggests that the enhancement of the effect observed after treatment with the anti-neutrophil antibody may be due to secretion of MRP-14 by macrophages. It has also been demonstrated that endogenous opioids and glucocorticoids are not involved in the antinociception observed at the 4th hour after Cg injection. CONCLUSION: These data support the hypothesis that neutrophils and the calcium-binding protein MRP-14 are participants of the endogenous control of inflammatory pain in mice despite the model of acute inflammation used.     

Nucleolin is a calcium-binding protein

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.     

The calcium-binding protein SPARC is secreted by Leydig and Sertoli cells of the adult mouse testis

In mammals, polypeptides secreted by cells of the testis are believed to influence spermatogenesis and to affect the behavior of the resident somatic cell populations. The 43,000-MW, secreted, calcium-binding glycoprotein SPARC (Secreted Protein, Acidic and Rich in Cysteine) is synthesized by a number of embryonic, fetal, and adult somatic cells and is associated with areas of cellular differentiation, proliferation, and morphological reorganization. Here, we report on the expression of SPARC in the testes of adult mice. By immunohistochemistry, SPARC was observed in the cytoplasm of Leydig cells and of Sertoli cells bearing late-stage, elongate spermatids. Testicular mRNA, translated in vitro, yielded a polypeptide of approximately 42,000 MW that bound anti-SPARC antibodies. Northern blot analysis revealed 2.3 kilobase (kb) SPARC mRNA in the testis, a size comparable to that of SPARC mRNA in nongonadal cells. Western blot assays of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an immunoreactive polypeptide of 43,000 MW in purified mouse Sertoli cells and their culture supernatants. Similar assays of testis interstitial fluid revealed 43,000 MW and 30,000 MW immunoreactive polypeptides. By indirect immunofluorescence, purified mouse Leydig cells cultured 24-48 h expressed SPARC in cytoplasmic granules. Cultured Leydig cells incorporated [35S]methionine into a secreted polypeptide of 43,000 MW that was recognized by anti-SPARC antibodies. In metal binding assays, purified SPARC bound Ca2+, Fe2+ and Cu2+. The function of SPARC in testes may be to sequester or transport certain metallic cations. Our recent discovery that SPARC induces changes in shape of certain nongonadal cell types also suggests that this glycoprotein may influence the functions of both Leydig and Sertoli cells by affecting their morphology.     

Spinach ferredoxin is a calcium-binding protein

Spinach-leaf ferredoxin was identified as a calcium-binding protein by ⁴⁵Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (stains-all). Binding of ⁴⁵Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl₂. At the higher MgCl₂ concentration the Ca²⁺-binding capacity is reduced. Only micromolar concentrations of LaCl₃, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - stains-all 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naptho[1,2-d]thiazolium bromide     

3C8 antigen is a novel protein expressed by human follicular dendritic cells

Although the pivotal role of follicular dendritic cells (FDCs) in humoral immune responses has been demonstrated, little is known at the molecular level of how FDCs contribute to the organogenesis, B cell differentiation, and regulation of T cell functions in the germinal center. By immunizing with FDC-like cells, we have developed a monoclonal antibody (MAb), which stains the germinal centers in tonsil section. In the current study, the target cell of MAb 3C8 was identified as FDC by confocal scanning fluorescence microscopy. Unlike other MAbs against FDC, MAb 3C8 does not cross-react with bone marrow-derived blood cells. Amino acid sequencing of NH(2)-terminal region of immunoprecipitated 3C8 Ag reveals that 3C8 is a novel FDC protein. Further studies of 3C8 molecule will shed light on the cellular origin of FDC and reveal unknown functions of FDC.     

Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.     

The calcium-binding activity of a vacuole-associated,dehydrin-like protein is regulated by phosphorylation

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 +/- 0.142 mM and 0.64 +/- 0.08 mM, and has an estimated 24.7 +/- 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.     

Characterization and site-specific mutagenesis of the calcium-binding protein oncomodulin produced by recombinant bacteria

A bacterial expression system for the parvalbumin-like calcium-binding protein oncomodulin has been constructed. This system can yield 50-fold more oncomodulin than the richest known mammalian source, the rat Morris hepatoma 5123. The bacterially produced protein folded correctly as monitored by UV, fluorescence, and 1H nuclear magnetic resonance spectroscopy and is immunologically identical to rat hepatoma oncomodulin. A calcium-specific conformational change is observed in the bacterial oncomodulin similar to that of the hepatoma protein. A modification of the putative calcium-specific CD loop by site-directed mutagenesis, which changed Asp-59 to Glu-59, eliminates calcium-specific effects. In contrast to the native molecule, the mutant Glu-59 now exhibits a magnesium-induced conformational change when monitored by UV difference or fluorescence excitation spectroscopy. The availability of large amounts of bacterially produced oncomodulin combined with the ability to modify at will the metal-binding ligands should now allow dissection of the unusual pairing in oncomodulin of one calcium-specific calmodulin-like site with one calcium/magnesium parvalbumin-like site.     

Rat skin calcium-binding protein is parvalbumin.

Only one major low-Mr calcium-binding protein could be isolated by h.p.l.c. procedures from aqueous extracts of homogenized adult rat skin. This was shown by tryptic peptide mapping and independent amino acid sequence analysis to be identical in all 109 residues with the parvalbumin from rat skeletal muscle. This calcium-binding protein was not in skin epidermis, but was confined to the dermal layer. Skin calcium-binding protein is therefore parvalbumin.     

Tetrahymena calcium-binding protein is indeed a calmodulin

We previously isolated a Ca2+-binding protein from a ciliate, Tetrahymena, and designated it as TCBP (Tetrahymena Ca2+-binding protein). The present paper reports that TCBP, which has two high affinity Ca2+-binding sites (Kd=4.6 X 10(-6) M), could activate porcine brain cyclic nucleotide phosphodiesterase at a concentration of over 10(-6) M free Ca2+, with the same mode of activation as that of authentic (porcine brain) calmodulin. In addition, the amino acid composition of TCBP was essentially the same as that of brain calmodulin. Therefore, we conclude that TCBP as an activator of Tetrahymena guanylate cyclase is indeed a calmodulin.     

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

The tumor marker human placental protein 11 is an endoribonuclease

Human PP11 (placental protein 11) was previously described as a serine protease specifically expressed in the syncytiotrophoblast and in numerous tumor tissues. Several PP11-like proteins were annotated in distantly related organisms, such as worms and mammals, suggesting their involvement in evolutionarily conserved processes. Based on sequence similarity, human PP11 was included in a protein family whose characterized members are XendoU, a Xenopus laevis endoribonuclease involved in small nucleolar RNA processing, and Nsp15, an endoribonuclease essential for coronavirus replication. Here we show that the bacterially expressed human PP11 displays RNA binding capability and cleaves single stranded RNA in a Mn(2+)-dependent manner at uridylates, to produce molecules with 2',3'-cyclic phosphate ends. These features, together with structural and mutagenesis analyses, which identified the potential active site residues, reveal striking parallels to the amphibian XendoU and assign a ribonuclease function to PP11. This newly discovered enzymatic activity places PP11-like proteins in a completely new perspective.     

The apoptosis-inducing protein kinase DRAK2 is inhibited in a calcium-dependent manner by the calcium-binding protein CHP

Calcineurin homologous protein (CHP) is an EF-hand Ca(2+)-binding protein capable of interacting with various cellular proteins including Na(+)/H(+) exchangers, kinesin-related proteins, and apoptosis-inducing protein kinase DRAK2. We investigated the role of CHP on the DRAK2 protein kinase in vitro. CHP significantly reduced (approximately 85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phosphorylation of exogenous substrate (myosin light chain). The inhibitory effect of CHP was dependent on the presence of Ca(2+), whereas the interaction between CHP and DRAK2 was not Ca(2+)-dependent. These observations suggest that CHP negatively regulates the apoptosis-inducing protein kinase DRAK2 in a manner that depends on intracellular Ca(2+)-concentration.     

Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR

Human translationally controlled tumor protein (TCTP) is a growth-related, calcium-binding protein. We determined the solution structure and backbone dynamics of human TCTP, and identified the calcium-binding site of human TCTP using multi-dimensional NMR spectroscopy. The overall structure of human TCTP has a rather rigid well-folded core and a very flexible long loop connected by a short two-strand β-sheet, which shows a conserved fold in the TCTP family. The C-terminal portions of loop L_α3β8 and strand β9 and the N-terminal region of strand β8 may form a calcium-binding site in the human TCTP structure, which is largely conserved in the sequence alignment of TCTPs. The K_d value for the calcium binding is 0.022-0.025 M indicating a very weak calcium-binding site.     

The stimulation of tumor cell growth by a substance produced by normal and tumor cells

It has been shown that a dialyzable substance produced by normal and tumor cells can stimulate the growth of a myeloid, erythroid, and two lymphoid leukemias, and a sarcoma. The growth stimulation of the tumor cells was observed as an increase in cloning efficiency and number of cells per colony. Rat granulocytes stimulated the growth of mouse tumor cells as efficiently as a variety of mouse cells. The stimulating substance was found in conditioned medium, and it was produced by all the normal and tumor cells tested.     

The adiponectin paralog CORS-26 has anti-inflammatory properties and is produced by human monocytic cells

The adiponectin paralog CORS-26 (collagenous repeat-containing sequence of 26kDa protein) is a member of the C1q/TNF-alpha molecular superfamily. CORS-26 is a secreted protein and baculovirus-produced CORS-26 released in the supernatant of insect cells forms stable trimers. Adiponectin exerts anti-inflammatory effects in LPS-treated monocytic cells and CORS-26 also reduces IL-6 and TNF-alpha secretion but does not increase IL-10. Suppression of NFkappaB signalling may explain the anti-inflammatory actions of CORS-26. Furthermore CORS-26 protein was detected in human monocytic and dendritic cells. The present data demonstrate for the first time that CORS-26 forms trimers, exerts anti-inflammatory properties and that it is expressed in monocytic cells. Therefore CORS-26 may provide a new target for pharmacological drugs in inflammatory diseases like the metabolic syndrome.     

ANGPTL4 is produced by entero-endocrine cells in the human intestinal tract

Gut hormones produced by entero-endocrine cells (EEC) located throughout the gastrointestinal tract play a major role in the regulation of glucose and energy homeostasis. Angiopoietin-like 4 (ANGPTL4, also referred to as fasting induced adipose factor) is a secreted factor involved in regulation of lipid homeostasis and has been proposed as circulating mediator between the gut microbiota and fat storage in adipose tissue, although discordant data exist. Currently, little is known about the site and regulation of ANGPTL4 production in the intestine. Here, we show using immunohistochemistry and immunofluorescence that cells positive for ANGPTL4 are scattered along the epithelial layer in the human small and large intestine. ANGPTL4-positive cells exhibit typical features of EEC characterized by large ANGPTL4-positive secretory granules directed towards the basolateral side. In support, extensive overlap was observed between ANGPTL4-positive cells and cells positive for the entero-endocrine marker chromogranin A. Higher resolution images revealed that ANGPTL4 and chromogranin A are partially present in distinct intracellular vesicles. Using entero-endocrine HuTu-80 cells, ANGPTL4 secretion was shown to be induced by short chain fatty acids and reduced by bile acids. Finally, levels of ANGPTL4 in human plasma were significantly decreased following meal consumption. In conclusion, ANGPTL4 is produced by EEC in human intestine and expression may be regulated by short chain fatty acids and bile acids.     

Microtubule-associated protein, MAP2, is a calcium-binding protein

Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region).