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As a member of the S100 protein family, S100A10 has already been purified. However, its purity, or even yield, have often not been reported in the literature. To facilitate future biophysical experiments with S100A10, we aimed to obtain it at a purity of at least 95% in a reasonably large amount. Here, we report optimized conditions for the transformation, overexpression and purification of the protein. We obtained a purity of 97% and performed stability studies by circular dichroism. Our data confirmed that the S100A10 obtained is suitable for experiments to be performed at room temperature up to several days.

S100 proteins are small, mostly dimeric, EF-hand Ca 2+ -binding proteins. Upon Ca 2+ binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.

The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

The examination was applied to enterprise workers laboring in conditions of vinyl chloride (79 patients), caustic soda (24 patients) and 10 patients with professional chronic mercury intoxication. The differences are established concerning manifestation of autoimmune reactions of personnel working in conditions of chronic effecting of vinyl chloride distinct of parameters characterizing autoimmune reactions of personnel working under impact of another neuro-toxicants (vapors of metallic mercury). The increasing of auto-antibodies to MAG was detected in healthy personnel and increasing of concentrations of auto-antibodies to protein S-100 and DNA was detected in personnel with initial manifestations of neuro-intoxication. These occurrences testify availability, of different mechanisms underlying formation of neurological disorders. The study data confirms involvement of auto-antibodies to neuronal antigens into derangement of neural activity in personnel working in conditions of effect of vinyl chloride and vapors of metallic mercury. Hence, the new possibilities are opened in studying pathogenesis of occupational neuro-intoxications. The detection of auto-antibodies to proteins of neural tissue can be recommended as a criterion of early identification of damage of neural system in personnel working in conditions of chemical industry.

Objective: To prepare S100A10 protein and its specific polyclonal antibody., Methods: The full-length gene fragment of S100A10 was amplified by PCR, and then cloned into pET28a(+) prokaryotic expression vector. After transformation, the vector was induced to express the recombinant (S100A10)(2) protein by IPTG in E.coli BL21 (DE3). The recombinant (S100A10)(2) was then purified by Ni-NTA resin. (S100A10)(2)-specific polyclonal antibody was prepared using the purified recombinant (S100A10)(2) protein as antigen to inoculate rabbit intradermally. The title and specificity of the polyclonal antibody were determined by ELISA and Western blotting., Results: The study successfully constructed the prokaryotic recombinant expression vector pET28a(+)-(S100A10)(2), and obtained the purified recombinant (S100A10)(2) protein and polyclonal antibody with high titer and specificity., Conclusion: The prokaryotic expression and purification system for S100A10 has been established and polyclonal antibody of (S100A10)(2) been prepared, which provides helpful fools for further researches on S100A10.

Aim: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites., Methods: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN)., Results: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ., Conclusion: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

X-ray crystallography is a potent and meanwhile fast technique to obtain detailed structural information of S100 proteins in their apo or metal ion-loaded state. S100 proteins crystallize in the absence or presence of Ca(2+) and Zn(2+) and the obtained crystals often diffract to high resolution yielding information on the ion-binding sites, conformation, and target interaction sites of the proteins. Here, I describe a general scheme to isolate and crystallize S100 proteins and the analysis of protein crystals using a modern synchrotron source.

High-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass -spectrometry (MS) is a relevant technique for the detection and relative quantitation of naturally occurring peptides and proteins. The peptide/protein mass is determined by deconvolution of the ESI-MS spectrum, and the resolution can be better than 1:10,000 with the instruments currently available. Accurate mass measurement, coupled with sufficient resolution, makes it possible to greatly restrict the enormous number of possible molecular formulas that might be represented by a specific molecular mass. As soon as the protein mass has been unequivocally attributed to a specific structure by means of different enzymatic and chemical treatments, the m/z values detected in the ESI spectrum can be utilized to reveal the protein and to perform its relative quantitation, by the extracted ion current (XIC) procedure, in an unlimited number of samples. This chapter describes the HPLC-ESI-MS experimental conditions which allow detecting and quantifying-in human saliva-different S100 proteins and their isoforms.

High quantity and quality of recombinant Ca(2+)-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca(2+)-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca(2+)-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn(2+)-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

S100A6, as a member of S100 protein family, have biological functions in cell proliferation, differentiation, morphology, cytoskeletal organization and apoptosis. In the last three decades, S100A6 has been caught more and more attention. Here, we introduced a simple and efficient method for producing high-purity recombinant human S100A6 from Escherichia coli culture with low level of endotoxin. We further demonstrated its biological activities for triggering SH-SY5Y cells apoptosis in vitro. These results can facilitate the study of physiological and pathological roles of S100A6 and other members of S100 family proteins., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

The S100 proteins are a large family of 10-12 kDa EF-hand signaling proteins that bind calcium, and in some cases zinc and copper, functioning as central regulators in a diversity of cellular processes. These proteins have tissue, cell, and subcellular-specific expression patterns, and many have an extracellular function. Altogether, these properties underlie their functional diversity and involvement in several pathological conditions including cancer, inflammation, and neurodegeneration. S100 proteins exhibit considerable structural plasticity, being able to exist as monomers or assemble into dimers, higher oligomers, and amyloids, frequently in a metal-dependent manner. Many of these oligomers are functionally relevant, and S100 amyloids have been recently found in prostatic inclusions. Here, we report experimental procedures for the isolation and quantitation of S100 oligomers from tissues, purification of recombinant human S100 protein for assays and use as standards, and an amyloidogenesis assay that allows monitoring the formation of S100 β-oligomers and amyloids in apo- and metal-bound S100 proteins.

Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca(2+)-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radioligand [(18)F]fluorobenzoyl-S100A4 ((18)F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of (18)F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.

S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Immunosubtraction is a powerful and resource-intensive laboratory medicine assay that reports both protein mobility and binding specificity. To expedite and automate this electrophoretic assay, we report on advances to the electrophoretic immunosubtraction assay by introducing a homogeneous, not heterogeneous, format with integrated sample preparation. To accomplish homogeneous immunosubtraction, a step-decrease in separation matrix pore-size at the head of a polyacrylamide gel electrophoresis (PAGE) separation channel enables "subtraction" of target analyte when capture antibody is present (as the large immune-complex is excluded from PAGE), but no subtraction when capture antibody is absent. Inclusion of sample preparation functionality via small pore size polyacrylamide membranes is also key to automated operation (i.e., sample enrichment, fluorescence sample labeling, and mixing of sample with free capture antibody). Homogeneous sample preparation and assay operation allows on-the-fly, integrated subtraction of one to multiple protein targets and reuse of each device. Optimization of the assay is detailed which allowed for ~95% subtraction of target with 20% non-specific extraction of large species at the optimal antibody-antigen ratio, providing conditions needed for selective target identification. We demonstrate the assay on putative markers of injury and inflammation in cerebrospinal fluid (CSF), an emerging area of diagnostics research, by rapidly reporting protein mobility and binding specificity within the sample matrix. We simultaneously detect S100B and C-reactive protein, suspected biomarkers for traumatic brain injury (TBI), in ~2 min. Lastly, we demonstrate S100B detection (65 nM) in raw human CSF with an estimated lower limit of detection of 3.25 nM, within the clinically relevant concentration range for detecting TBI in CSF. Beyond the novel CSF assay introduced here, a fully automated immunosubtraction assay would impact a spectrum of routine but labor and time-intensive laboratory medicine assays.

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Human skin is a rich source of human antimicrobial peptides. Its cellular source is the keratinocyte, which terminally differentiates in the uppermost parts of the skin, eventually forming the stratum corneum, the horny layer. The easy availability of human stratum corneum makes it possible to identify and characterize human antimicrobial peptides with a biochemical approach. Moreover, the availability of lesional scales of patients with psoriasis, an inflammatory skin disease, allows the identification of human-inducible peptide antibiotics, which are absent in healthy skin. With this strategy, the beta-defensins hBD-2 and hBD-3, RNase-7 as well as psoriasin/S100A7 have been discovered as human antimicrobial peptides and proteins. A detailed description of the strategies and methods is presented, which allowed a successful identification and characterization of human antimicrobial peptides. We used various HPLC techniques, combined with antimicrobial testing as read-out system. In parallel, SDS-PAGE analyses as well as electrospray ionization mass spectrometry were used for further biochemical characterization as well as purity assessment.

S100B and S100A10 are dimeric, EF-hand proteins. S100B undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N-terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by (1)H-(15)N HSQC spectra showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments ((1)H, (15)N, and (13)C) showed that residues T102-S108 of annexin A2 formed a well-defined alpha-helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N-terminus with a single turn of alpha-helix from D108-K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.

S100A5 is a calcium binding protein of the S100 family, with one canonical and one S100-specific EF-hand motif per subunit. Although its function is still unknown, it has recently been reported to be one of the S100 proteins able to interact with the receptor for advanced glycation end products. The homodimeric solution structures of S100A5 in both the apo and the calcium(II)-loaded forms have been obtained, and show a conformational rearrangement upon calcium binding. This rearrangement involves, in particular, the hinge loop connecting the N-terminal and the C-terminal EF-hand domains, the reorientation of helix III with respect to helix IV, as common to several S100 proteins, and the elongation of helix IV. The details of the structural changes are important because they must be related to the different functions, still largely unknown, of the different members of the S100 family. For the first time for a full-length S100 protein, relaxation measurements were performed on both the apo and the calcium-bound forms. A quite large mobility was observed in the hinge loop, which is not quenched in the calcium form. The structural differences resulting upon calcium binding change the global shape and the distribution of hydrophobic and charged residues of the S100A5 homodimer in a modest but significantly different manner with respect to the closest homologues S100A4 and S100A6.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

Background: S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets., Methods: S100A4 was immuoprecipitated from a panel of in vitro and in vivo sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF., Results: A characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots., Conclusion: Endogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses.

Data concerning the pathophysiological role of the interaction of circulating S100 proteins, a multigenic family of Ca(2+)-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) in cardiovascular diseases, inflammatory processes, and tumorigenesis in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A1, S100B, and S100A12 as potential probes for molecular imaging of this interaction. Therefore, human S100 proteins were cloned as GST fusion proteins in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100 proteins were radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiolabeled recombinant S100 proteins ((18)F-S100) were used in biodistribution experiments and small animal positron emission tomography (PET) studies in rats. The tissue-specific distribution of (18)F-S100 proteins in vivo correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. These findings indicate circulating S100A1, S100B, and S100A12 proteins to be ligands for RAGE in rats in vivo. The approach allows the use of small animal PET and provides novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease.

S100A1, a Ca(2+)-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca(2+) homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca(2+) homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F(1)-ATPase, which affects F(1)-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the alpha- and beta-chain of F(1)-ATPase to interact with S100A1 in a Ca(2+)-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F(1)-ATPase and the analysis of the S100A1-F(1)-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F(1)-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.

S100A2 is a Ca(2+)-binding EF-hand protein that is mainly localized in the nucleus. There, it acts as a tumour suppressor by binding and activating p53. Wild-type S100A2 and a S100A2 variant lacking cysteines have been purified. CD spectroscopy showed that there are no changes in secondary-structure composition. The S100A2 mutant was crystallized in a calcium-free form. The crystals, with dimensions 30 x 30 x 70 microm, diffract to 1.7 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.5, b = 57.8, c = 59.8 A, alpha = beta = gamma = 90 degrees. Preliminary analysis of the X-ray data indicates that there are two subunits per asymmetric unit.

S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1alpha, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 A resolution and the space group was assigned as primitive orthorhombic P2(1)2(1)2(1).

Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

The EF-hand protein, S100A4, binds calcium ions and interacts specifically in vitro with protein targets. Elevated levels of S100A4 have been shown to produce a metastatic phenotype in independent models of breast cancer. The presence of S100A4 in the carcinoma cells of patients with different carcinomas is associated with reduced patient survival. In order to identify the region of the S100A4 molecule that is responsible for its metastasis-inducing properties, specific mutant S100A4 genes and proteins have been produced which contain targeted mutations to the two calcium-binding sites and a deletion of the last 15 amino-acid residues of the protein. The ability of the mutant proteins to bind to a potential specific target in vitro, nonmuscle myosin heavy chain, is correlated with their ability to cause motile, invasive and metastatic phenotypes. Mutation of the C-EF hand of S100A4 virtually abolished calcium binding, and motility/invasion in vitro, abolished interaction with a molecular target, and reduced metastasis induction by 2.5-3-fold. However, deletion of the last 15 amino acids of S100A4 reduced motility/invasion, target binding and metastasis-induction to similar extents as the C-EF-hand mutant, but reduced calcium binding by only 26%. The results suggest that the ability to interact with protein target(s) is important in S100A4-induced metastasis.

Immunohistochemical localization of S-100 protein was studied in anterior, intermediate and posterior lobe of the pig pituitary gland. Two immunopositive cells for S-100 protein were identified: the folliculo-stellate cells (FSc) in the glandular lobes and the pituicytes in the neural lobe. In the anterior lobe, immunoreactive folliculo-stellate cells were scattered among secretory cells. In the area where the secretory cells form strands and follicle-like groups the positive cells were concentrated in groups. In the intermediate lobe, S-100 protein-positive cells were located sparsely among secretory cells and next to secretory follicles and the pituitary cleft. These FSc were more voluminous and displayed fewer cytoplasmic processes. In the neurohypophysis, positive reaction for S-100 protein was seen in the pituicytes. These cells were distributed singly or concentrated in groups. The distribution, and morphologic characteristics of the FSc in the glandular lobes and the pituicytes in the neural lobe in the pig indicate different origin of both S-100 protein-positive cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a widespread environmental contaminant that causes multiple effects in vertebrates. TCDD elicits its toxicity through aryl hydrocarbon receptor (AhR)-mediated modulation of gene regulation, increasing intracellular free calcium, and inducing calcium-mediated apoptosis in cell culture. Two TCDD-responsive cDNAs, which encode putative calcium-binding proteins, have been isolated from zebrafish and rainbow trout. The zebrafish and rainbow trout sequences are 88% similar to each other at the amino acid level and are orthologs of the human S100A4 calcium-binding protein. In zebrafish liver cell culture, treatment with TCDD increases S100A4a mRNA abundance. In juvenile rainbow trout, S100A4 mRNA was constitutively expressed in the heart, kidney, intestine, and spleen, but not in the liver. Exposure to TCDD significantly increased rainbow trout S100A4 mRNA abundance in the rainbow trout kidney. Taken together, these findings demonstrate in zebrafish and rainbow trout that dioxin increases expression of this EF-hand calcium-binding protein gene in a tissue-dependent fashion. However, demonstration that the encoded S100A4 proteins actually bind calcium and play a role in dioxin toxicity will require further study.

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun.285, 1317-1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b.

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.

By photoaffinity labeling with a tritiated azido derivative of phenylarsine oxide (PAO), 4[N-(4-azido-2-nitrophenyl)amino-[(3)H]acetamido]phenylarsine oxide ([(3)H]azidoPAO), we demonstrate that PAO binds selectively to the S100 A8/A9 complex of bovine neutrophil cytosol (previously known as p7/p23, homologous to the MRP-8/MRP-14 complex of human phagocytes). Using a semirecombinant cell free assay of oxidase activation and the determination of oxidase activity by the production of the superoxide anion O(-)(2), we found that the PAO binding protein (p7/p23) was able to potentiate the activation of NADH oxidase and that this effect was synergized by PAO. The p7/p23 protein complex of bovine neutrophils can therefore be considered as a positive regulator of NADPH oxidase activation in neutrophils., (Copyright 2001 Academic Press.)

Dendritic cells seem to be of major importance for the pathogenesis of psoriasis. They are increased in number in lesional psoriatic skin which is thought to be due to an increased influx from the peripheral blood regulated by chemotaxins. Using a biological/biochemical approach we have addressed the question whether psoriasis scale extracts contain proteinaceous chemotaxins for dendritic cells. Human monocytes differentiated into dendritic cells by culture with GM-CSF and IL-4 (MoDC) served as responder cells. Chemotactic activity for MoDC was purified by several HPLC-steps. The results of our study show that C5a/C5adesarg is the major chemotactic peptide for MoDC in psoriasis scale extracts. In comparison to other stimuli such as fMLP or monocyte chemotactic peptide 1 (MCP-1) C5a proved to be a most potent and efficient chemotaxin for MoDC. C5a co-eluted with MRP14/calgranulin B which is present in large amounts in psoriasis scale extracts as identified by amino acid sequencing. However, MRP14/calgranulin B did not possess any chemotactic activity for MoDC. Our results provide evidence that C5a/C5adesarg although not specific for dendritic cells seems to be the major chemoattractant for these cells in lesional psoriasis skin.

MRP14 is a protein that is specifically expressed in myeloid and epithelial cells during the stages of acute or chronic inflammatory states such as rheumatoid arthritis or sarcoidosis. MRP14 has EF-hand motifs as Ca(2+)-binding sites and belongs to the S100 family of proteins. This paper deals with the sample preparation (cloning, overexpression and purification), crystallization and preliminary crystallographic analysis of recombinant human MRP14. Crystals of MRP14 were obtained by the hanging-drop vapour-diffusion method. MRP14 crystals belong to space group P2(1), with unit-cell parameters a = 57.59, b = 178.44, c = 61.23 A, beta = 113.17 degrees, and diffract to 2.1 A resolution.

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells. A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J. (1998) J. Neurochem. 70, 1793-1798). Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP. A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin. CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% alpha-helix, 15% beta -sheet, and 81% random coil. Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 microm for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions. NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of (15)N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

S100A2 is a calmodulin-like protein of unknown function, whose transcription is positively regulated in response to ErbB and p53 signaling. Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epidermal hyperplasia, and decreased in the context of hypofunctional p53 mutations in carcinoma cell lines and tumors. This bimodal pattern of regulation suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis. Taking the biochemical approach to the determination of S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes. S100A2 in hypotonic lysates remained soluble after centrifugation at 100 000 x g, indicating that it is not associated with cell membranes. Permeabilization experiments confirmed the lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy. Pulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening revealed that S100A2 displays a strong propensity to homodimerize. Naturally expressed S100A2 dimers in normal human keratinocytes readily underwent intermolecular disulfide cross-linking unless a strong denaturant was present during cell lysis. Treatment of intact normal human keratinocytes with hydrogen peroxide strongly promoted S100A2 cross-linking. These results demonstrate that native S100A2 is a homodimer that does not depend on disulfide cross-linking for stability, but undergoes intermolecular cross-linking at cysteine residues in response to oxidative stress. Based on these findings, we propose that S100A2 may protect normal keratinocytes against carcinogens by participating in the cellular proof-reading response to oxidative stress.

S100 proteins became of major interest because of their divergent cell- and tissue-specific expression, their close association with a number of human diseases, and their importance for clinical diagnostics. Here, we report for the first time the purification and characterization of human recombinant S100A13. Flow dialysis revealed that the homodimeric S100A13 binds four Ca(2+) in two sets of binding sites, both displaying positive cooperativity but of very different affinity. Fluorescence and difference spectrophotometry indicate that the Trp/Tyr signal changes are almost complete upon binding of Ca(2+) to the two high affinity sites, which probably correspond to the C-terminal EF-hands in each subunit. The far-UV circular dichroic signal also changes upon binding of the first two Ca(2+). So far, the tissue distribution of S100A13 has not been well characterized. Here, we show that S100A13 is widely expressed in various types of tissues with a high expression level in thyroid gland. Using specific antisera against S100A13, high protein expression was detected in follicle cells of thyroid, Leydig cells of testis, and specific cells of brain. In human smooth muscle cells, which co-express S100A2 in the nucleus and S100A1 in stress fibers, S100A13 shows a unique subcellular localization in the perinuclear area. These data suggest diverse functions for this protein in signal transduction.

S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg(2+)-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca(2+), with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca(2+)-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg(2+)-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca(2+)-dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg(2+)-ATPase activity through its Ca(2+)-dependent interaction with actin filaments., (Copyright 2000 Academic Press.)

Expression of S100A6 (Calcyclin), a member of the S100 family and of Zn(2+)-binding proteins is elevated in a number of malignant tumors. In vitro the protein associates with several actin-binding proteins and annexins in a Ca(2+)-dependent manner. We have now studied the subcellular localization of S100A6 using a new, specific monoclonal antibody. Immunofluorescence microscopy of unfixed, ultrathin, frozen sections demonstrated a dual localization of S100A6 at the nuclear envelope and the plasma membrane of porcine smooth muscle only in the presence of Ca(2+). The same localization was found by immunofluorescence and immunogold electron microscopy as well as by confocal laser scanning microscopy with cultured, fixed, human CaKi-2 and porcine ST interphase cells. Upon cell division, however, S100A6 was found exclusively in the cytoplasm. Cell fractionation studies showed that S100A6 was present in the microsomal fraction in the presence of Ca(2+) and was released from this fraction by the addition of EGTA/EDTA but not by Triton X-100. The data demonstrate that S100A6 is localized both at the plasma membrane and the nuclear envelope in vivo and suggest a Ca(2+)-dependent interaction with annexins or other components of the nuclear envelope.

In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types., (Copyright 1999 Academic Press.)

The expression of inducible nitric oxide synthase (iNOS), major histocompatibility class II molecules (MHC-II), CD68, and the calcium-binding proteins S100A8 and S100A9 (also called MRP8 and MRP14, respectively) was assessed in lung tissues from cattle that succumbed to pneumonia. Expression patterns of these markers were related to the types of lung lesion. iNOS expression was only observed in lungs infected with Arcanobacterium pyogenes or Pasteurella haemolytica but not in lungs from cattle with subacute chronic interstitial pneumonia and acute interstitial pneumonia due to Escherichia coli infection. High levels of iNOS were expressed by cells (probably leukocytes) surrounding necrotic foci. Occasionally, iNOS was expressed by intraalveolar macrophages in viable parenchyma, by leukocytes within the airways, and by some chondrocytes in the supporting cartilage of bronchi. Cells expressing MHC-II were distributed relatively evenly throughout areas of inflammation and did not display any clear association with necrotic foci. Cell types expressing MHC-II included type II alveolar epithelial cells, spindle-shaped cells of the interstitium, cells in bronchus-associated lymphoid tissue, and leukocytes in lymph and blood vessels but largely excluded iNOS-positive cells. Likewise, CD68-positive cells were rarely positive for iNOS and were not confined to the areas surrounding necrotic tissue. As with MHC-II and CD68, there was little if any coexpression of iNOS and either of the S100 proteins tested. Thus, in cattle with necrotizing bronchopneumonia, iNOS-expressing cells were largely restricted to the cellular zone surrounding necrotic areas.

While previous studies have identified target proteins that interact with S100A1 in a calcium-dependent manner as well as target proteins that interact in a calcium-independent manner, the molecular mechanisms of S100A1-target protein interaction have not been elucidated. In this study, point and deletion mutants of S100A1 were used to investigate the contribution of carboxyl terminal amino acids to S100A1 interaction with calcium-dependent and calcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properties indistinguishable from native S100A1. Next, proteins lacking the carboxyl-terminal nine residues of recS100A1 (Delta85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 88 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins interacted with phenyl-Sepharose in a calcium-dependent manner. However, the Delta85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. The F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent interaction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 comprise the PSBR of S100A1. Fluorescence studies showed that the Delta85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl CapZ peptide, TRTK, while W90A bound TRTK with a Kd of 5.55 microM. These results demonstrate that the calcium-dependent target protein-binding site and the PSBR are indistinguishable. In contrast to the calcium-dependent target TRTK, activation of the calcium-independent target protein aldolase A by the point and deletion mutant S100A1s was indistinguishable from native S100A1. These results demonstrate that carboxyl-terminal residues are not required for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanisms for interaction with calcium-independent and calcium-dependent target proteins.

Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.

The S100 Ca(2+)-binding proteins recently became of major interest because of their differential expression in neoplastic tissues, their involvement in metastatic processes, and the clustered organization of at least 10 S100 genes on human chromosome 1q21, a region frequently rearranged in several tumors. As a first attempt towards a specific and differentiated immunohistochemical classification of human tumors, we produced, purified and characterized a number of human recombinant S100 proteins and raised specific polyclonal antibodies. Their distinct cellular and intracellular localization was examined by immunohistochemical methods in normal and cancerogenic human tissues and cell lines. S100A1 and S100A2 can be detected in a few normal tissues only, whereas S100A4, S100A6, and S100B are expressed at higher levels in cancer tissues. In the future, these S100 antibodies will potentially be of great value in cancer diagnosis and therapy.