Abstract Results of chemical, physical, and enzymatic analyses have established that the product of a cell envelope-associated polyglutamyl synthetase, isolated from an encapsulated strain of Bacillus licheniformis ATCC 9945A, is poly(γ-d-glutamic acid). Proof that the optical configuration of the glutamyl residues in the enzymatically synthesized polymer and the native capsular polymer is d, has been provided by (a) chromatographic separation of diastereoisomeric dipeptides obtained by derivatization with l-leucine-N-carboxyanhydride, and (b) gas-liquid chromatographic separation of N-trifluoroacetyl-l-prolylglutamyl dipeptide methyl esters. The amide linkage has been shown to be γ by (a) kinetics of mild acid catalyzed hydrolysis and (b) LiBH4 reduction of the methyl ester of the polymer followed by acid hydrolysis which yielded γ-amino-δ-hydroxyvaleric acid. Identical treatment of an authentic poly(α-d-glutamyl) polymer yielded α-amino-δ-hydroxyvaleric acid. Neither the enzymatically synthesized polymers nor the native capsular polymers synthesized in vivo contained any ester, anhydride, or lactone linkages. Detailed ultracentrifugal studies have established that the biosynthetic polymers are polydisperse with weight-average molecular weight distribution values ranging from 1.72 x 105 to 3.63 x 105. Similar studies carried out on native γ-d-glutamyl capsular polymers synthesized in vivo showed a much greater degree of polydispersity with apparent weight-average molecular weight distribution values ranging from 8.4 x 104 to 1.15 x 106. The biosynthetic polymers are susceptible to enzymatic hydrolysis by a poly(γ-d-glutamyl) depolymerase. This enzyme, which readily hydrolyzed the native capsular polymer, showed no activity against authentic α-l-glutamyl polymers. These data provide additional evidence that the product of the membrane-mediated reaction is structurally identical with the native capsular γ-d-glutamyl polymers synthesized in vivo.
Epstein-Barr virus (EBV)-genome-negative human lymphoma lines, Ramos and BJAB, can be converted by EBV in vitro into EBV-genome-positive virus nonproducer lines. These cell lines have been used to identify surface antigens unique to EBV, with the expectation that such determinants might be related to the antigenic target responsible for EBV-specific immunosurveillance. Antisera prepared in rabbits immunized with either whole cells or purified plasma membranes were used in immunoblot experiments to analyze antigenic differences resulting from expression of the resident EBV genome. Unexpectedly, an increase in polypeptides of 32 and 70 kilodaltons was consistently observed in the EBV-converted Ramos lines. In contrast, these antigens were not expressed in BJAB or in its EBV-converted lines. These data suggested that p32 and gp70 might be murine leukemia virus (MuLV)-coded antigens because Ramos, but not BJAB, had been passaged in athymic nude mice during establishment of this cell line. This conclusion was confirmed by using antisera specific for MuLV p30 and gp70. Retroviral antigens were expressed constitutively at low levels in Ramos. Quantitative immunoblotting showed that EBV conversion of Ramos amplified the expression of MuLV proteins 3- to 5-fold. The molecular mechanism responsible for the enhanced expression is unknown. The biological relevance of this phenomenon is also not clear, but the interaction between a DNA and a RNA tumor virus in a Burkitt lymphoma line that carries both viruses may have important biological consequences in relation to retrovirus latency and tumor induction. These results also show that caution must be used when ascribing "uniqueness" to EBV-determined antigens, particularly in the Ramos lines. This warning extends also to the use of Ramos cell lines as immunogens, because immunization of rabbits elicited antibodies that recognized proteins of the same size as the retroviral antigens.
A sensitive target binding assay has recently been shown to detect natural killer (NK) cells in the mouse. Preincubation of NK cells with detergent-solubilized cell-surface proteins of YAC lymphoma cells prevented subsequent binding to intact YAC targets. The NK target structures (NK-TS) consisted of three molecular species tentatively assigned molecular weights of 130,000, 160,000, and 240,000 based on electrophoretic mobility in sodium dodecyl sulfate/polyacrylamide gels. Moloney cell surface antigen (MCSA), gp71, p30, H-2, and NK-TS were localized in distinct fractions of gels. The NK-TS bound to concanavalin A-Sepharose columns and could be eluted with the specific sugar, suggesting that the target structures may be glycosylated. NK-TS molecules could not be detected in gels of NK-insensitive target cells such as P815, A9HT, YWA, or EL-4. The quantity obtained from the gels varied directly with the NK sensitivity of YAC which is more sensitive when grown in vitro than when grown in vivo . The NK-TS molecules specifically inhibited the binding of NK cells but not alloimmune T cells to their appropriate targets. Additional NK-sensitive tumor cells also expressed some or all of the target molecules exhibited by YAC. Some of these structures shared specificities in the case of MPC-11 or were unique in the case of Molt-4 and K562, as shown by cross-inhibition studies. These results suggest that NK-sensitive cell lines express distinct target structures with possible relevance to natural tumor resistance.
The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule. The capsule is a neurovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules. While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E. coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl acceptors. A structural feature common to the preferred sialyl acceptors (GD3 > GT1a > GQ1b = GT1b > GD2 = GD1b = GD1a > GM1) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasaccharide with a terminal Sia residue (e.g., GD3) was the minimum length oligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sia residues were transferred to the disialyllactose moiety of GD3. The significance of these findings is two-fold. (i) The E. coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus creating oligo- or polysialylated GSLs. Such "designer" GSLs may have potentially important biological and pharmacological properties. (ii) The use of GSLs as exogenous sialyl acceptors increases the sensitivity of detecting polyST activity. The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTParamagnetic isoprenoid carrier lipids. 1. Chemical synthesis and incorporation into model membranesMichael A. McCloskey and Frederic A. TroyCite this: Biochemistry 1980, 19, 10, 2056–2060Publication Date (Print):May 13, 1980Publication History Published online1 May 2002Published inissue 13 May 1980https://pubs.acs.org/doi/10.1021/bi00551a008https://doi.org/10.1021/bi00551a008research-articleACS PublicationsRequest reuse permissionsArticle Views110Altmetric-Citations31LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.
Abstract Treatment of 14C-uniformly labeled cell walls of Penicillium chrysogenum with chitobiase-containing chitinase resulted in 73% solubilization. The solubilized components were isolated, identified, and determined quantitatively. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose ((GlcNAc)2) accounted for 42% of the cell wall. Since kinetic analysis showed that GlcNAc arose from (GlcNAc)2, at least 42% of the cell wall contains GlcNAc in β-d-(1 → 4)-glycosidic linkage. When the same experiment was performed with a chitobiase-free chitinase preparation nearly identical results were obtained, except, as expected, essentially all of the GlcNAc was present as (GlcNAc)2. Small amounts of glucose and laminaritriose were also released. In addition, a nondialyzable component, Fraction 5A, accounting for 14% of the wall, was isolated. This fraction, devoid of GlcNAc, contains 20 times the amount of galactose and 2 times the amount of mannose with respect to glucose found in cell wall hydrolysates. An additional 13% of the wall was solubilized when the cell wall residue, following treatment with chitinase, was treated with chitinase-free β-d-(1 → 3)-glucanase. Therefore, 86% of the wall was solubilized when treated first with chitinase, then with β-d-(1 → 3)-glucanase. Initial treatment of cell walls with β-d-(1 → 3)-glucanase resulted in 48% solubilization. From an analysis of the solubilized components the conclusion was reached that at least 40% of the wall contains glucose in β-d-(1 → 3)-glycosidic linkage. An additional 44% of the wall was solubilized when the residue, following treatment with β-d-(1 → 3)-glucanase, was treated with chitinase. Thus, 92% of the wall was solubilized when treated first with β-d-(1 → 3)-glucanase, then with chitinase. The observations that β-d-(1 → 3)-glucanase-free chitinase released in addition to GlcNAc and (GlcNAc)2 also glucose, laminaritriose, and a fraction containing mannose, galactose, and glucose (Fraction 5A) and that treatment of this fraction with β-d-(1 → 3)-glucanase released glucose and laminaribiose suggest that glucose may exist in cell walls in one or more forms other than the major glucan. From the available data it is not clear whether Fraction 5A contains one or several polymers. An analysis of the monomeric cell wall constituents released by acid hydrolysis is in excellent agreement with the results obtained by enzymatic digestion. These results showed the cell wall to contain about 48% glucosamine and about 52% neutral hexoses, which consisted of glucose, galactose, and mannose in the molar proportion of 10:1:2. Thus, if 40% of the wall is glucose, then galactose and mannose account for 4% and 8% of the wall, respectively.
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