Catherine Ronin

Enzymatic N-glycosylation of synthetic ASN-X-THR containing peptides

The role of polyprenol sugar derivatives in the biosynthesis of certain eukaryotic glycoproteins has been partly described [ 11. Recently lipid pyrophospho-oligosaccharides, postulated as necessary intermediates to initiate N-glycosylation, were shown, when supplied to microsomes, to be effective donors to proteinic acceptors presenting at least one vacant Asn-X-zk sequence: unfolded forms of proteins [Z], polypeptide fragments from CNBr cleavages [3] or a synthetic heptapeptide [4]. It was proposed [S] on the grounds of structural examination of numerous glycoproteins, that this basic tripeptide sequence was a necessary, although probably not sufficient, requirement for an asparagine residue to be N-glycosylated. The data we report here extend our work [4] showing that thyroid rough microsomes catalyze the transfer of oligosaccharide from [Mrfa-r4C] oligosaccliaride-lipids to a synthetic human thyroglobulin heptapeptide and further to its d~itropl~enylated derivative. Our aim has been to further investigate specificity requirements of the oligosaccharide transferase, using as tools the following series of synthetic peptides: (I) H-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH, _~_._ (II) DNP-Ala-Leu-GluAsn-Ala-Thr-Arg-NH, (III) H Asn-Ala-Thr-NH, (IV) DNPAsn-Ala-Thr-NH, (V) DNP-Ala-Let.-Glu Asn-Pro-Thr-Arg-NH, __~_._ (VI) H-Tyr-Gln-SerAsn-Ser-~r-~et-NH~ ~--

Localization in a Glogi-rich thyroid fraction of sialyl-, galactosyl- and N-acetylglucosaminyltransferases(*)

Summary The present report provides evidence for the association of three nucleotide-sugar : glycoprotein glycosyltransferases involved in terminal glycosylation of thyroglobulin with a Golgi-rich subcellular fraction of the thyroid. Enzyme activities were measured after membrane disruption, either with endogenous acceptors or with added exogenous acceptors such as specifically deglycosylated thyroglobulin or fetuin. Enzyme assays were conducted in conditions under which sugar transfer was proportional to the amount of enzyme protein. Sheep thryoids were homogenized in a medium favoring the preservation of Golgi structures. Homogenates were fractionated by differential centrifugation, and the microsomal pellet subjected to an already described ultracentrifugation in a discontinuous sucrose density gradient. RNA, DNA and « markerenzymes were determined. The results indicated that N-acetylneuraminyl-, galactosyl- and N-acetylglucosaminyltransferases were preferentially located in the low density fraction previously found enriched in identifiable Golgi fragments.

Cell-free peptide synthesis and carbohydrate incorporation by various thyroid particles.

Abstract Various sheep thyroid particles were prepared and used in cell-free systems to study either peptide synthesis or carbohydrate incorporations of endogenous acceptors including thyroglobulin precursors. Rough microsomes (i.e. microsomes enriched in granular vesicles), total polyribosomes and free polyribosomes were purified from a post-mitochondrial supernatant in Tris-HCl, MgCl2 and sucrose and were incubated, in conditions allowing peptide synthesis, with either 14C-labeled amino acids, UDP -N-[ 14 C]acetylglucosamine or GDP-[14C]mannose. Amino acid-incorporating efficiencies of rough microsomes, prepared according to several slightly different procedures, and of polysomes were compared on an RNA basis and found roughly similar. Only microsomes, not polyribosomes, were able to incorporate N- acetylglucosamine and mannose from their nucleotides. Several facts are consistent with a membranous extra-ribosomal sugar incorporation to completed and released peptide chains: the lack of significant inhibition by cycloheximide or puromycin, the almost total extractability by sodium deoxycholate of the rough microsomal carbohydrate labels, contrary to only about 20% of the peptide label, and finally the efficiency of rough microsomes to incorporate N- acetylglucosamine and mannose in incomplete systems (i.e. without peptide synthesis). Golgi-rich agranular microsomes were purified from a post-mitochondrial supernatant in sodium phosphates, Dextran and sucrose and were incubated in conditions avoiding membrane rupture, with either UDP -N-[ 14 C]acetylglucosamine , UDP-[14C]galactose or CMP -N- acetyl-[ 14 C]neuraminic acid, to study incorporation of relatively peripheral sugars in the more complex heterosaccharide units of thyroglobulin. Sucrose gradient ultracentrifugation of recently prepared digitonin extracts from variously labeled rough and agranular microsomes, and of an amino acid-labeled polyribosomal supernatant, exhibited quite distinctive patterns, suggestive of multistep events.

Transfer of glucose in the biosynthesis of thyroid glycoproperties I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar. Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1 microM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them. Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to act as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was found that the presence of glucose did not significantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.

Immunochemical characterization of thyroglobulin-related components synthesized in cell-free systems

Abstract Various sheep thyroid subcellular fractions enriched either in rough microsomes or in Golgi structures (agranular microsomes) and isolated total polyribosomes have been incubated in the presence of various labeled precursors: 14C-labeled amino acids and nucleotides of 14C-labeled monosaccharides. Digitonin extracts of the microsomal fractions contained an acid-insoluble 14C-labeled material, a part of which can always be specifically bound by anti-19-S sheep thyroglobulin antibodies: 20 to 50% in the rough microsomal extracts labeled with 14C-labeled amino acids, [14C]mannose or N-[ 14 C ] acetylglucosamine ; 45 to 70% in the agranular microsome extracts labeled with [14C]galactose, N-[ 14 C ] acetylglucosamine or [14C]sialic acid. Polyribosomes incubated with 14C-labeled amino acids secreted into the medium an acid-insoluble labeled material containing up to 30% of thyroglobulin immunoreactive material. Sucrose gradient centrifugation showed that 14C-labeled proteins extracted from rough microsomes were essentially located in a 6–8-S peak accompanied, in the case of labeling with 14C-labeled amino acids, by a 2–4-S peak. Proteins synthesized by polyribosomes were in a 3–5-S peak. In agranular microsomal extracts a 17- to 19-S species was predominant. The proportion of 14C-labeled thyroglobulin immunologically detectable increased with increasing sedimentation coefficient (5–10% in the 2–4-S zone, up to 90% in the 17–19-S zone), whatever labeled precursor was used. In the case of rough microsomal extracts, an evolution has been observed in vitro: storage of the dialyzed extracts at −20°C or incubation in conditions favoring disulfide interchange generally increased the percent of immunologically-precipitable material, whereas 10- to 12-S and 17- to 19-S species often appeared. A parallelism between the acquisition of immunoreactive determinants, the evolution of the three-dimensional structure and the incorporation of carbohydrates, during the first steps of synthesis and secretion of the thyroglobulin is suggested and discussed.

Evidence for mannolipids as intermediates in mannose transfer to thyroid rough microsomal glycoproteins

Thyroid rough microsomes catalyzed the transfer of mannose from GDP-mannose to endogeneous glycoprotein(s) and to glycolipids comprising a recently described dolichol phosphomannose extractable with usual organic solvents and a material tentatively identified as an oligosaccharide lipid. The labeling of the two lipids was consistent with a role in mannose transfer to glycoprotein(s). When partially purified dolichol phospho[14C] mannose was incubated with rough microsomes, a part of the label appeared in the second lipid, suggesting a role as intermediate, and less rapidly in glycoprotein(s). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis did not allow to ascertain whether or not the glycoproteins receiving label from these sugar lipids comprised thyroglobulin precursors.

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. I. Mannosylated units.

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [14C]Man material specifically soluble in CHCl3/CH3OH/H2O, 10:10:3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some dolichol-P-[14C]Man). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to alpha-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.

Enzymatic transfer of oligosaccharide from oligosaccharide-lipids to an Asn-Ala-Thr containing heptapeptide.

Abstract Thyroid rough microsomes catalyzed the transfer of oligosaccharide from previously labeled thyroid [Man- 14 C] oligosaccharide-lipids to a synthetic dinitrophenylated heptapeptide containing a sequence Asn-Ala-Thr. The resulting product revealed an N-glycosidic attachment, probably to the asparaginyl residue in this sequence. The reaction which was time-dependent up to 1 h and exhibited an apparent Km of 94 μM for the DNP-heptapeptide was favoured by dimethylsul-foxide and inhibited by EDTA.

Effect of dimethylsulfoxide on two synthetic Asn-X-Thr containing substrates of the oligosaccharyltransferase

Abstract The enzymatic transfer of oligosaccharides from oligosaccharide lipids to synthetic Asn-X-Thr containing peptides of various length was studied in presence and absence of dimethylsulfoxide. Up to 13.5%, this solvent was found to specifically enhance the efficiency of a ribonuclease heptapeptide to be substrate of the thyroid oligosaccharyltransferase and in contrast, not that of a tripeptide. Circular dichroic analysis performed in various dimethylsulfoxidewater mixtures showed that only the heptapeptide underwent conformational modifications when increasing the concentration in dimethylsulfoxide. Some ordered structure in the immediate vicinity of the Asn-X-Thr signal sequence thus appears of importance in the N-glycosylation process.

Solubilization of oligosaccharide tranferase and glucosidase activities from thyroid rough microsomes

In previous studies from this laboratory [ 1,2], an oligosaccharide transferase activity was demonstrated in thyroid rough microsomes: the membranous enzyme catalyzes the transfer of oligosaccharides from exogenously supplied oligosaccharide lipids to the asparagine residue of synthetic Asn-X-Thr containing peptide acceptors. This transferase was also recently described in hen oviduct [3], rat liver [4] and yeast [5] membranes. As a prerequisite for purification, this paper examines the conditions which allow total extraction of the enzyme from membranes while maintaining full activity. Although a few other glycosyltransferases involved in the early lipid-linked pathway of N-glycosylation have already been solubilized [6,7], this is the first report dealing with the oligosaccharide transferase. Several authors have suggested that glucose-containing oligosaccharide lipids constitute the physiological donor of the oligosaccharide transferase [8,9]. Glucose residues might be a signal in the transfer reaction before being removed by specific glucosidases. The present study points out that the oligosaccharide transferase is extracted together with two glucosidases of distinct specificities.