Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry.

A method for the analysis of the rViscumin heterodimer (recombinant mistletoe lectin) based on two-dimensional (2-D) polyacrylamide gel electrophoresis and mass spectrometry was developed and used for quality control concerning purity and homogeneity of the recombinant protein processed under Good Manufacturing Practice (GMP) conditions. A series of spots with different pI-values in the pH-gradient of both rViscumin A- and B-chain were observed independently from the experimental conditions like urea concentration, heat treatment or the use of cysteine alkylating agents. Comparative studies of the major spots using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography-electrospray ionization (LC-ESI)-MS and LC-ESI-tandem MS (MS/MS) after tryptic in-gel digestion resulted in a sequence coverage of 92% for the A-chain and 95% for the B-chain. No molecular differences like common chemical or post-translational modifications or nonenzymatic deamidation were found to cause the different charge values of the separated spots. Therefore, these protein spots were extracted from the 2-D gel and separated again by 2-D gel electrophoresis (termed Re-2-DE). Each of the single spots tested in the Re-2-DE experiment split up in the same heterogeneous pattern concerning the pI-values. We suggest that the observed charge variants of rViscumin are the result of conformational protein variants, existing in an equilibrium during sample preparation and/or isoelectric focusing and are not caused from microheterogeneity in the primary structure of rViscumin.

Ligand motif of the autoimmune disease-associated mouse MHC class II molecule H2-A(s).

The MHC class II molecule H2-A(s), expressed in the SJL mouse strain, is the principle restriction element of autoreactive CD4(+) T cells mediating experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We deduced the H2-A(s) ligand motif from the analysis of naturally processed self peptides and from peptide binding studies. Major anchor residues were identified using various sets of substituted and truncated peptides, derived from natural peptide ligands and known H2-A(s) binders like myelin basic protein 81 - 99. The nine-residue H2-A(s) core binding motif comprises an arrangement of anchors in relative positions P1, P4, P6, P7, and P9. The P1 pocket is relatively unspecific and the P6 pocket favors hydrophobic-aliphatic side chains. The P1 pocket contributes little to peptide binding. Primary anchors were identified in P4, P7, and in particular in P9. The preferred anchor residues are Lys (P4), His/Arg (P7), and Pro (P9), respectively. Ala-polysubstituted peptides containing only one of these dominant anchor residues still retain the capacity to bind to H2-A(s). Thus, the presence of only one suitable anchor side chain in P4, P7, or P9 is sufficient for high-affinity peptide binding, at least in the absence of negatively charged side chains nearby. The identified ligand motif facilitates the analysis of immunogenic peptides interacting with H2-A(s) and will allow a better prediction of pathogenetically relevant peptide antigens in the autoimmune mouse model.

Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD and Edman degradation.

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at -80 degrees C to prevent peptide losses.

The blue-colored linker polypeptide L55 is a fusion protein of phycobiliproteins in the cyanobacterium synechocystis sp. strain BO 8402.

The cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, lacks phycobilisomes but instead forms inclusion bodies containing remnants of phycobiliproteins. The inclusion bodies are surrounded by a proteinaceous capsule and contain alpha-phycocyanin and beta-phycocyanin, the rod linker polypeptide L35RPC and a novel blue-colored protein L55 with an apparent molecular mass of 55 kDa. An antibody raised against beta-phycocyanin showed a strong cross-reaction with L55. Mass spectrometry analysis of proteolytic peptides from L55 revealed mass identity to proteolytic peptides derived from L35RPC and beta-phycocyanin. However, analysis of the genome of strain BO 8402 revealed only one cpcBACE operon, encoding the apoproteins of beta-phycocyanin and alpha-phycocyanin, L35RPC and a subunit of the phycocyanin alpha subunit phycocyanobilin lyase, respectively. The gene structure, sequence and transcription of these genes were identical to that of a revertant strain, Synechocystis sp. strain BO 9201, which formed phycobilisomes and did not express L55. Based on these observations, we concluded that L55 did not derive from a particular gene or from a special form of mRNA-processing. We propose that L55 is formed by post-translational fusion of L35RPC and beta-phycocyanin. Cross-linking may stabilize the formation of the large paracrystalline phycocyanin aggregates unique to Synechocystis sp. strain BO 8402.

Defensins are dominant HLA-DR-associated self-peptides from CD34(-) peripheral blood mononuclear cells of different tumor patients (plasmacytoma, chronic myeloid leukemia).

The HLA-DR-associated peptides from peripheral blood mononuclear cells of 2 patients with plasmacytoma and 1 with chronic myeloid leukemia were isolated, identified, and compared. Several were identified as derivatives of the defensin family. Defensins (or human neutrophil peptides [HNP]) are antimicrobial, cationic peptides of 29 to 35 amino acids in length and are the major constituents of the azurophilic granules of human neutrophils. Using peripheral blood cells from leukapheresis, containing about 90% of polymorphonuclear cells, we could identify HNP-1, -2, and -4 and propeptides of up to 49 amino acids in length, eluted from HLA class II molecules. Binding of isolated and synthetic defensin peptides to various HLA-DR alleles using an in vitro binding/competition assay based on size exclusion chromatography revealed that defensin may bind into the peptide-binding groove. In a T-cell competition assay, defensins were able to reduce the proliferation of an HLA-DR-restricted T-cell line after preincubation of stimulating cells (CHO-DRB1*0401 transfectants) with defensin. Therefore, binding of defensins might prevent T-cell recognition of HLA class II molecules expressed on different blood precursor cells (all of which are "nonprofessional" antigen-presenting cells) by blocking the HLA peptide-binding groove or, alternatively, might protect defensin-expressing cells from self-destruction. (Blood. 2000;95:2890-2896)

Evidence for a copper-coordinated histidine-tyrosine cross-link in the active site of cytochrome oxidase.

Following hints from X-ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547-10553; Yoshikawa S et al., 1998, Science 280: 1723-1729), chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copperB-coordinated His240-Tyr244) cross-link at the O2-activating Heme Fea3-CuB center in the catalytic subunit 1 of the enzyme. The early evolutionary invention of this unusual structure may have prevented damaging *OH-radical release at e(-)-transfer to dioxygen and thus have enabled O2 respiration.

Direct identification of the agonist binding site in the human brain cholecystokininB receptor.

In investigating the agonist binding site of the human brain cholecystokininB receptor (CCKBR), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (KD = 1.2 nM) to the CCKBR and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCKBR. Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a protein core of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequent chemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified by Edman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. The position of the radioactive probe within the identified peptide was determined using combined tandem electrospray mass spectrometry and peptide mapping. The probe was covalently attached within the sequence L52ELAIRITLY61 that represents the transition between the N-terminal domain and predicted transmembrane domain 1. Using this interaction as a constraint to orientate the ligand within the putative receptor binding site, a model of the CCK-8s-occupied CCKBR was constructed. The hormone was found to be placed in a binding pocket built from both extracellular and transmembrane domains of CCKBR with its N-terminus mainly interacting with residues Arg57 and Tyr61.

Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif.

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase.

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.

X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.