Heating and storage of structured acylglycerols with succinyl-linked stigmasterol residue does not cause negative chemical or biological changes.

Four structured acylglycerols with stigmasterol bonded by a succinyl linker were investigated and their stability were analyzed. Samples were heated to 60 °C and kept at that temperature to simulate storage, and to 180 °C to simulate frying conditions. The degradation of the synthesized compounds and formed derivatives was determined, and their cytotoxicity and genotoxicity on normal human cells from the digestive system was determined. Holding at 180 °C resulted in greater degradation of the compounds than holding at 60 °C. The most stable compound in each sample proved to be one with oleic acid in its structure-1,3-dioleoyl-2-stigmasterylsuccinoyl-sn-glycerol (DO2SSt) at 60 °C and 1,2-dioleoyl-3-stigmasterylsuccinoyl-sn-glycerol (DO3SSt) at 180 °C. These results indicate that the type of fatty acid in the molecule is more important than its position in the glycerol structure. None of the diacylmonostigmasterylsuccinoyl-sn-glycerols (DASStGs) before or after heating exhibited cytotoxic or genotoxic potential to small intestine and colon mucosa cells.

Thermo-oxidative stability and safety of new acylglycerols with stigmasterol residue: Effects of fatty acids saturation and position in the glycerol backbone.

The safety and thermoxidative stability of new diacyl-stigmasterylcarbonoyl-sn-glycerols (DAStGs) with two molecules of palmitic or oleic acids and one molecule of stigmasterol at the sn-2 or sn-3 position were studied. After heating to 60 °C, the compounds with stigmasterol at the sn-2 position were more stable than those with stigmasterol at the sn-3 position. The lowest level of degradation of stigmasterol after heating to 180 °C was detected for both compounds with oleic acid, followed by the samples with palmitic acid. The high content of SOPs, especially triolSt, as well as the high level of dimers showed the most effect on the cytotoxicity of DAStGs heated at both temperatures. DAStGs with oleic acid at sn-1,3 and stigmasterol at sn-2 position were the most stable compounds. Both oleic acid and the location of stigmasterol in the middle of the glycerol molecule play an important role in increasing the thermoxidative stability of stigmasterol.

Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols.

The study investigated the thermo-oxidative stability of distigmasterol-modified acylglycerols as a new structured acylglycerols. Samples were heated at 60 and 180 °C for 8 h. Their percentage degradation and products formed during heating were compared with free stigmasterol and stigmasteryl esters. The remaining of stigmasterol and fatty acid parts, the formation of stigmasterol oxidation products and the composition of polar and non-polar fractions were analysed using chromatographic methods. The cytotoxicity and genotoxicity were determined with the use of an MTT test and a comet assay, respectively. The highest stability during heating was observed for 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) and the lowest for 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA). Data showed that the formation of thermo-oxidative degradation products is affected by the temperature and chemical structure of lipids present in the molecule. The dStigMAs bonded by a succinate linker and products formed during their thermo-oxidation showed no cytotoxic or genotoxic activity to normal human cells.

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.

Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end.

Recent studies on the mechanism of degradation of newly synthesized glycoproteins suggest the involvement of a retrotranslocation of the glycoprotein from the lumen of the rough endoplasmic reticulum into the cytosol, where a deglycosylation process takes place. In the studies reported here, we used a glycosylation mutant of Chinese hamster ovary cells that does not synthesize mannosylphosphoryldolichol and has an increased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein synthesis, we observed an accumulation of glucosylated oligosaccharide-lipid donors (Glc3Man5GlcNAc2-PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough endoplasmic reticulum glucosidase(s) by castanospermine led to the formation of Glc3Man5GlcNAc2(OSGn2) (in which OSGn2 is an oligomannoside possessing two GlcNAc residues at its reducing end), which was then retained in the lumen of intracellular vesicles. Thus they were protected during an 8 h chase period from the action of cytosolic chitobiase, which is responsible for the conversion of OSGn2 to oligomannosides possessing one GlcNAc residue at the reducing end (OSGn1). In contrast, when protein synthesis was maintained in the presence of castanospermine, glucosylated oligomannosides (Glc1-3Man5GlcNAc1) were recovered in cytosol. Except for monoglucosylated Man5 species, which are potential substrates for luminal calnexin and calreticulin, the pattern of oligomannosides was similar to that observed on glycoproteins. The occurrence in the cytosol of glucosylated species with one GlcNAc residue at the reducing end implies that the deglycosylation process that generates glucosylated OSGn1 from glycoproteins occurs in the cytosol.

'Glyco-deglyco' processes during the synthesis of N-glycoproteins.

For the past 15 years, it has appeared increasingly evident that the N-glycosylation process was accompanied by the release of oligomannoside type oligosaccharides. This material is constituted of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn1) or two GlcNAc (OS-Gn2) at the reducing end. It has been demonstrated that oligosaccharide-phosphates originated from the cleavage by a specific pyrophosphatase, of non-glycosylated cytosolic faced oligosaccharide-PP-Dol and chiefly the Man5GlcNAc2-PP-Dol. The Man5GlcNAc2-P, as the main product, is recovered in the cytosolic compartment and is further degraded to Man5GlcNAc1 by as for yet not depicted enzymes. In contrast, OS-Gn2 produced from hydrolysis of oligosaccharide-PP-Dol (presumably as a transfer reaction onto water) when the amount of protein acceptor is limiting, are generated into the lumen of rough endoplasmic reticulum (ER). They are further submitted to processing alpha-glucosidases and rough ER mannosidase and are (mainly as Man8GlcNAc2) exported into the cytosolic compartment. This material is further degraded into a single component, the Man5GlcNAc1: Man alpha 1-2Man alpha 1-2Man alpha 1-3 (Man alpha 1-6)Man beta 1-4GlcNAc by the sequential action of a cytosolic neutral chitobiase followed by cytosolic mannosidase. Furthermore, OS-Gn1 could have a dual origin: on the one hand, they originate from OS-Gn2 by the cytosolic degradation pathway indicated above; on the other hand, we will discuss a possible origin from the degradation or remodeling of newly synthesized glycoproteins. Considered first as a minor phenomenon, these observations have lead to the concept of intracellular oligomannoside trafficking, a process which results from more fundamental phenomena such as the control of the dolichol cycle, and the so-called quality-control of glycoprotein. In this review, we would like to describe the evolution of ideas on the origin, intracellular trafficking and putative roles of these oligomannosides released during the N-glycosylation process. We propose that these early stage 'glyco-deglyco' processes represent a way of control of N-glycosylation and of the fate of N-glycoproteins.

Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase.

We have previously reported the substrate specificity of the cytosolic alpha-D-mannosidase purified from rat liver using Man9GlcNAc, i.e. Man alpha 1-2Man alpha 1-3(Man alpha 1-2Man alpha 1-6)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3) Man beta 1-4G1cNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99-106]. Man9 G1cNAc is hydrolysed giving Man5GlcNAc, i.e. Man alpha 1-2 Man alpha 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc, possessing the same structure as the oligosaccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here the activity of the purified cytosolic alpha-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the biosynthesis of N-glycans, and also towards soluble oligosaccharides released from the endoplasmic reticulum which are glucosylated or not and possessing at their reducing end either a single N-acetylglucosamine residue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dolichol pyrophosphate oligosaccharide substrates are poorly hydrolysed by the cytosolic alpha-D-mannosidase; (2) oligosaccharides with a terminal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucosamine are the real substrates for the enzyme. These results suggest a role for alpha-D-mannosidase in the catabolism of glycans released from the endoplasmic reticulum rather than in the regulation of the biosynthesis of asparagine-linked oligosaccharides.

Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: a study with Madin-Darby bovine kidney (MDBK) cells.

Neutral oligomannosides possessing one GlcNAc (OS-Gn1) and two GlcNAc (Os-Gn2) at the reducing end have been reported to be released during the N-glycosylation process in various biological models. To investigate which enzyme is responsible for OS-Gn1 formation, we used the Madin-Darby bovine kidney (MDBK) cell line which exhibits neither lysosomal chitobiase nor endoglucosaminidase activities. However, these cells produced OS-Gn1 and we showed that a neutral chitobiase is responsible for the transformation of OS-Gn2 into OS-Gn1. Using streptolysin O-permeabilized MDBK cells, we demonstrated that this neutral chitobiase activity is located in the cytosolic compartment and is active on oligomannoside species released during the N-glycosylation process.

Catabolism of glycan moieties of lipid intermediates leads to a single Man5GlcNAc oligosaccharide isomer: a study with permeabilized CHO cells.

This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides. Permeabilized cells released 30 times more oligosaccharide material than metabolically labelled cells, normalized to the amount of labelled glycoprotein acceptor, making this an amenable system for study. Fifteen to forty per cent of the oligosaccharide material released by permeabilized cells was oligosaccharide-phosphate, depending on the nature and amount of the oligosaccharide-lipids synthesized. The oligosaccharide-phosphates released were recovered in the cytosol, and were exclusively Man2Glc-NAc2P and Man5GlcNAc2P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral oligosaccharide material were similar to those attached to newly synthesized glycoproteins, indicating that the oligosaccharides were subjected to the same processing enzymes whether or not they were protein bound. Importantly, the kinetics of the transfer to protein and the release of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses demonstrated that the same Man5GlcNAc2 structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosaccharides with one GlcNAc residue at the reducing end (OS-Gn1) were found exclusively in the supernate. The major neutral oligosaccharide produced after 2 h of metabolic labelling was Man5GlcNAc and it was found in the cytosol.

Application of electrospray and fast atom bombardment mass spectrometry to the identification of post-translational and other chemical modifications of proteins and peptides.

Mass spectrometry is a very powerful tool in the identification of chemical modifications of proteins and peptides. Often these modifications cannot be determined by conventional techniques. This report describes the combined use of electrospray ionization mass spectrometry and fast atom bombardment mass spectrometry to complete the primary structure of proteins and peptides. Examples illustrate how mass spectrometry is used to locate sites of phosphorylation, methylation and acetylation, and identify blocking groups and unexpected side reactions such as deamidation or alkylation.

Complete sequence of Sipunculus nudus erythrocyte histone H2B and its gene. Identification of an N,N-dimethylproline residue at the amino-terminus.

The complete amino acid sequence (122 residues) of histone H2B from erythrocytes of the marine worm Sipunculus nudus, has been established from sequence analysis of peptides generated by highly specific cleavage of the protein and from the nucleotide sequence of the encoding gene. The isolation of the H2B gene was facilitated by using a highly specific nucleotide probe, devised from amino acids 58-68 of the protein. The presence of an N,N-dimethylproline residue at the amino-terminus of the protein was established from data provided by mass spectrometry and NMR spectroscopy. This unusual post-translational modification of histone H2B generates a stable positive charge which could strongly interact with the linker DNA.

Primary structure of the two variants of a sperm-specific histone H1 from the annelid Platynereis dumerilii.

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b. The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1). H1a and H1b have short N- and C-terminal basic domains but the size of the globular domain (approximately equal to 80 residues) is similar to that of other H1s. In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.

Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin.

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.

Complete amino-acid sequences of DNA-binding proteins HU-1 and HU-2 from Escherichia coli.

The DNA-binding protein HU from Escherichia coli is a heterodimer constituted of two polypeptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116--120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395--398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes.

Primary structure of chicken erythrocyte histone H2A.

The complete amino acid sequence (128 residues) of the chicken erythrocyte histone H2A was deduced from the data provided by structural studies on the tryptic peptides from the maleylated histone and of the peptides obtained by thermolysin digestion of the native protein. The sequence of chicken histone H2A differs from the calf homologous histone by the deletion of one residue of histidine at position 123 or 124 and three conservative substitutions: a residue of serine replaces a residue of threonine at position 16, a residue of aspartic acid replaces a residue of glutamic acid at position 121 and a residue of alanine replaces a residue of glycine at position 128.