A Comparative Study of Avidin-Biotin-Peroxidase Complexes for the Immunohistochemical Detection of Antigens in Neural Tissue

It has been suggested that the use of avidin-biotin immunohistochemical techniques for antigen detection in neural tissue produces nonspecific background staining. For this reason neural tissue was used to test the quality, sensitivity and specificity of four commercially available antibody detection kits which use avidin or streptavidin binding to biotin. Free-floating, thick-section immunohistochemistry on perfusion fixed rat central nervous system revealed variability among staining kits for all parameters analyzed under the same experimental conditions. The reagents from the Vector 'Elite' kit were the most sensitive and specific, and received the highest overall rating for quality. Most commercial products tested could be used at greater dilutions than those recommended by the manufacturers without compromising specific staining. No staining was evident when the primary and secondary antibodies were omitted. This suggests that nonspecific binding is unlikely to be due to endogenous ligands, charge of hydrophilic reactions between these tertiary complexes and the tissue sections.     

A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina

Iron deficiency induces two major transferrin-like proteins in the plasma membrane (Pm) of the halotolerant alga Dunaliella salina. TTf, a 150-kDa protein, previously identified as a salt-induced triplicated transferrin, having iron-binding characteristics resembling animal transferrins, and a 100-kDa protein designated idi-100 (for iron-deficiency-induced 100 kDa protein). According to the predicted amino acid sequence of idi-100, it is only 30% identical to TTf and differs from it in having two, rather than three, homologous internal repeats and in a lower conservation of canonical iron/bicarbonate binding residues. Both are localized in the outer surface of the membrane; however, TTf can be dissociated from the membrane by treatment with EDTA, whereas release of idi-100 requires detergents. The accumulation of idi-100 under iron deficiency lags behind that of TTf and in contrast to TTf, it is not induced by high salinity, suggesting that induction of idi-100 requires lower Fe threshold levels than that of TTf. In contrast to TTf, idi-100 does not bind Fe; however, there are indications for interactions with bicarbonate ions. These results suggest that despite their common resemblance to transferrins, their similar subcellular localization and their induction by iron deficiency, idi-100 and TTf fulfill different functions.     

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.     

Two-dimensional separation of the membrane protein sarcoplasmic reticulum Ca-ATPase for high-performance liquid chromatography-tandem mass spectrometry analysis of posttranslational protein modifications

Oligosaccharyltransferase (OST) catalyzes the en bloc transfer of dolichylpyrophosphate oligosaccharides to an asparagine residue found in the sequon Asn-Xaa-Thr/Ser of newly synthesized proteins. Currently the method most commonly used to monitor this reaction, involving multiple solvent extractions and HPLC, is extremely time consuming and tedious. Herein, we present the use of a biotinylated peptide as the acceptor substrate and dolichylpyrophosphate [3H]chitobiose as the donor substrate for the OST-catalyzed reaction. This allows for separation (avidin-agarose beads) and quantitative analysis (scintillation counting) of only the biotinylated glycopeptide product of the OST-catalyzed reaction. This new assay yields highly reproducible results in a rapid manner.     

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.     

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.     

Turnover of the transferrin receptor is not influenced by removing most of the extracellular domain

We treated intact cells with trypsin to remove most of the external domain of the transferrin receptor and investigated what effect the absence of the external domain had on the turnover of the fragment that remained associated with the cells. To detect the cell-associated tryptic fragment, which contains a small amount of the external domain, the transmembrane domain, and the cytoplasmic domain, we prepared an anti-peptide antibody against a segment of the cytoplasmic domain. This antibody specifically immunoprecipitated the intact transferrin receptor as well as a 21-kDa peptide from trypsin-treated HeLa cells. Several lines of evidence indicated that the 21-kDa peptide was the cell-associated tryptic fragment of the transferrin receptor. The fragment was only present in trypsin-treated cells; the fragment migrated as a dimer in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as it should if it were derived from the transferrin receptor; a goat antibody prepared to the purified human transferrin receptor also precipitated the 21-kDa peptide from trypsinized cells. In addition, treating the tryptic fragment with neuraminidase increased the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting the fragment contained O-linked carbohydrate. When cells were trypsinized and then incubated at 37 degrees C, the half-life of the tryptic fragment (15 +/- 4 h) was not significantly different than the half-life of the intact receptor (19 +/- 6 h). This indicates that removing 95% of the external domain of the transferrin receptor has little effect on processes operating in the turnover of the receptor.     

RNase A oligomerization through 3D domain swapping is favoured by a residue located far from the swapping domains

Bovine pancreatic ribonuclease A forms 3D domain-swapped oligomers by lyophilization from 40% acetic acid solutions or if subjected to various thermally-induced denaturation procedures.Considering that the intrinsic swapping propensity of bovine seminal RNase, the only member of the pancreatic-type RNase super-family that is dimeric in nature, is decreased from 70 to 30% if Arg80 is substituted by Ser (the corresponding residue in native RNase A), we introduced the opposite mutation in position 80 of the pancreatic enzyme. Our aim was to detect if the RNase A tendency to aggregate through domain swapping could increase.Aggregation of the S80R-RNase A mutant was induced either through the ‘classic’ acetic acid lyophilization, or through a thermally-induced method. The results indicate that the S80R mutant aggregates to a higher extent than the native protein, and that the increase occurs especially through N-terminal swapping.Additional investigations on the dimeric and multimeric species formed indicate that the S80R mutation increases their stability against regression to monomer, and does not significantly change their structural and functional features.     

c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.     

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin αvβ3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

Structured summary

MINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071)     

An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.     

Lateral dimerization of the E-cadherin extracellular domain is necessary but not sufficient for adhesive activity

Cadherins are transmembrane glycoproteins involved in Ca(2+)-dependent cell-cell adhesion. Using L cells coexpressing E-cadherin constructs with different epitope tags, we examined the lateral dimerization of E-cadherin and its adhesive activity by co-immunoprecipitation and aggregation assays, respectively. Although the transmembrane domain is required for dimerization, tail-less constructs possessing the transmembrane domain of either N-cadherin or CD45 show dimerization and are active in aggregation assays. Two mutant constructs having either of two amino acid substitutions, W2A or substitutions that disrupt the recognition sequence for endoproteolytic enzymes involved in removal of the precursor segment, cannot form dimers and are inactive in aggregation. These monomeric proteins, like their wild-type dimerizing counterparts, retain their Ca(2+)-dependent resistance to trypsin digestion, suggesting that dimerization per se does not induce a large conformational change. Two other constructs, having either an amino acid substitution, D134A, or a C-terminal deletion of 70 amino acid residues, retain the ability to associate laterally but are inactive in aggregation assays. Staurosporine treatment of cells expressing the latter construct increases aggregation but does not increase the extent of lateral dimerization. Thus, lateral dimerization is necessary, but not sufficient for adhesive activity.     

Ribosomal protein S1 is not essential for the trans-translation machinery

In eubacteria, ribosome stalling during protein synthesis is rescued by a tmRNA-derived trans-translation system. Because ribosomal protein S1 specifically binds to tmRNA with high affinity, it is considered to be involved in the trans-translation system. However, the role of S1 in trans-translation is still unclear. To study the function of S1 in the trans-translation system, we constructed an S1-free cell-free translation system. We found that trans-translation proceeded even in the absence of S1. Addition of S1 into the S1-free system did not affect trans-translation efficiency. These results suggest that S1 does not play a role in the trans-translation machinery.     

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.