The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers.

The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

A distinct member of the aspartic proteinase gene family from the human malaria parasite Plasmodium falciparum.

A gene (hap) transcribed during the intra-erythrocytic life cycle stages of the human malaria parasite Plasmodium falciparum was cloned and sequenced. It was found to encode a protein belonging to the aspartic proteinase family but which carried replacements of catalytically crucial residues in the hallmark sequences contributing to the active site of this type of proteinase. Consideration is given as to whether this protein is the first known parasite equivalent of the pregnancy-associated glycoproteins that have been documented in ungulate mammals. Alternatively, it may be operative as a new type of proteinase with a distinct catalytic mechanism. In this event, since no counterpart is known to exist in humans, it affords an attractive potential target against which to develop new anti-malarial drugs.

Sciatic nerve of normal and T200x5R Swiss white mice fails to support multiplication of intraneurally injected M. leprae.

In a preliminary study we have shown that freshly harvested Mycobacterium leprae, when injected into the sciatic nerve in normal and immunosuppressed (TR) mice, induce massive but localized epithelioid and macrophage granuloma, respectively, in 3-4 weeks. In order to determine the fate of M. leprae injected intraneurally into normal and TR mice, in the present study we measured sequentially the viability, fold increase and clearance, if any, using semi-quantitative methods. The average M. leprae yield per nerve assessed at regular intervals, beginning at 24 hr and including 72 hr, 1 week, 2, 3, 4, 12, 24 and 48 weeks, showed neither a significant increase nor a decrease in either the normal or the TR mice. The viability of M. leprae, assessed using the standard mouse foot pad method, showed a significant decrease as compared to baseline growth effective at 24 hr and remained static until approximately 4 weeks. A further decline and total loss of viability was noted by 12 months. The results show that injection of M. leprae via the intraneural route in both normal and TR mice failed to sustain the viability and failed to support the multiplication of the organisms.

Cloning, expression and characterization of the proteinase from human herpesvirus 6.

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.

Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B.

COUP-TFs (Chicken Ovalbumin Upstream Promoter Transcription Factors) have been proposed to be negative regulators of retinoid receptor-mediated transcriptional activation. In a previous paper we reported the cloning of a Xenopus (x) COUP-TF (Matharu, P.J. and Sweeney, G.E. (1992) Biochim. Biophys. Acta 1129, 331-334). Here we describe the cloning of a second xCOUP-TF. Amino acid sequence comparison between these two Xenopus COUP-TFs revealed a high level of similarity. Extensive amino acid sequence conservation was found among all Drosophila, Xenopus, zebrafish and mammalian COUP-TF genes examined. Phylogenetic tree analyses indicate that the vertebrate COUP-TFs fall into three classes. The two Xenopus COUP-TF genes show similar temporal expression patterns: both are expressed from the end of gastrulation. In situ hybridization studies reveal complex expression patterns in the developing central nervous system (CNS), besides expression in the eye and in some mesodermal tissues. Retinoic acid (RA) treatment enhances xCOUP-TF-A expression in neurula stage embryos, whereas the expression of xCOUP-TF-B is inhibited during the same developmental period. The strictly conserved amino acid sequences and the strong similarities between the expression patterns of the two different xCOUP-TFs on the one hand, and other vertebrate COUP-TF homologues on the other, make it likely that COUP-TFs have a conserved role in patterning the nervous system.

Cloning and sequencing of a Xenopus homologue of the inducible orphan receptor NGFI-B.

A cDNA clone encoding the NGFI-B transcription factor, a growth factor inducible member of the steroid/thyroid receptor superfamily, has been isolated from a Xenopus neurula (stage 17 embryo) library. Sequencing of this clone reveals an open reading frame encoding a 577 amino acid protein. Comparisons with its counterparts in rat, mouse and human show that the Xenopus protein has a well conserved DNA binding domain whereas homology in the carboxy terminal region, which includes the putative ligand binding domain, is lower than that typically observed in members of the steroid/thyroid receptor superfamily. This relative lack of homology suggests that, in Xenopus, the as-yet uncharacterized ligand may have subtle distinctions from its mammalian counterparts.

Cloning and sequencing of a COUP transcription factor gene expressed in Xenopus embryos.

A cDNA clone encoding COUP transcription factor, a member of the steroid/thyroid receptor superfamily, has been isolated from a Xenopus neurula (stage 17 embryo) library. Sequencing of this clone reveals an open reading frame encoding a 397 amino acid protein. The amino acid sequence of Xenopus COUP has been compared with its human and Drosophila homologues showing that there are few similarities within the amino-terminal region, whereas the remainder of the protein, including the putative DNA and ligand binding domains, is very well conserved.