Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay

Profiling of carbohydrate structures on cell membranes has been difficult to perform because of the complexity and the variations of such structures on cell surface glycans. This study presents a novel method for rapid profiling of cell surface glycans for terminal N-acetyllactosamines (Galβ1-(3)4GlcNAc-R) that are uncapped, capped with sialic acid as SA-Galβ1-(3)4GlcNAc-R, or with α1,3galactosyls as the α-gal epitope- Galα1-3Galβ1-(3)4GlcNAc-R. This method includes two enzymatic reactions: (1) Terminal sialic acid is removed by neuraminidase, and (2) α-gal epitopes are synthesized on the exposed N-acetyllactosamines by α1,3galactosyltransferase. Existing and de novo synthesized α-gal epitopes on cells are quantified by a modification of radioimmunoassay designated as “ELISA inhibition assay,” which measures binding of the monoclonal anti-Gal antibody M86 to α-gal epitopes. This binding is proportional to the number of cell surface α-gal epitopes. The amount of free M86 antibody molecules remaining in the solution is determined by ELISA using synthetic α-gal epitopes linked to albumin as solid phase antigen. The number of α-gal epitopes on cells is estimated by comparing binding curves of M86 incubated with the assayed cells, at various concentrations of the cells, with the binding of M86 to rabbit red cells expressing 2 × 10⁶ α-gal epitopes/cell. We could demonstrate large variations in the number of sialic acid capped N-acetyllactosamines, α-gal epitopes and uncapped N-acetyllactosamines on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species. This method may be useful for rapid identification of changes in glycosylation patterns in cells subjected to various treatments, or in various states of differentiation.     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA.     

Coexpression of the Soybean Vegetative Storage Protein β Subunit (S-VSPβ) Either with the Bacterial Feedback-Insensitive Dihydrodipicolinate Synthase or with S-VSPα Stabilizes the S-VSPβ Transgene Protein and Enhances Lysine Production in Transgenic Tobacco Plants

Soybean vegetative storage proteins (S-VSPs) are lysine-rich leaf proteins, originally found to accumulate to high levels in depodded soybean plants. In the present study, we overexpressed S-VSP, the ruminant stable subunit of the S-VSP genes, in transgenic tobacco plants. The S-VSP protein accumulated in all organs studied, but its level declined drastically with leaf age. This instability of S-VSP could be overcome either by elevating free lysine levels or by coexpressing S-VSP with S-VSP. High levels of rumen-stable, lysine-rich proteins is expected to improve absorption of lysine by ruminants. Furthermore, the expression of S-VSPs in heterologous plants led to a significant increase in total soluble lysine, suggesting that these proteins may also permit better assimilation of lysine by humans and monogastric animals.     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.     

Engineering hypervirulence in a mycoherbicidal fungus for efficient weed control

Agents proposed for biocontrol of major weeds in arable row-crop agriculture have not met expectations because an evolutionary balance has developed between microorganism and weed, even when the mycoherbicide is used inundatively at very high levels (>10(4)spores/cm<(2)). Sufficient virulence can be achieved by transferring genes to the microorganism, tipping the evolutionary balance. Virulence was increased ninefold and was more rapidly effected; furthermore, the requirement for a long duration at high humidity was decreased by introducing NEP1 encoding a phytotoxic protein, to an Abutilon theophrasti-specific, weakly mycoherbicidal strain of Colletotrichum coccodes. The parent strain was at best infective on juvenile cotyledons of this intransigent weed. The transgenic strain was lethal through the three-leaf stage, a sufficient time window to control this asynchronously germinating weed. Strategies of coupling virulence genes with fail-safe mechanisms to prevent spread (due to broadened host range) and to mitigate transgene introgression into crop pathogens could be very useful in the biocontrol of major weeds in row crops.     

In vitro induction of cell-mediated immunity to murine leukemia cells

Summary An adoptive chemoimmunotherapeutic model based on the use of chemotherapy and lymphocytes specifically sensitized against tumor cells in vitro was tested in mice transplanted with syngeneic leukemia cells. C57BL/6 and A strain mice were inoculated i.p. or i.v. (day 0) with lethal doses (1×10³–1×10⁵) of EL4 and YAC leukemia cells, respectively. Leukemic mice were subsequently treated (day 1 or day 3) with partially curative doses (80–140 mg/kg) of cyclophosphamide (Cy), followed by i.p. or i.v. administration of 1–3×10⁷ cytotoxic lymphocytes (CL) induced in macro-mixed leukocyte-tumor cell cultures (MLTC). The following results were obtained: untreated mice died with tumor within 20 days; mice receiving sensitized lymphocytes only showed a modest prolongation of survival and only 5–15% of the animals were cured; treatment with Cy alone or with Cy and normal lymphocytes prolonged survival considerably and cured 20–60% of the mice; mice subjected to Cy in conjunction with in vitro-sensitized lymphoid cells, either syngeneic or allogeneic, had survival rates of 80–100% (100 days). Under the conditions employed, no severe manifestations of clinical graft-versus-host (GVH) reaction were observed. These findings imply that in vitro-sensitized immunocytes and cytoreductive drugs can operate cumulatively.     

Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay

The study of the expression of Gal alpha 1----3Gal beta 1----4GlcNAc residues on mammalian glycoconjugates is of particular interest since as many as 1% of circulating IgG antibodies in man (the natural anti-Gal antibody) interact specifically with this carbohydrate residue. In recent studies, we have found that Gal alpha 1----3Gal beta 1----4GlcNAc residues are abundant on red cells and nucleated cells of nonprimate mammals, prosimians, and New World monkeys, but their expression is diminished in Old World monkeys, apes, and humans. In the present work, we have analyzed the expression of these residues on secreted mammalian glycoproteins. For this purpose, we have developed a radioimmunoassay (RIA) which enables the quantification of Gal alpha 1----3Gal beta 1----4GlcNAc residues on the secreted glycoproteins. Purified biotinylated anti-Gal was used as the antibody in the RIA, and bovine thyroglobulin enriched for Gal alpha 1----3Gal beta 1----4GlcNAc residues served as a solid-phase antigen. In this study, it is reported for the first time that the evolutionary pattern of Gal alpha 1----3Gal beta 1----4GlcNAc residue distribution in in vivo secreted glycoproteins is similar to that observed in membranes of cell lines and of red cells. Thyroglobulin, fibrinogen, or IgG molecules from nonprimate mammals and from New World monkeys express varying amounts of Gal alpha 1----3Gal beta 1----4GlcNAc residues ranging between 0.01 and 11 residues per molecule, whereas no such residues are present on any of these glycoproteins of human or Old World monkey origin.(ABSTRACT TRUNCATED AT 250 WORDS)