Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl++ +) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid

The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.     

Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular Slime Mold, Polysphondylium pallidum

The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistanttypes of cell-cell adhesion. The major component of them hasbeen identified as a glycoprotein with a molecular mass of 64kDa on SDS-PAGE (referred to as gp64). We found that a substantialamount of the gp64 run as dimer, when gp64 was dissolved inSDS-sample buffer without 2-mercaptoethanol and then subjectedto electrophoresis. The occurrence of a homophilic dimer wasdemonstrated by analyzing the dimer-like band on a gel for itsamino acid sequence and amino acid composition. The dimer-likeband also was analyzed by three sorts of monoclonal antibodies,two of which recognize respectively a conforniational epitopeand a denatured epitope of the protein moiety of gp64. The dataindicate that the native conformation of gp64 is necessary fordimer formation. ²Present address: Institute of Immunological Science, HokkaidoUniversity, Sapporo, 060 Japan     

Novel POMGnT1 mutations cause muscle-eye-brain disease in Chinese patients

Muscle-eye-brain (MEB) disease is a congenital muscular dystrophy (CMD) phenotype characterized by hypotonia at birth, brain structural abnormalities and ocular malformations. To date, few MEB cases have been reported in China where clinical recognition and genetic confirmatory testing on a research basis are recent developments. Here, we report the clinical and molecular genetics of three MEB disease patients. The patients had different degrees of muscle, eye and brain symptoms, ranging from congenital hypotonia, early-onset severe myopia and mental retardation to mild weakness, independent walking and language problems. This confirmed the expanding phenotypic spectrum of MEB disease with varying degrees of hypotonia, myopia and cognitive impairment. Brain magnetic resonance imaging showed cerebellar cysts, hypoplasia and characteristic brainstem flattening and kinking. Four candidate genes (POMGnT1, FKRP, FKTN and POMT2) were screened, and six POMGnT1 mutations (four novel) were identified, including five missense and one splice site mutation. Pathogenicity of the two novel variants in one patient was confirmed by POMGnT1 enzyme activity assay, protein expression and subcellular localization of mutant POMGnT1 in HeLa cells. Transfected cells harboring this patient’s L440R mutant POMGnT1 showed POMGnT1 mislocalization to both the Golgi apparatus and endoplasmic reticulum. We have provided clinical, histological, enzymatic and genetic evidence of POMGnT1 involvement in three unrelated MEB disease patients in China. The identification of novel POMGnT1 mutations and an expanded phenotypic spectrum contributes to an improved understanding of POMGnT1 structure–function relationships, CMD pathophysiology and genotype–phenotype correlations, while underscoring the need to consider POMGnT1 in Chinese MEB disease patients.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

Element analysis of the Polysphondylium pallidum gp64 promoter

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.     

Contribution of the Distal Pocket Residue to the Acyl-Chain-Length Specificity of (R)-Specific Enoyl-Coenzyme A Hydratases from Pseudomonas spp.

(R)-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid β-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1_Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1_Pa from Pseudomonas aeruginosa, despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1_Pp, the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1_Pa, the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1_Pp and PhaJ1_Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1_Pp than in PhaJ1_Pa. Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1_Pp and PhaJ1_Pa, all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1_Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1_Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity.     

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.