Identification of nucleolin as a new L-selectin ligand.

Apart from leucocyte-endothelial interactions, the adhesion molecule L-selectin mediates the homotypic adhesion of leucocytes during recruitment at sites of acute inflammation, as well as intercellular adhesion of haematopoietic progenitor cells during haematopoiesis. There is evidence that, in addition to P-selectin glycoprotein ligand-1, other as-yet-unidentified proteins function as L-selectin ligands on human leucocytes and haematopoietic progenitor cells. In the present study, we show: (i) by affinity chromatography on L-selectin-agarose; (ii) by protein identification using MS; and (iii) by covalent cell-surface labelling with sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate that the multifunctional nuclear protein nucleolin is partly exposed on the cell surface, and is a ligand of L-selectin in human leucocytes and haematopoietic progenitor cells.

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs.

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.

Precise determination of RNA-protein contact sites in the 50 S ribosomal subunit of Escherichia coli.

RNA-protein cross-linked complexes were isolated and purified to obtain precise data about RNA-protein contact sites in the 50 S ribosomal subunit of Escherichia coli. N-terminal microsequencing and matrix-assisted laser desorption ionization MS were used to identify the cross-linking sites at the amino acid and nucleotide levels. In this manner the following contact sites of five ribosomal proteins with the 23 S rRNA were established: Lys-67 of L2 to U-1963, Tyr-35 of L4 to U-615, Lys-97 of L21 to U-546, Lys-49 of L23 to U-139 or C-140 and Lys-71 and Lys-74 of L27 to U-2334.

Cartography of ribosomal proteins of the 30S subunit from the halophilic Haloarcula marismortui and complete sequence analysis of protein HS26.

By two-dimensional polyacrylamide gel electrophoresis of 30S ribosomal subunit proteins (S proteins) from Haloarcula marismortui we identified 27 distinct spots and analyzed all of them by protein sequence analysis. We demonstrated that protein HmaS2 (HS2) is encoded by the open reading frame orfMSG and has sequence similarities to the S2 ribosomal protein family. The proteins HmaS5 and HmaS14 were identified as spots HS7 and HS21/HS22, respectively. Protein HS4 was characterized by amino-terminal sequence analysis. The spot HS25 was recognized as an individual protein and also characterized by sequence analysis. Furthermore, the complete primary sequence of HS26 is reported, showing similarity only to eukaryotic ribosomal proteins. The sequence data of a further basic protein shows a high degree of similarity to ribosomal protein S12, therefore, it was designated HmaS12. Slightly different results compared to published sequence data were obtained for the protein HS12 and HmaS19. The putative 'ribosomal' protein HSH could not be localized in the two-dimensional pattern of the total 30S ribosomal subunit proteins of H. marismortui. Therefore, it seems to be unlikely that this protein is a real constituent of the H. marismortui ribosome.