M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted

Apoptosis of lymphocytes induced by chromium(VI/V) is through ROS-mediated activation of Src-family kinases and caspase-3

Mechanistic insights into Cr(VI)-induced carcinogenicity and possible implication of Cr(V) species formed by the redox reactions of chromium-bearing species have attracted interest. We have previously demonstrated that when human peripheral blood lymphocytes are exposed to the Cr(V) complexes, viz., sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[CrνO(ehba)2] and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[CrνO(hmba)2], apoptosis and formation of reactive oxygen species (ROS) are observed. The molecular mechanisms involving cellular signaling pathways leading to apoptosis are addressed in the present study. Treatment of lymphocytes with Na[Crν;O(ehba)2] and K2Cr2O7 leads to the activation of the Src=family protein tyrosine kinases namely, p56lck, p59fyn, and p56/53lyn, which then activates caspase-3, both of which are under the partial influence of ROS. Inhibition of the Src-family tyrosine kinases activity by PP2 and of caspase-3 by Z-DEVD-FMK reverses apoptosis, thereby suggesting their importance. Antioxidants only partially reverse the apoptosis induced by Cr(VI/V), suggesting that pathways other than those induced by ROS cannot be ruled out. Although the complex, Na[CrVO(ehba)2] is known to be relatively stable in aqueous solutions, previous studies have shown that the Cr(V) complex, Na[CrνO(ehba)2] disproportionates to Cr(VI) and Cr(III) forms at pH 7.4 through complex mechanistic processes. Dynamics studies employing EPR data show that the Cr(V) state in Na[CrνO(ehba)2] is relatively more stable in RPMI-1640 medium containing plasma. Formation of ROS during the reaction of redox partners with Na[CrνO(ehba)2] is an early event and compares favorably in kinetic terms with the reported rate processes for disproportionation. This investigation presents evidence for the direct implication of Cr(V) in Cr(VI)-induced apoptosis of lymphocytes

Hydroxopentaamminechromium(III) promoted phosphorylation of bovine serum albumin: its potential implications in understanding biotoxicity of chromium

Evidence for chromium(III) induced phosphorylation of a biomarker protein bovine serum albumin (BSA) is presented. Radiolabelled adenosine 5'-triphosphate (ATP) was reacted with BSA in the presence of various Cr(III) salts. While [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(H<SUB>2</SUB>O)]<SUP>3+</SUP> brought about phosphorylation of BSA, several Cr(III) complexes, viz. [Cr(bpy)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(phen)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(en)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(salen)(H<SUB>2</SUB>O)<SUB>2</SUB>]<SUP>+</SUP> and [Cr(salprn)(H<SUB>2</SUB>O)<SUB>2</SUB>]<SUP>+</SUP>, did not phosphorylate BSA. The Cr(III) mediated the transfer of γ- and α-phosphates but not the adenine and the sugar moieties of the ATP molecule to BSA. The observed stoichiometry was 0.75 mol P<SUB>i</SUB> to mol BSA for the γ-phosphate and 0.5 mol P<SUB>i</SUB> to mol BSA for the α-phosphate of ATP. The presence of serine phosphate and threonine phosphate was detected in the hydrolysate of phosphorylated BSA by means of comparison of R<SUB>f</SUB> values with authentic samples of phosphoserine and phosphothreonine after chromatographic separation and autoradiography. [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(H<SUB>2</SUB>O)]<SUP>3+</SUP> at pH 7.4 is known to exist as the conjugate base [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(OH)]<SUP>2+</SUP> and is capable of ligand substitution involving metal-oxygen bond retention. Such anation reaction of [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(OH)]<SUP>2+</SUP> with ATP subsequently leads to the esterification of alcoholic hydroxyl groups of serine and threonine of BSA. Possible consequences of chromium(III) induced in vivo phosphorylation of proteins are discussed

Apoptosis of lymphocytes in the presence of Cr(V) complexes: role in Cr(VI)-induced toxicity

Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through epidemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxidation states such as Cr(V) and Cr(IV) in Cr(VI)-induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human peripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr<SUP>ν</SUP>O(ehba)<SUB>2</SUB>], 1 and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr<SUP>ν</SUP>O(hmba)<SUB>2</SUB>], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time-and concentration-dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before exposure to chromium(V) complexes reverse apoptosis partially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible

Non-enzymatic phosphorylation of bovine serum albumin by Cr(V) complexes: Role in Cr(VI)-induced phosphorylation and toxicity

Evidence for the non-enzymatic phosphorylation of bovine serum albumin (BSA) by sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[CrνO(ehba)2], 1, sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[CrνO(hmba)2], 2 and potassium dichromate, K2Cr2O7, 3 in the presence of labeled adenosine-5'-triphosphate (ATP) under conditions of physiological pH is presented. Aggregation and extent of phosphorylation of BSA mediated by 1, 2 or 3 seems to increase with the concentration and time of incubation of the reaction mixture containing all the reactants. The [γ-32P] label in ATP is incorporated into aggregates of BSA in the in vitro reaction of the protein with ATP in the presence of 1, 2 or 3. Phosphorylation of BSA by ATP in the absence of 1, 2 or 3 is negligible. Addition of EDTA reverses aggregation of protein and liberates partially the incorporated phosphate label. The stoichiometry of phosphorylation is found to be the highest and is equal to 12.25 mol PO43−/mol BSA in the presence of 500 μM of 1, which decreases to 10.56 mol PO43−/mol BSA after EDTA treatment. Resistance to the removal of phosphate label by EDTA increases with increase in time of incubation. Dialysis of phosphorylated BSA reverses the incorporated [γ-32P] label only partially, indicating the formation of covalent links of phosphate groups to BSA. Evidence for the site of phosphorylation in the reaction mediated by 1, 2 or 3 being hydroxyl side groups of tyrosine and serine/threonine residues has been gained. Based on the results, a possibility that 1, 2 and 3 mimic the function of tyrosine and serine/threonine kinases has been invoked

Mutations In Ripk4 Cause The Autosomal-Recessive Form Of Popliteal Pterygium Syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-kappa B (NF-kappa B) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.WoSScopu

Rare genetic variants of SIAE.^§§

§§<p>The subjects from Surolia et al. (4) and Hirschfield et al. (5) include controls and autoimmune subjects of European ancestry. EVS (Exome variant server) data comprises unannotated American subjects (disease status is unknown) of European and African ancestry and is current as of June 20th 2012. Rare variants reported in both African-Americans and European-Americans are marked with a single asterisk (*n = 6500). Rare variants seen only in European-Americans or only in African-Americans are marked with double (**n = 4299) and triple asterisks (***n = 2201) respectively.</p>nf<p>These variants were found in dbSNP and/or 1000 genomes project but frequency data is not available. The dbSNP data is not ethnically stratified and was derived from the 1000 genomes project.</p

K71R SIAE and A467V SIAE proteins are functionally normal.

<p>The <i>K71R</i> and <i>A467V SIAE</i> variants were re-created by site-directed mutagenesis. Wild type (<i>WT) SIAE</i>, a known catalytic site mutant (<i>S127A SIAE</i>), and the two <i>SIAE</i> common variants were transfected into HEK 293T cells. Half of each cell lysate was immunoprecipitated with anti-Flag antibodies and revealed in a quantitative Western blot assay (A) and the other half was similarly immunoprecipitated and examined for esterase activity, presented following normalization for lysate SIAE protein content (B). Each mutant was separately transfected and analyzed on three occasions. A representative experiment is shown. Error bars reflect esterase assays performed in triplicate in a single experiment.</p

Frequency of the subset of catalytically defective <i>SIAE</i> variants genotyped by Hunt et al. [7].

<p>Frequency of the subset of catalytically defective <i>SIAE</i> variants genotyped by Hunt et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053453#pone.0053453-Hunt1" target="_blank">[7]</a>.</p