Recent progress in the molecular biology of the clonedN-acetylglucosaminyltransferases

Several genes which code for theN-acetylglucosaminyltransferases have been cloned and characterized. Physiological and pathophysiological roles of the genes still remain to be elucidated but accumulated evidence suggests that theN-acetylglucosaminyltransferase genes are implicated in differentiation, morphogenesis and cancer metastasis.     

红花石蒜苯丙氨酸解氨酶催化合成N-甲基-L-苯丙氨酸

[背景] N-甲基-L-苯丙氨酸是一种N-烷基化芳香氨基酸,是重要的手性合成单元/中间体/组成成分,在医药、农业、食品等领域有重要应用价值的代谢产物中广泛存在。N-烷基化芳香氨基酸的合成与制备仍具有巨大的挑战。[目的] 在研究加兰他敏的生物合成过程中,我们从产加兰他敏的红花石蒜中克隆并表征苯丙氨酸解氨酶LrPAL3。LrPAL3催化区域及对映选择性的氢胺化反应得到L-苯丙氨酸。通过生物信息学分析,推测LrPAL3可能催化反式-肉桂酸的一步N-甲基胺化反应得到N-甲基-L-苯丙氨酸。[方法] 将反式-肉桂酸与甲胺,以及表达LrPAL3的大肠杆菌全细胞一起孵育。HPLC-DAD及HRESIMS分析表明,上述反应产物为N-甲基-苯丙氨酸。为确定该产物的立体构型,将上述催化反应放大,通过分离纯化得到该酶催化反应产物。[结果] 该化合物的氢谱数据及比旋光数据与N-甲基-L-苯丙氨酸标准品的数据一致。由此说明,LrPAL3能够催化反式-肉桂酸和甲胺发生N-烷基胺化反应,区域和立体专一性地生成N-甲基-L-苯丙氨酸。[结论] 本研究为手性N-烷基氨基酸的不对称合成提供了一种全新的绿色、高效生物催化剂。通过对LrPAL3的蛋白质定向进化及代谢工程,将会进一步扩展LrPAL3的催化反应范围,以多种N-烷基胺类及取代的苯基丙烯酸为底物,实现手性N-烷基-芳基氨基酸的高效区域及立体选择性生物合成。     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Effect of N-linked glycosylation on glycopeptide and glycoprotein structure

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

The three-subunit cytochromebc 1 complex ofParacoccus denitrificans. Its physiological function,structure, and mechanism of electron transfer and energy transduction

The cytochromebc ₁ complex purified fromP. denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromesb, c ₁, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria. However, the bacterialbc ₁ complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary poly-peptides, which have no obvious electron-transfer function. TheP. denitrificans complex is a paradigm for thebc ₁ complexes of all gram-negative bacteria. In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, theP. denitrificans bc ₁ complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer.     

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian β1,4-galactosyltransferase

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine -1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.     

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related specie*

Summary The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 by region immediately upstream of the TATA box is highly conserved in theZea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5 region, which is common to the four genes and is probably essential for their expression. The 3 end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.The sequences reported in this paper have been deposited in the EMBL/GenBank Database (Bolt, Beranek, and Newman Laboratories, Cambridge, Mass., and EMBL, Heidelberg), accession nos. M36635 (maize Ac1503), X63134 (maize W22), X64173 (teosinte) and X56010 (sorghum)     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

The hypothalamus of Lacerta sicula R.

Summary The posterior (caudal) hypothalamus of the lizard, Lacerta sicula R. was investigated by means of Golgi methods. The periventricular grey is formed mainly by isodendritic bipolar and multipolar neurons, while in the lateral hypothalamus a more stellate form of neuronal elements is encountered. CSF-contacting neurons are restricted to the tuberal area and to the paraventricular organ. In the latter area they are highly differentiated and endowed with laterally branched processes. The overall pattern of the lizard hypothalamus (organization of neuropil, lateral nuclei, appearance of cell clusters, morphology of the neuronal elements) represents an intermediate stage in the phylogenetic development of the hypothalamus, being more advanced than the amphibian stage.     

The hypothalamus of Lacerta sicula R.

Summary An analysis of the preoptic area of the lizard, Lacerta sicula R., with the use of the Golgi method revealed that: 1)in principle, the dendritic pattern of its neurons is relatively simple; 2) the supraoptic nucleus contains large- to medium-sized bipolar or multipolar neurons together with small, usually multipolar nerve cells; 3) the preoptic periventricular gray and the paraventricular nucleus exhibit a varied neuronal typology, including large multipolar or bipolar elements, abundant CSF-contacting neurons, and some tufted elements; and 4) the lateral regions display some conspicuous multipolar neurons.With a financial contribution from Ministero della Pubblica Istruzione     

The occurrence of non-ring producing branches in Abies lasiocarpa

Lack of annual growth ring production at the base of branches in the lower portion of the crown is a commonly observed phenomenon. In this study, branches with missing rings were found on 47 of 49 Abies lasiocarpa trees sampled. The number of missing rings on the lowest live branch in the crown averaged 12, and ranged as high as 28. The lower one-third of the live crown tended to consist of branches without rings; these branches contained an average of 30% of the total crown leaf area. The ratio of foliage weight to total branch weight, in combination with position of the branch in the canopy, was an effective discriminator of non-ring producing branches. This suggests that both structural and environmental factors influence the cessation of ring production. The potential ecological implications of branches that fail to produce rings are discussed.     

The development and ultrastructure of gum ducts inCitrus plants formed as a result of brown-rot gummosis

Summary Young stems ofCitrus plants were infected with the fungusPhytophthora citrophthora. The effect of the infection on gum duct development was studied. The following sequence of structural changes was observed in the cambial zone: 1. The middle lamellae between layers of xylem mother cells dissolve forming duct cavities. 2. The cells around the duct cavities differentiate into epithelial cells rich in cytoplasm. 3. The amount of Golgi bodies and associated vesicles increases. The vesicles and small vacuoles, some of which seem to originate from the fusion of Golgi vesicles, contain fibrillar material that stains for polysaccharides. Vesicles and vacuoles appear to fuse with the plasmalemma. Material staining positively for polysaccharides accumulates between the plasmalemma and cell wall, and penetrates the latter. 4. The protoplast shrinks and the space below the cell wall, which contains polysaccharides, increases in volume. 5. After a period of 10 days or more the gum ducts become embedded in the xylem, and the activity of the epithelial cells ceases. The cell walls of many of them break, and the gum still present in the cells is released.     

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5′-diphospho-N-[²H]-acetyl-glucosamine and the chemical preparation of N-[²H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.     

Enzymic glycosphingolipid synthesis on polymer supports.

Two synthetic approaches leading toN-4-carboxymethyl-2-nitrobenzyloxycarbonyl derivatives of model compounds and of glycosylsphingosines are described. The carboxymethyl group can be converted into a hydrazido derivative and the resulting products react with an active ester polymer yielding light-sensitive polymeric products that may subsequently serve as acceptors in glycosyltransferase reactions.Author for correspondence. Address until September 1990; Graduate Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02254, USA     

The nematode Caenorhabditis elegans as a model to study the roles of proteoglycans

The nematode Caenorhabditis elegans is a powerful animal model for exploring the genetic basis of metazoan development. Recent genetic and biochemical studies have revealed that the molecular machinery of glycosaminoglycan (GAG) biosynthesis and modification is highly conserved between C. elegans and mammals. In addition, genetic studies have implicated GAGs in vulval morphogenesis and zygotic cytokinesis. The extensive knowledge of C. elegans biology, including its elucidated cell lineage, together with the completed and well annotated DNA sequence and availability of reverse genetic tools, provide a platform for studying the functions of proteoglycans and their GAG modification. Published in 2003.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

N-acetylaspartate in the vertebrate brain: metabolism and function

N-Acetyl-l-aspartate (NAA) is an amino acid that is present in the vertebrate brain. Its concentration is one of the highest of all free amino acids and, although NAA is synthesized and stored primarily in neurons, it cannot be hydrolyzed in these cells. Furthermore, neuronal NAA is dynamic and turns over more than once each day by virtue of its continuous efflux, in a regulated intercompartmental cycling via extracellular fluids, between neurons and a second compartment in oligodendrocytes. The metabolism of NAA, between its anabolic compartment in neurons and its catabolic compartment in oligodendrocytes, and its possible physiological role in the brain has been the subject of much speculation. There are two human inborn errors in metabolism of NAA. One is Canavan disease (CD), in which there is a buildup of NAA (hyperacetylaspartia) and associated spongiform leukodystrophy, caused by a lack of aspartoacylase activity. The other is a singular human case of lack of NAA (hypoacetylaspartia), where the enzyme that synthesizes NAA is apparently absent. There are two animal models currently available for studies of CD. One is a rat with a natural deletion of the catabolic enzyme, and the other a gene knockout mouse. In addition to the presence of NAA in neurons, its prominence in ¹H nuclear magnetic resonance spectroscopic studies has led to its wide use in diagnostic human medicine as both an indicator of brain pathology and of disease progression in a variety of CNS diseases. In this review, various hypotheses regarding the metabolism of NAA and its possible role in the CNS are evaluated. Based on this analysis, it is concluded that although NAA may have several functions in the CNS, an important role of the NAA intercompartmental system is osmoregulatory, and in this role it may be the primary mechanism for the removal of intracellular water, against a water gradient, from myelinated neurons.