Peter Kleinert

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patients fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.

Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer A PUTATIVE Ca2+ MODULATOR MATURES HUMAN HAIR CUTICLE

S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.

Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities.

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.

Mass Spectrometric Analysis of Human Transferrin in Different Body Fluids

Abstract In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

Filter paper cards contaminated with EMLA cream produce artefacts on acylcarnitine analysis.

Summary: Electrospray ionization tandem mass spectrometry is a widely applied method for the analysis of acylcarnitines in blood samples spotted on filter paper cards (Guthrie cards). When the filter paper cards are contaminated by EMLA cream, highly intense signals at m/z 221 and 235 are detected under ESI-MS/MS conditions, monitoring for precursors of m/z 85. These signals correspond to the active ingredients prilocaine and lidocaine in EMLA and overlap with the signals from the isotopically labelled internal standards (2H3)propionyl carnitine and (2H3)butyrylcarnitine. This interference prevents the proper quantification of the two short-chain acylcarnitines when samples are analysed without derivatization.

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.

Advances in hemoglobinopathy detection and identification

Hemoglobin disorders consist of two different groups, the structural hemoglobin variants and the thalassemias. The structural hemoglobin variants typically are based on the point mutations in the alpha- or beta-globin chain that results in a single-amino acid substitution in the corresponding globin chain, whereas thalassemias are caused by quantitative reduction in globin chain synthesis. Various techniques are applied for the laboratory investigation of these diseases, among them mass spectrometry (MS) for the detection and identification of structural hemoglobin variants and array techniques for the thalassemias. In this review, we present in the first part the most important mass spectrometric techniques applied in hemoglobin variant detection and identification and discuss several important aspects of this analysis technique in hematology. In the second part, the DNA analysis techniques used in hemoglobin analysis, such as reverse hybridization or microarray-based comparative genomic hybridization (CGH) techniques, are briefly discussed.

A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

A new α-globin mutation [α138(H21)Ser→Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an α-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the α2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser→Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.