Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

Identification of Normal and Abnormal Human Megakaryocytes Based on Acid Fast Metachromasia after Staining with Basic Black MSP

Megakaryocytes from normal persons and from patients with immune thrombocytopenic purpura, myelodysplastic disorders, Hypersplenism, and essential thrombocythemia displayed vivid magenta metachromatic staining of the cytoplasm when stained with basic black MSP followed by brief exposure to dilute hydrochloric acid. Under the same conditions, other hematopoietic cells were completely decolorized. Acid fast metachromasia of megakaryocytes facilitates their identification, particularly in cases of small and atypical megakaryocytes found in disease states.     

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

In a previous report [Yanget al., (1987a),J. Biol Chem. 262, 7034–7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C₁₈-reversed phase highper-formance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser¹¹⁵, one of thein vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio 0.9) and TT(p)HYGSLPQK (molar ratio 0.8) as the major phosphorylation site sequences in³²P-MBP phosphorylated by autokinase, further indicating that -Arg-XSer/Thr-(neutral amino acid)₃-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)₂-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/ multifunctional protein kinase in the brain.     

Poly(ADP-ribose) polymerase: Structural conservation among different classes of animals and its implications

Poly(ADP-ribose) polymerase cDNAs have been isolated from different classes of animals. Cloning of genes from lower eukaryotes has allowed us to investigate directly the biological functions of poly(ADP-ribosyl)ationin vivo. The conservation of specific regions among mammals, chicken,Xenopus laevis, andDrosophila melanogaster reveals the essential structural elements required for recognition of breaks in DNA and for catalytic activity. Cys, His and basic residues in the zinc-finger consensus region are conserved. The carboxyl terminal region corresponding to an NAD-binding domain is strongly conserved. The dinucleotide-binding consensus sequence and 1-A-2, Rossmann fold structure, and -sheet structures are completely conserved from mammals to insect. InDrosophila, a putative leucine-zipper motif has been identified, and other poly(ADP-ribose) polymerases also contain an -helical, amphipathic structure in the auto-modification domain. In this article, we review the recent structural analyses of the functional domains of poly(ADP-ribose) polymerase in phylogenetically divergent species, and discuss the implications of structural conservation for its biological functions.Abbreviations aa amino acid(s) - D. melanogaster Drosophila melanogaster - PARP poly(ADP-ribose) polymerase [EC 2.4.2.30] - PCR polymerase chain reaction - X. laevis Xenopus laevis     

Protein Kinase FA/Glycogen Synthase Kinase-3 Predominantly Phosphorylates the In Vivo Site Thr97-Pro in Brain Myelin Basic Protein: Evidence for Thr-Pro and Ser-Arg-X-X-Ser as Consensus Sequence Motifs

Abstract: In a previous study, protein kinase F_A/glycogen synthase kinase-3 ( F_A/GSK-3 ) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F_A/GSk-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F_A/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr⁹⁷-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase F_A/GSK-3, implicating a physiologically relevant role of F_A/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT⁹⁴(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [³²P]MBP phosphorylated by kinase F_A/GSK-3, further indicating that kinase F_A/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.     

Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain

Abstract: A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca²⁺, a temperature optimum at ～50°C, and two K_mvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher K_mhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]₂₀₀. At 5°C, the [θ]₂₀₀of the deiminated protein was -70 × 10³ compared with -30 × 10³ deg cm²/dmol for the native protein. When the temperature was increased to 70°C, the [θ]₂₀₀ was -44 × 10³ for the deiminated protein and -20 × 10⁷ deg cm²/ dmol for the native C-1. When plotted as a function of temperature, [θ]₂₀₀ decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.