Jan Pawel Jastrzebski

Glycans of myelin proteins

Human P0 is the main myelin glycoprotein of the peripheral nervous system. It can bind six different glycans, all linked to Asn93, the unique glycosylation site. Other myelin glycoproteins, also with a single glycosylation site (PMP22 at Asn36, MOG at Asn31), bind only one glycan. The MAG has 10 glycosylation sites; the glycoprotein OMgp has 11 glycosylation sites. Aside from P0, no comprehensive data are available on other myelin glycoproteins. Here we review and analyze all published data on the physicochemical structure of the glycans linked to P0, PMP22, MOG, and MAG. Most data concern bovine P0, whose glycan moieties have an MW ranging from 1,294.56 Da (GP3) to 2,279.94 Da (GP5). The pI of glycosylated P0 protein varies from pH 9.32 to 9.46. The most charged glycan is MS2 containing three sulfate groups and one glucuronic acid; whereas the least charged one is the BA2 residue. All glycans contain one fucose and one galactose. The most mannose rich are the glycans MS2 and GP4, each of them has four mannoses; OPPE1 contains five N‐acetylglucosamines and one sulfated glucuronic acid; GP4 contains one sialic acid. Furthermore, human P0 variants causing both gain and loss of glycosylation have been described and cause peripheral neuropathies with variable clinical severity. In particular, the substitution T95→M is a very common in Europe and is associated with a late‐onset axonal neuropathy. Although peripheral myelin is made up largely of glycoproteins, mutations altering glycosylation have been described only in P0. This attractive avenue of research requires further study.

High-resolution structural model of porcine P2 myelin membrane protein with associated fatty acid ligand: Fact or artifact?

Myelin membrane is a biological complex of glial cells origin; it is composed of 25% (w/w) proteins and 75% lipids, and more than 300 proteins are associated with central nervous system myelin (for peripheral nervous system myelin, such data are lacking). Myelin plays an important role in maintaining propagation of nerve signals. To uncover the nature of propagation phenomena, it is essential to study biochemistry of myelin proteins and lipids, myelin composition, and myelin structure. Nearly all myelin proteins are like antigens, causing clinically well‐defined devastating diseases; multiple sclerosis and Guillain‐Barré syndrome are two of them. In this article, a high‐resolution study (1.8 Å) of porcine myelin P2 protein is presented. Myelin was purified from porcine intradural spinal roots, which were stored at −80°C for 10 years before myelin and P2 protein were purified (spinal roots were a gift of Prof. Kunio Kitamura, Saitama Medical School). The three‐dimensional structural analysis uncovered embedded 18‐carbons‐long fatty acid. Some speculative interpretation is presented, to uncover how this ligand of fatty acid may form cholesterol ester and stabilize the myelin structure or form simple raft microdomain. Protein crystallography indicates that the ligand may be 18‐carbons‐long fatty acid. This is unlike previous work with mass spectrometry, in which three ligands were determined. In other protein crystallography‐based studies of P2 (bovine), an oleic fatty acid was suggested, but, for recombinant (human) protein, palmitic acid was found. There is no fatty acid ligand in equine P2 protein.

Structural-functional adaptations of porcine CYP1A1 to metabolize polychlorinated dibenzo-p-dioxins.

Polychlorinated dibenzo-p-dioxins (PCDDs) are widespread by-products of human industrial activity. They accumulate in tissues of animals and humans, exerting numerous adverse effects on different systems. In living organisms, dioxins are metabolized by enzymes of the cytochrome P450 family, including CYP1A1. Particular dioxin congeners differ in their toxicity level and ability to undergo biodegradation. Since the molecular mechanisms underlying dioxin susceptibility or resistance to biodegradation are unknown, in the present study the molecular interactions between five selected dioxins and porcine CYP1A1 protein were investigated. It was found that the ability of a dioxin to undergo CYP1A1-mediated degradation is associated mainly with the number and position of chlorine atoms in the dioxin molecule. Among all examined congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrated the highest affinity to CYP1A1 and, at the same time, the greatest distance to the active site of the enzyme. Interestingly, in contrast to other dioxins, the binding of the TCDD molecule to the porcine CYP1A1 active site resulted in a rapid and continuous closure of substrate channels. All the information may help to explain the extended half-life of TCDD in living organisms as well as its high toxicity.

Sequence motifs of myelin membrane proteins : Towards the molecular basis of diseases

The shortest sequence of amino acids in protein containing functional and structural information is a “motif.” To understand myelin protein functions, we intensively searched for motifs that can be found in myelin proteins. Some myelin proteins had several different motifs or repetition of the same motif. The most abundant motif found among myelin proteins was a myristoylation motif. Bovine MAG held 11 myristoylation motifs and human myelin basic protein held as many as eight such motifs. PMP22 had the fewest myristoylation motifs, which was only one; rat PMP22 contained no such motifs. Cholesterol recognition/interaction amino‐acid consensus (CRAC) motif was not found in myelin basic protein. P2 protein of different species contained only one CRAC motif, except for P2 of horse, which had no such motifs. MAG, MOG, and P0 were very rich in CRAC, three to eight motifs per protein. The analysis of motifs in myelin proteins is expected to provide structural insight and refinement of predicted 3D models for which structures are as yet unknown. Analysis of motifs in mutant proteins associated with neurological diseases uncovered that some motifs disappeared in P0 with mutation found in neurological diseases. There are 2,500 motifs deposited in a databank, but 21 were found in myelin proteins, which is only 1% of the total known motifs. There was great variability in the number of motifs among proteins from different species. The appearance or disappearance of protein motifs after gaining point mutation in the protein related to neurological diseases was very interesting.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.)

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

Seasonal differences in the testicular transcriptome profile of free-living European beavers (Castor fiber L.) determined by the RNA-Seq method.

The European beaver (Castor fiber L.) is an important free-living rodent that inhabits Eurasian temperate forests. Beavers are often referred to as ecosystem engineers because they create or change existing habitats, enhance biodiversity and prepare the environment for diverse plant and animal species. Beavers are protected in most European Union countries, but their genomic background remains unknown. In this study, gene expression patterns in beaver testes and the variations in genetic expression in breeding and non-breeding seasons were determined by high-throughput transcriptome sequencing. Paired-end sequencing in the Illumina HiSeq 2000 sequencer produced a total of 373.06 million of high-quality reads. De novo assembly of contigs yielded 130,741 unigenes with an average length of 1,369.3 nt, N50 value of 1,734, and average GC content of 46.51%. A comprehensive analysis of the testicular transcriptome revealed more than 26,000 highly expressed unigenes which exhibited the highest homology with Rattus norvegicus and Ictidomys tridecemlineatus genomes. More than 8,000 highly expressed genes were found to be involved in fundamental biological processes, cellular components or molecular pathways. The study also revealed 42 genes whose regulation differed between breeding and non-breeding seasons. During the non-breeding period, the expression of 37 genes was up-regulated, and the expression of 5 genes was down-regulated relative to the breeding season. The identified genes encode molecules which are involved in signaling transduction, DNA repair, stress responses, inflammatory processes, metabolism and steroidogenesis. Our results pave the way for further research into season-dependent variations in beaver testes.

The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species

Colobanthus apetalus is a member of the genus Colobanthus, one of the 86 genera of the large family Caryophyllaceae which groups annual and perennial herbs (rarely shrubs) that are widely distributed around the globe, mainly in the Holarctic. The genus Colobanthus consists of 25 species, including Colobanthus quitensis, an extremophile plant native to the maritime Antarctic. Complete chloroplast (cp) genomes are useful for phylogenetic studies and species identification. In this study, next-generation sequencing (NGS) was used to identify the cp genome of C. apetalus. The complete cp genome of C. apetalus has the length of 151,228 bp, 36.65% GC content, and a quadripartite structure with a large single copy (LSC) of 83,380 bp and a small single copy (SSC) of 17,206 bp separated by inverted repeats (IRs) of 25,321 bp. The cp genome contains 131 genes, including 112 unique genes and 19 genes which are duplicated in the IRs. The group of 112 unique genes features 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs). A total of 12 forward repeats, 10 palindromic repeats, five reverse repeats and three complementary repeats were detected. In addition, a simple sequence repeat (SSR) analysis revealed 41 (mono-, di-, tri-, tetra-, penta- and hexanucleotide) SSRs, most of which were AT-rich. A detailed comparison of C. apetalus and C. quitensis cp genomes revealed identical gene content and order. A phylogenetic tree was built based on the sequences of 76 protein-coding genes that are shared by the eleven sequenced representatives of Caryophyllaceae and C. apetalus, and it revealed that C. apetalus and C. quitensis form a clade that is closely related to Silene species and Agrostemma githago. Moreover, the genus Silene appeared as a polymorphic taxon. The results of this study expand our knowledge about the evolution and molecular biology of Caryophyllaceae.

Preliminary RNA-Seq Analysis of Long Non-Coding RNAs Expressed in Human Term Placenta

Development of particular structures and proper functioning of the placenta are under the influence of sophisticated pathways, controlled by the expression of substantial genes that are additionally regulated by long non-coding RNAs (lncRNAs). To date, the expression profile of lncRNA in human term placenta has not been fully established. This study was conducted to characterize the lncRNA expression profile in human term placenta and to verify whether there are differences in the transcriptomic profile between the sex of the fetus and pregnancy multiplicity. RNA-Seq data were used to profile, quantify, and classify lncRNAs in human term placenta. The applied methodology enabled detection of the expression of 4463 isoforms from 2899 annotated lncRNA loci, plus 990 putative lncRNA transcripts from 607 intergenic regions. Those placentally expressed lncRNAs displayed features such as shorter transcript length, longer exon length, fewer exons, and lower expression levels compared to messenger RNAs (mRNAs). Among all placental transcripts, 175,268 were classified as mRNAs and 15,819 as lncRNAs, and 56,727 variants were discovered within unannotated regions. Five differentially expressed lncRNAs (HAND2-AS1, XIST, RP1-97J1.2, AC010084.1, TTTY15) were identified by a sex-bias comparison. Splicing events were detected within 37 genes and 4 lncRNA loci. Functional analysis of cis-related potential targets for lncRNAs identified 2021 enriched genes. It is presumed that the obtained data will expand the current knowledge of lncRNAs in placenta and human non-coding catalogs, making them more contemporary and specific.

Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and /or different activation pathways.

The complete mitogenome of Colletotrichum lupini var. setosum

Abstract The structure of Colletotrichum lupini mitogenome is typical of a fungus from the genus Colletotrichum similar to C. acutatum and C. lindemuthianum. The sequenced mitogenome has a total length of 36 554 bp. The nucleotide composition in the following genome is: 35.7% – A, 16.5% – C, 13.4% – G and 29.9% – T. In the C. lupini mitogenome we identified 46 genes: 15 protein coding genes, two ribosomal RNAs and 29 tRNA genes.