Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D.

Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglycerol signaling pathways with unique domain composition and enzymatic properties, still awaiting identification of its specific substrate(s). Here we have isolated, cloned, and characterized a novel protein from PC12 cells, termed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first identified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in brain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD co-immunoprecipitates and phosphorylates endogenous Kidins220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12, 13-dibutyrate treatment. A constitutively active PKD mutant (PKD-S744E/S748E) phosphorylates recombinant Kindins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosphorylation is abolished when a dominant negative mutant of PKD (PKD-D733A) is used. Moreover, a peptide within the Kidins220 sequence, containing serine 919 in a consensus motif for PKD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abrogated peptide phosphorylation. Furthermore, by generating an antibody recognizing Kidins220 phosphorylated on serine 919, we show that phorbol ester treatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis--preliminary findings.

Proteomics-based studies offer a powerful complementary approach to DNA/RNA-based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 microm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein-based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two-dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.

Fragmentation of complex carbohydrates following ionization by matrix-assisted laser desorption with an instrument fitted with time-lag focusing.

Matrix-assisted laser desorption/ionization time-of-flight spectra of carbohydrates recorded with an instrument using time-lag focusing (delayed extraction) were found to exhibit fragment ions, whereas, in the non time-lag-focusing mode, fragment ions were not observed. Fragment ions consisted both of glycosidic and cross-ring cleavage products whose absolute, but not relative abundance increased with the delay time and indicated that they were formed within the ion source. Only the glycosidic cleavage ions were present in post-source decay spectra and their abundance was inversely correlated with the delay time. The results indicated that the cross-ring cleavages were predominately the products of fast reactions whereas the glycosidic cleavages occurred over a longer time frame.

Rapid approach for sequencing neutral oligosaccharides by exoglycosidase digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A new way of combining exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved: (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da); (iii) exoglycosidase digestion in a volume of 1 microliter; (iv) removal of the incubation buffer by droplet dialysis; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques: (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques; (ii) no derivatization of the analyte is required; (iii) exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the Km of the enzyme; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.

Effect of structure on the signal strength of oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments.

The signal strength of the MNa+ ion from 25 underivatized oligosaccharides (linear, and both O- and N-linked oligosaccharides from glycoproteins) was measured by matrix-assisted laser desorption/ionization mass spectrometry on a time-of-flight and on a magnetic sector instrument with 2,5-dihydroxybenzoic acid as the matrix, in order to examine the influence of structure on ion abundance. Oligosaccharides with masses greater than about 1000 Da exhibited similar signal strengths, irrespective of structure, when examined on the time-of-flight instrument. Oligosaccharides with masses below 1000 Da displayed a progressive reduction in signal intensity with decreasing molecular weight, an effect which was probably due to temporary saturation of the detector by the intense matrix ions (m/z 130-200 region). Similar studies performed on a magnetic sector instrument revealed that all oligosaccharides studied produced signals of equivalent intensity and that no reduction in signal strength occurred with the smaller sugars. The compounds could be measured with a precision of +/- 5% on this instrument. The nature of the matrix did not appear to exert a differential effect on the ionization of sugars of different structural types although some matrices appeared to have a preference for ionization of smaller molecules.

Effect of the reducing-terminal substituents on the high energy collision-induced dissociation matrix-assisted laser desorption/ionization mass spectra of oligosaccharides.

High-energy collision-induced dissociation (CID) matrix-assisted laser desorption/ionization mass spectra of N-linked oligosaccharides bearing different, commonly encountered, reducing terminal modifications (hydroxyl, 2-aminobenzamide, asparagine and a tetrapeptide) were recorded on a magnetic sector instrument equipped with an orthogonal-acceleration time-of-flight (OA-TOF) analyser. All the compounds formed abundant molecular (MNa+) and fragment ions, the latter corresponding to glycosidic and cross-ring cleavages as well as to internal fragment ions, all of which provided much insight into the oligosaccharide structure. The nature of the modification considerably influenced the CID behaviour. The strongest and most complete series of glycosidic cleavage ions (mainly Y and B--Domon and Costello nomenclature) was formed by the underivatized oligosaccharide whereas most cross-ring fragment ions, diagnostic of linkage, were found in the spectra of the glycopeptides. A-type cross-ring cleavage ions were particularly abundant in the spectrum of the asparagine derivative. Reductive amination using 2-aminobenzamide resulted in an opened reducing-terminal sugar ring and suppression of the cross-ring fragment ions carrying information associated with that ring. This information was present in the spectra of the free carbohydrate and the peptide derivatives.

Comparison of fragmentation modes for the structural determination of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry.

Fragment ions from underivatized N-linked oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry were obtained by spontaneous fragmentation on a magnetic sector mass spectrometer, by post-source decay (PSD) on a reflectron time-of-flight (TOF) instrument and by collision-induced dissociation on a magnetic sector instrument fitted with an orthagonal-TOF analyser. Spontaneous fragmentation on the magnetic sector instrument produced ions mainly by glycosidic cleavage together with two abundant ions formed by cross-ring cleavage of the reducing-terminal residue. The PSD spectra were similar, the majority of ions being formed by glycosidic cleavage. Internal fragment ions were abundant. High-energy collision-induced dissociation spectra recorded with the orthagonal-TOF analyser, differed considerably from the other types of spectra, particularly in the appearance of major fragment ions produced by cross-ring cleavages of most of the constituent monosaccharide residues. These ions allowed much sequence and branching information to be obtained from the oligosaccharide.