Immunocytochemical detection of the growth-associated protein B-50 by newly characterized monoclonal antibodies in human brain and muscle

The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissuespecific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain.

Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein.     

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.     

A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

Magnetosomes are membrane-enveloped bacterial organelles containing nano-sized magnetic particles, and function as a cellular magnetic sensor, which assist the cells to navigate and swim along the geomagnetic field. Localized with each magnetosome is a suite of proteins involved in the synthesis, maintenance and functionalization of the organelle, however the detailed molecular organization of the proteins in magnetosomes is unresolved. MamA is one of the most abundant magnetosome-associated proteins and is anchored to the magnetosome vesicles through protein-protein interactions, but the identity of the protein that interacts with MamA is undetermined. In this study, we found that MamA binds to a magnetosome membrane protein Mms6. Two different molecular masses of Mms6, 14.5-kDa and 6.0-kDa, were associated with the magnetosomes. Using affinity chromatography, we identified that the 14.5-kDa Mms6 interacts with MamA, and the interaction was further confirmed by pull-down, immunoprecipitation and size-exclusion chromatography assays. Prior to this, Mms6 was assumed to be strictly involved with biomineralizing magnetite; however, these results suggest that Mms6 has an additional responsibility, binding to MamA.     

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics.With well-developed algorithms and computational tools for mass spectrometry (MS)¹ data analysis, peptide-based bottom-up proteomics has gained considerable popularity in the field of systems biology (1–9). Nevertheless, the bottom-up approach is suboptimal for the analysis of protein posttranslational modifications (PTMs) and sequence variants as a result of protein digestion (10). Alternatively, the protein-based top-down proteomics approach analyzes intact proteins, which provides a “bird''s eye” view of all proteoforms (11), including those arising from sequence variations, alternative splicing, and diverse PTMs, making it a disruptive technology for the comprehensive analysis of proteoforms (12–24). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for processing data from bottom-up proteomics experiments, the data analysis tools in top-down proteomics remain underdeveloped.The initial step in the analysis of top-down proteomics data is deconvolution of high-resolution mass and tandem mass spectra. Thorough high-resolution analysis of spectra by horn (THRASH), which was the first algorithm developed for the deconvolution of high-resolution mass spectra (25), is still widely used. THRASH automatically detects and evaluates individual isotopomer envelopes by comparing the experimental isotopomer envelope with a theoretical envelope and reporting those that score higher than a user-defined threshold. Another commonly used algorithm, MS-Deconv, utilizes a combinatorial approach to address the difficulty of grouping MS peaks from overlapping isotopomer envelopes (26). Recently, UniDec, which employs a Bayesian approach to separate mass and charge dimensions (27), can also be applied to the deconvolution of high-resolution spectra. Although these algorithms assist in data processing, unfortunately, the deconvolution results often contain a considerable amount of misassigned peaks as a consequence of the complexity of the high-resolution MS and MS/MS data generated in top-down proteomics experiments. Errors such as these can undermine the accuracy of protein identification and PTM localization and, thus, necessitate the implementation of visual components that allow for the validation and manual correction of the computational outputs.Following spectral deconvolution, a typical top-down proteomics workflow incorporates identification, quantitation, and characterization of proteoforms; however, most of the recently developed data analysis tools for top-down proteomics, including ProSightPC (28, 29), Mascot Top Down (also known as Big-Mascot) (30), MS-TopDown (31), and MS-Align+ (32), focus almost exclusively on protein identification. ProSightPC was the first software tool specifically developed for top-down protein identification. This software utilizes “shotgun annotated” databases (33) that include all possible proteoforms containing user-defined modifications. Consequently, ProSightPC is not optimized for identifying PTMs that are not defined by the user(s). Additionally, the inclusion of all possible modified forms within the database dramatically increases the size of the database and, thus, limits the search speed (32). Mascot Top Down (30) is based on standard Mascot but enables database searching using a higher mass limit for the precursor ions (up to 110 kDa), which allows for the identification of intact proteins. Protein identification using Mascot Top Down is fundamentally similar to that used in bottom-up proteomics (34), and, therefore, it is somewhat limited in terms of identifying unexpected PTMs. MS-TopDown (31) employs the spectral alignment algorithm (35), which matches the top-down tandem mass spectra to proteins in the database without prior knowledge of the PTMs. Nevertheless, MS-TopDown lacks statistical evaluation of the search results and performs slowly when searching against large databases. MS-Align+ also utilizes spectral alignment for top-down protein identification (32). It is capable of identifying unexpected PTMs and allows for efficient filtering of candidate proteins when the top-down spectra are searched against a large protein database. MS-Align+ also provides statistical evaluation for the selection of proteoform spectrum match (PrSM) with high confidence. More recently, Top-Down Mass Spectrometry Based Proteoform Identification and Characterization (TopPIC) was developed (http://proteomics.informatics.iupui.edu/software/toppic/index.html). TopPIC is an updated version of MS-Align+ with increased spectral alignment speed and reduced computing requirements. In addition, MSPathFinder, developed by Kim et al., also allows for the rapid identification of proteins from top-down tandem mass spectra (http://omics.pnl.gov/software/mspathfinder) using spectral alignment. Although software tools employing spectral alignment, such as MS-Align+ and MSPathFinder, are particularly useful for top-down protein identification, these programs operate using command line, making them difficult to use for those with limited knowledge of command syntax.Recently, new software tools have been developed for proteoform characterization (36, 37). Our group previously developed MASH Suite, a user-friendly interface for the processing, visualization, and validation of high-resolution MS and MS/MS data (36). Another software tool, ProSight Lite, developed recently by the Kelleher group (37), also allows characterization of protein PTMs. However, both of these software tools require prior knowledge of the protein sequence for the effective localization of PTMs. In addition, both software tools cannot process data from liquid chromatography (LC)-MS and LC-MS/MS experiments, which limits their usefulness in large-scale top-down proteomics. Thus, despite these recent efforts, a multifunctional software platform enabling identification, quantitation, and characterization of proteins from top-down spectra, as well as visual validation and data correction, is still lacking.Herein, we report the development of MASH Suite Pro, an integrated software platform, designed to incorporate tools for protein identification, quantitation, and characterization into a single comprehensive package for the analysis of top-down proteomics data. This program contains a user-friendly customizable interface similar to the previously developed MASH Suite (36) but also has a number of new capabilities, including the ability to handle complex proteomics datasets from LC-MS and LC-MS/MS experiments, as well as the ability to identify unknown proteins and PTMs using MS-Align+ (32). Importantly, MASH Suite Pro also provides visualization components for the validation and correction of the computational outputs, which ensures accurate and reliable deconvolution of the spectra and localization of PTMs and sequence variations.     

An Acinetobacter baumannii,Zinc-Regulated Peptidase Maintains Cell Wall Integrity during Immune-Mediated Nutrient Sequestration

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Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells

B-50 (GAP-43) is an axonal, plasma membrane-associated protein involved in growth cone morphology and function. We have conducted immunocytochemical, electron microscopic, and time-lapse experiments to visualize morphological consequences of local accumulations of B-50 at the plasma membrane of B-50-transfected PC-B2 cells, a clonal PC12 cell line with very low expression of endogenous B-50. The distribution of the transfected B-50 within these cells was inhomogeneous. At sites where the B-50 concentration was locally increased up to twofold, numerous filopodia were present in growth cone-like, substrate-attached regions. When local B-50 concentrations were even higher (up to 6.2-fold), blebs were formed, often containing vesicular structures, heavily decorated with B-50 immunoreactivity. Double labeling with f-actin binding phalloidin revealed that local B-50 accumulations were accompanied by increased actin filament concentrations. Colocalization of B-50 with actin filaments was prominent in filopodia, but was virtually absent in blebs, suggesting a disconnection of the bleb plasma membrane from the actin cytoskeleton. We conclude that B-50 evokes distinct effects on cell-surface activity in PC12 cells depending on its local concentration.