Molecular evolution of a novel marsupial S100 protein (S100A19) which is expressed at specific stages of mammary gland and gut development.

S100 proteins are calcium-binding proteins involved in controlling diverse intracellular and extracellular processes such as cell growth, differentiation, and antimicrobial function. We recently identified a S100-like cDNA from the tammar wallaby (Macropus eugenii) stomach. Phylogentic analysis shows wallaby S100A19 forms a new clade with other marsupial and monotreme S100A19, while this group shows similarity to eutherian S100A7 and S100A15 genes. This is also supported by amino acid and domain comparisons. We show S100A19 is developmentally-regulated in the tammar wallaby gut by demonstrating the gene is expressed in the forestomach of young animals at a time when the diet consists of only milk, but is absent in older animals when the diet is supplemented with herbage. During this transition the forestomach phenotype changes from a gastric stomach into a fermentation sac and intestinal flora changes with diet. We also show that S100A19 is expressed in the mammary gland of the tammar wallaby only during specific stages of lactation; the gene is up-regulated during pregnancy and involution and not expressed during the milk production phase of lactation. Comparison of the tammar wallaby S100A19 protein sequence with S100 protein sequences from eutherian, monotreme and other marsupial species suggest the marsupial S100A19 has two functional EF hand domains, and an extended His tail. An evolutionary analysis of S100 family proteins was carried out to gain a better understanding of the relationship between the S100 family member functions. We propose that S100A19 gene/protein is the ancestor of the eutherian S100A7 gene/protein, which has subsequently modified its original function in eutherians. This modified function may have arisen due to differentiation of evolutionary pressures placed on gut and mammary gland developmental during mammal evolution. The highly regulated differential expression patterns of S100A19 in the tammar wallaby suggests that S100A19 may play a role in gut development, which differs between metatherians and eutherians, and/or include a potential antibacterial role in order to establish the correct flora and protect against spiral bacteria in the immature forestomach. In the mammary gland it may protect the tissue from infection at times of vulnerability during the lactation cycle.

Structural stability and reversible unfolding of recombinant porcine S100A12.

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.

The S100 proteins for screening and prognostic grading of bladder cancer.

The S100 gene family, which is composed of at least 24 members carrying the Ca2+ binding EF-hand motif, has been implicated in both intracellular and extracellular functions, including enzyme activities, immune responses, cytoskeleton dynamics, Ca2+ homeostasis, cell growth and cell differentiation. Altered S100 protein levels are associated with a broad range of diseases, including cardiomyopathy, inflammatory and immune disorders, neurodegenerative disorders and cancer. Although the precise role of S100 protein in carcinogenesis is poorly understood, it seems that formation of homo- and hetero-dimers, binding of Ca2+ and interaction with effector molecules are essential for the development and progression of many cancers. Several studies have suggested that S100 proteins promote cancer progression and metastasis through cell survival and apoptosis pathways. In animal models of bladder cancer, several S100 proteins are differentially expressed in bladder tumors relative to normal urothelium. In human bladder cancer, overexpression of S100A4, S100A8 or S100A11 are associated with stage progression, invasion, metastasis and poor survival. This review summarizes these findings and evaluates their implications for human bladder cancer management.

An update of the S100 nomenclature.

The plethora of names given to S100 proteins resulted in considerable confusion. Here we present the official and updated nomenclature of this protein family, approved by the HGNC (HUGO gene nomenclature committee) and ECS (European Calcium Society).

S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature).

The S100 protein family is the largest subgroup within the superfamily of proteins carrying the Ca2+-binding EF-hand motif. Despite their small molecular size and their conserved functional domain of two distinct EF-hands, S100 proteins developed a plethora of tissue-specific intra- and extracellular functions. Accordingly, various diseases such as cardiomyopathies, neurodegenerative and inflammatory disorders, and cancer are associated with altered S100 protein levels. Here, we review the different S100 protein functions and related diseases from an evolutionary point of view. We analyzed the structural variations, which are the basis of functional diversification, as well as the genomic organization of the S100 family in human and compared it with the S100 repertoires in mouse and rat. S100 genes and proteins are highly conserved between the different mammalian species. Moreover, we identified evolutionary related subgroups of S100 proteins within the three species, which share functional similarity and form subclusters on the genomic level. The available S100-specific mouse models are summarized and the consequences of our results are discussed with regard to the use of genetically engineered mice as human disease models. An update of the S100 nomenclature is included, because some of the recently identified S100 genes and pseudogenes had to be renamed.

Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family.

In an effort to identify psoriasis-associated genes, we compared gene expression in normal and psoriatic skin, using differential display RT-PCR technique. Sequence analysis of a 650-bp cDNA fragment (clone 110) that was highly up-regulated in lesional skin revealed homology to a noncoding cDNA (NICE-2). By subsequent cDNA cloning, using RNA from psoriatic skin, we have identified two alternatively spliced mRNA-isoforms (0.5 and 4.4 kb), which differ in composition of their untranslated regions. By sequence comparison, we have mapped the novel gene, named S100A15, to the S100 gene cluster within the epidermal differentiation complex (chromosome 1q21). Analysis of the deduced amino acid sequence revealed a protein of 101 amino acids containing two potential EF-hand motifs with high homology to the S100A7. Northern blot hybridization and semiquantitative RT-PCR analysis confirmed the S100A15 overexpression in psoriasis, showing different levels of expression of the S100A15 mRNA isoforms. In situ hybridization of the S100A15 revealed a markedly increased staining of basal and suprabasal epidermal layers of psoriatic skin compared with healthy tissue. Our data suggest an involvement of the novel S100A15 in epidermal differentiation and inflammation and might therefore be important for the pathogenesis of psoriasis and other diseases.

The EH1 domain of Eps15 is structurally classified as a member of the S100 subclass of EF-hand-containing proteins.

The Eps15 homology (EH) domain is a protein-protein interaction module that binds to proteins containing the asparagine-proline-phenylalanine (NPF) or tryptophan/phenylalanine-tryptophan (W/FW) motif. EH domain-containing proteins serve important roles in signaling and processes connected to transport, protein sorting, and organization of subcellular structure. Here, we report the solution structure of the apo form of the EH1 domain of mouse Eps15, as determined by high-resolution multidimensional heteronuclear NMR spectroscopy. The polypeptide folds into six alpha-helices and a short antiparallel beta-sheet. Additionally, it contains a long, structured, topologically unique C-terminal loop. Helices 2-5 form two EF-hand motifs. Structural similarity and Ca(2+) binding properties lead to classification of the EH1 domain as a member of the S100 subclass of EF-hand-containing proteins, albeit with a unique set of interhelical angles. Binding studies using an eight-residue NPF-containing peptide derived from RAB, the cellular cofactor of the HIV Rev protein, show a hydrophobic peptide-binding pocket formed by conserved tryptophan and leucine residues.

Rapid desensitization of PC12 cells stimulated with high concentrations of extracellular S100.

Undifferentiated PC12 cells undergo apoptosis, via a calcium-induced calcium release mechanism, when the calcium-binding protein purified from bovine brain (native S100) is present in micromolar concentration in the medium. This process begins when S100 binds to specific membrane binding sites and involves up to 50% of the cell population. In the experiments reported here, we demonstrate that, by utilizing [3H]S100, the S100 protein can be displaced from its binding sites only during the first 10 min of incubation. This fact is due to an internalization mechanism, having a time-course with a plateau after 10-20 min of incubation. The native form of S100 is a mixture of two different S100 isoforms: S100A1 (20%) and S100B (80%). Using confocal microscopy and monoclonal antibodies, we demonstrated that only one of these isoforms, S100A1, was autoexpressed in more than 50% of the PC12 cells analysed. After cell incubation with 2 microM native S100, S100B also appears in PC12 cells, with a maximum presence after 10 min of incubation. This fact seems to indicate that this isoform, at least, is effectively translocated when stimulated with external native S100. From the data reported, it is possible to hypothesize that, in PC12 cells, a possible homeostatic mechanism is present that can counteract the effect of a continuously applied lethal stimulus (stimuli) on cell viability.

Immunohistochemical evaluation of the Ca(2+)-binding S-100 proteins S-100A1, S-100A2, S-100A4, S-100A6 and S-100B in salivary gland tumors.

The Ca(2+)-binding S-100 proteins are involved in the regulation of a number of cellular processes and an altered expression has been reported in several neoplastic tissues. Tissue specimens of normal salivary glands (n = 23), pleomorphic adenomas (n = 60), basal cell adenomas (n = 6), canalicular ademomas (n = 2), myoepitheliomas (n = 2), adenoid cystic carcinomas (n = 26) and adenocarcinomas NOS (n = 11) were evaluated for the expression of S-100A1, S-100A2, A-100A4, S-100A6 and S-100B by using highly specific polyclonal and monoclonal antibodies generated against the recombinant human protein. In normal salivary glands, the ductal cells showed mild to intense immunoreactivity for S-100A1, S-100A2, S-100A4 and S-100A6, while S-100B was observed in nerve fibers in the connective tissue. The normal myoepithelial cells were unreactive. In pleomorphic adenoma, the luminal tumor cells of the duct-like structures showed moderate to intense immunoreactivity for S-100A2, while reactivity for S-100A1, S-100A4 and S-100A6 was relatively weak. The non-luminal cells, also termed neoplastic myoepithelial cells, showed immunoreactivity for S-100B, while tumor cells in the solid, myxoid and chondroid areas were immunoreactive for S-100A1, S-100A4, S-100A6 and S-100B. The non-luminally located tumor cells in basal cell adenomas and canalicular adenomas, and numerous tumor cells in clusters in myoepitheliomas were intensely reactive for S-100A2. In adenoid cystic carcinomas and in adenocarcinomas not otherwise specified, the luminal cells forming the tubular or cribriform structures were markedly positive for S-100A2 and/or S-100A6. Squamous metaplastic cells in salivary tumors showed intense immunoreactivity for S-100A2. The results of the present study suggest that the majority of the tumor cells in salivary neoplasms, despite the most heterogeneous tumor cell differentiation, express S-100 proteins more heterogeneously than the normal glandular ducts. The salivary ducts in normal glands, the luminal tumor cells and squamous metaplastic cells in the neoplastic lesions were intensely immunoreactive for S-100A2 as compared to S-100A1, S-100A4 or S-100A6. In contrast, the non-luminal tumor cells showed a rather heterogeneous expression of the S-100 proteins.

Psoriasin binds calcium and is upregulated by calcium to levels that resemble those observed in normal skin.

Recently, we described a small molecular weight protein termed psoriasin that showed sequence similarity with the S100 calcium-binding proteins and that is highly upregulated in psoriatic epidermis as well as in primary human keratinocytes undergoing abnormal differentiation. Here we present evidence showing that natural and recombinant psoriasin binds calcium, as judged by the calcium overlay assay, and that it contains all the sequence features characteristic of the S100 family. Furthermore, [35S]-methionine labeling experiments showed that psoriasin synthesis is upregulated by 2 mM Ca++ (ratio Ca++/control at 88 h = 2.56) to levels that resemble those observed in unfractionated keratinocyte populations obtained from normal skin.

Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.

The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.

Cloning of cDNAs encoding human S-100 alpha and beta subunits and their differential expression in human tumor cell lines.

We isolated nearly full-length clones of S-100 alpha and beta subunit cDNAs from a human brain cDNA library. The alpha subunit cDNA was 579 bp long and contained 99 bp of 5'-noncoding region, 282 bp of coding region, and 198 bp of 3'-noncoding region. The beta subunit cDNA was 743 bp long and contained 57 bp of 5'-noncoding region, 276 bp of coding region, and 410 bp of 3'-noncoding region. An amino acid sequence comparison between human and bovine alpha subunits and between human and rat beta subunits showed that both subunits were nearly entirely conserved. The amino acid sequences of human alpha and beta subunits were conserved at both Ca(2+)-binding domains. Northern blot analysis of brain RNA showed that human alpha and beta subunit cDNA probes discriminated between alpha and beta subunit mRNAs. By using these subunit-specific cDNA probes, it was demonstrated that alpha and beta subunit mRNAs were expressed in different manners among tumor cell lines: beta was detected in melanoma and some glioma cell lines, while alpha was detected only in a melanoma cell line. Southern blot analysis showed that there were no major deletions and rearrangements of alpha and beta subunit genes in these cell lines, regardless of the level of alpha and beta subunit expression, suggesting that the expression of these subunits may be regulated at the transcriptional or RNA stability level.

Large-scale, one-step purification of oxidized and reduced forms of bovine brain S100b protein by HPLC.

A rapid and simple method, using a reverse-phase column in a HPLC system, has been developed to purify high yields of both oxidized and reduced S100b proteins from a bovine brain S100 protein mixture. The final proteins were characterized by amino acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and absorbance and fluorescence spectroscopy. Both S100b subtypes appeared highly purified and differed only by their oxidation state: all four cysteinyl sulfhydryl groups were free in reduced S100b protein whereas two of them gave disulfide bridges in oxidized S100b protein. The stability of the oxidation state of the two isolated subtypes suggests that the two forms were not in rapid equilibrium and probably coexisted in vivo.

Tissue distribution of rat S100 alpha and S100 beta and S100-binding proteins.

To understand the physiological role of the calcium-binding proteins S100 alpha and S100 beta, it is necessary to determine the distribution of these proteins and detect their intracellular targets in various tissues. The distribution of immunoreactive S100 alpha and S100 beta in various rat tissues was examined by radioimmunoassay. All tissues examined contained detectable S100, but the S100 beta/S100 alpha ratio in each tissue differed. Brain, adipose, and testes contained 18- to 40-fold more S100 beta than S100 alpha; skin and liver contained approximately equivalent amounts and kidney, spleen, and heart contained 8- to 75-fold more S100 alpha than S100 beta. Analysis of S100-binding proteins by gel overlay showed that each tissue possessed its own complement of binding proteins. The S100 beta-binding profile was indistinguishable from the S100 alpha-binding profile and both of these profiles were distinct from the calmodulin-binding profile. These observations suggest that the differential distribution and quantity of the individual S100 polypeptides and their binding proteins in various tissues may be important factors in determining S100 function.