Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis . Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.

BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.

Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry.

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.

Glypican-2 levels in cerebrospinal fluid predict the status of adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis in vivo. Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.

Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry.

We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary. The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.

Identification of proteins by capillary electrophoresis-tandem mass spectrometry. Evaluation of an on-line solid-phase extraction device.

Capillary electrophoresis-tandem mass spectrometry has been used successfully for the analysis of complex peptide mixtures. The method is limited by a relatively high concentration limit of detection and by matrix effects. Here we describe on-line coupling of a solid-phase microextraction device to a capillary electrophoresis-tandem mass spectrometry system. The performance of the integrated instrument was evaluated for the identification of proteins by their amino acid sequence. We report that the concentration limit of detection was improved at least 1000 fold to the low attomole/microliter range and that matrix effects were minimized by extensive sample clean-up during solid-phase extraction. We demonstrate that the implementation of a solid-phase extraction device significantly enhances capillary electrophoresis-tandem mass spectrometry as a method for the identification of low abundance proteins isolated from high-resolution two-dimensional polyacrylamide gels.

Technetium 99m labelled heparin: pharmacokinetics and tissue distribution in rats after vascular surgery.

Pharmacokinetics of technetium 99m (99mTc) labelled heparin was studied after I.V. infusion (70 IU/kg) in normal rats and in a group of animals just after aortic longitudinal incision and suture. Anticoagulant activity measurements and radioactivity were compared on the same plasma samples. The pharmacokinetic decay of 99mTc-heparin followed a bi-exponential pattern. Half-lives (T1, T2) were significantly shortened after surgery using both techniques. Liver accumulation of labelled heparin was decreased in "sutured" rats and a high uptake of 99mTc-heparin was found on the operated area of the aorta.

Chiral high performance liquid chromatography resolution of ibuprofen esters.

Two cellulose-based chiral stationary phases (Chiralcel OD and Chiralcel OJ) were compared on their ability to resolve various aliphatic ibuprofen esters. Chiralcel OJ with hexane as the mobile phase allows for the separation of most of the esters. Observed changes in resolution depending on the solute nature (basicity of the solute, esterified alcohol chain length, presence of a double bond) are discussed. An example of the application of this method for following the kinetic resolution of racemic ibuprofen is presented.

Colloidal silver staining of electroblotted proteins for high sensitivity peptide mapping by liquid chromatography-electrospray ionization tandem mass spectrometry.

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level.

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.

High-sensitivity detection of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by capillary liquid chromatography-microelectrospray ion trap mass spectrometry.

We describe the separation and detection at the low-femtomole level of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins (311-PTHs) by capillary liquid chromatography-microelectrospray ion trap mass spectrometry. Highest sensitivity was obtained in the multiple-ion monitoring operating mode in which we detected 311-PTHs at the 5-fmol level with a signal-to-noise ratio of approximately 10. We investigated the fragmentation patterns of the isobaric 311-PTH isoleucine and 311-PTH leucine by electrospray ionization ion trap tandem mass spectrometry. The compounds could be differentiated by a fragment ion of mass m/z = 366.1 which was specific for the breakdown of 311-PTH leucine, thus allowing for the unambiguous identification of the 311-PTH derivatives of all 20 naturally occurring amino acids by their masses and fragmentation patterns.

The complete amino acid sequence of R-phycocyanin-I alpha and beta subunits from the red alga Porphyridium cruentum. Structural and phylogenetic relationships of the phycocyanins within the phycobiliprotein families.

We present here the complete primary structure of R-phycocyanin-I alpha and beta subunits from the red alga Porphyridium cruentum. The alpha chain is composed of 162 amino acid residues (18049 Da, calculated from sequence, including chromophore) and carries a phycocyanobilin pigment covalently linked to Cys84. The beta chain contains 172 amino acids (19344Da, calculated from sequence, including chromophores) and carries a phycocyanobilin pigment covalently linked at Cys82 and a phycoerythrobilin pigment at Cys153. A gamma-N-methyl asparagine residue was also characterised at position beta 72 similar to other phycobiliprotein beta subunits. R-phycocyanin-I from Porphyridium cruentum shares high sequence identity with C-phycocyanins (69-83%), R-phycocyanins (66-70%) and in a less extent with phycoerythrocyanins (57-65%) from various sources. The presented phylogenetic trees are based on a comparison of all phycobiliprotein amino acid sequences known so far and confirm the clear affiliation of the R-phycocyanins in the phycocyanin family. In spite of their particular phycobilin pattern, they do not represent intermediate forms between the phycocyanin and the phycoerythrin family. Phycoerythrocyanin, a phycocyanin-related phycobiliprotein adapted to green light harvesting, is also shown to belong to the phycocyanin family. However, the phycoerythrocyanins diverge from phycocyanins in their different function and it is suggested that they should be assigned to a separate group within the phycocyanin family.

Enzymatic preparation of biosurfactants from sugars or sugar alcohols and fatty acids in organic media under reduced pressure.

Biosurfactants were prepared by enzymatic esterification of sugars and sugar alcohols in nonaqueous media. Sorbitol monooleate was produced in pure molten substrates, with reduced pressure to remove water. The results were compared to synthesis in organic solvent, with and without water removal. Synthesis in organic solvent with water removal, obtained by refluxing through a desiccant under reduced pressure, proved to be the most efficient method in terms of total yield and side-products formation. This process was applied to the production of different surfactants, by changing the nature of the hydrophilic and hydrophobic moieties. Yields above 90% of monoesters were obtained after 24 h when the reaction was carried out in 2-methyl-2-butanol with Novozym 435 (Type B lipase from Candida antarctica) with an excess of hydroxyl donor.

Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp. PCC 7120.

In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus. Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathyway along the antenna. Upon dissociation, the phycobiliproteins were released from the phycobilisome. One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion-exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE. The amino-terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes. Partial amino-terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here. Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp. PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria. The phycobilisome of Anabaena sp. PCC 7120 was extensively analyzed by electron microscopy. It differs from the common hemidiscoidal tricylindrical, six-rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner. One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each. On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp. PCC 7120, M. laminosus and Anabaena variabilis is proposed. This model accounts more accurately for the 'grape' pattern typically exhibited by these phycobilisomes in electron micrographs.

Axonal proteins involved in myelination: characterization of a collagen-like protein.

An anti-axolemma monoclonal antibody, designated G21.3, has been isolated in order to understand molecular mechanisms involved in myelination. Both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures. On Western blots of axolemma preparations, the antibody recognized 140- and 120-kD proteins. The present study involves the isolation and characterization of the G21.3 antigen. The G21.3-immunoreactive proteins of 140 and 120 kD were purified from the adult rat sciatic nerve and amino acid sequencing of these proteins revealed significant homology to alpha I and alpha II chains of collagen type I. Biochemical and Western blot analysis using pure collagen, collagen I antibody and collagenase D suggest that the antigen isolated from sciatic nerve is collagen. However, immunofluorescence studies using the G21.3 antibody, collagen I antibody, collagenase D and Northern blot analysis using a collagen probe do not fully support the view that the G21.3 antigen in the CNS is also a collagen. We conclude that the G21.3 antigen is a collagen-like protein involved in CNS myelination.

A simplified gradient solvent delivery system for capillary liquid chromatography-electrospray ionization mass spectrometry.

We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low-cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.

RGSZ1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Galphaz phosphorylation, and relationship to a Gz gtpase-activating protein subfamily.

We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J., Song, X., and Ross, E. M. (1997) J. Biol. Chem. 272, 5732-5740) and a member of the RGS family of G protein GAPs. Its sequence is 83% identical to RET-RGS1 (except its N-terminal extension) and 56% identical to GAIP. Purified, recombinant RGSZ1, RET-RGS1, and GAIP each accelerated the hydrolysis of Galphaz-GTP over 400-fold with Km values of approximately 2 nM. RGSZ1 was 100-fold selective for Galphaz over Galphai, unusually specific among RGS proteins. Other enzymological properties of RGSZ1, brain Gz GAP, and RET-RGS1 were identical; GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Galphaz. RGSZ1, RET-RGS1, and GAIP thus define a subfamily of Gz GAPs within the RGS proteins. RGSZ1 has no obvious membrane-spanning region but is tightly membrane-bound in brain. Its regulatory activity in membranes depends on stable bilayer association. When co-reconstituted into phospholipid vesicles with Gz and m2 muscarinic receptors, RGSZ1 increased agonist-stimulated GTPase >15-fold with EC50 <12 nM, but RGSZ1 added to the vesicle suspension was <0.1% as active. RGSZ1, RET-RGS1, and GAIP share a cysteine string sequence, perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by Gz. Phosphorylation of Galphaz by protein kinase C inhibited the GAP activity of RGSZ1 and other RGS proteins, providing a mechanism for potentiation of Gz signaling by protein kinase C.

Characterization of human serum amyloid A protein isoforms separated by two-dimensional electrophoresis by liquid chromatography/electrospray ionization tandem mass spectrometry.

A detailed structural analysis of the serum amyloid A proteins (SAA) of an individual with highly active, chronic rheumatoid arthritis is reported. SAA isoforms were separated by high-resolution two dimensional (2-D) gel electrophoresis. Peptide mapping by reverse-phase chromatography/electrospray ionization tandem mass spectrometry was applied to correlate the protein(s) contained in each spot with their respective coding gene and to study the post-translational processing and modification events which might result in differential electrophoretic mobility. Nine protein spots were analyzed. The six major spots corresponded to the Arg and des-Arg forms of SAA1 alpha and SAA2 alpha, respectively, and to the glycosylated and nonglycosylated form of constitutive serum amyloid A protein (C-SAA). Two minor spots were identified as SAA1 alpha isoforms containing post-translational modifications. We suggest that these variants contained a gamma-N, N'-dimethylasparagine residue at position 83 and that one of them was additionally oxidized at Trp53 and Trp85. The ninth spot was shown to contain a mixture of SAA1 alpha and SAA2 alpha. To our knowledge, this is the first report in which analysis of peptides has been used to verify the presence of C-SAA in acute-phase serum. Furthermore, the data illustrate that extensive post-translational processing results in a structurally diverse class of acute-phase SAA proteins, which are derived from a small number of genes. Finally, the fast and conclusive technology used in this study promises to be generally useful for the comprehensive investigation of proteins at the level of the primary structure.