Phosprenyl: a novel drug with antiviral and immunomodulatory activity.

A novel antiviral drug with immunomodulatory activity (Phosprenyl) is presented. The main active ingredient of the preparation is polyprenyl phosphates. This medicine is highly efficient against a number of viruses, including HIV in vitro, and tick-borne encephalitis and rabies viruses in the experimental models in vivo. In veterinary practice Phosprenyl is now regarded as an effective therapeutic means for treatment of canine distemper, hepatitis, enteritis, etc.

[Dolichol-coupled biosynthesis of an oligosaccharide precursor for N-glycosylated proteins in the rough endoplasmic reticulum]. / Dolikhol-sopriazhennyi biosintez oligosakharidnogo predshestvennika dlia N-glikozilirovaniia belkov v sherokhovatom éndoplazmaticheskom retikulume.

Data from literature concerning the structure and function of enzymes, taking part in dolichol-coupled biosynthesis of oligosaccharide precursor, which is used for N-glycosylation of protein in endoplasmic reticulum of eukaryotic cells, are summarized and analyzed in the review. The mechanisms of translocation of dolichol and its derivatives through membrane are discussed. The data about topography of reactions of "dolichol cycle", taking place on cytoplasmic and luminal surfaces of endoplasmic reticulum membrane, are considered.

Regulation of dolichyl phosphate-mediated protein glycosylation: estrogen effects on glucosyl transfers in oviduct membranes.

Regulation of Glc transfer from UDP-Glc via Glc-P-Dolichol to form Glc3-Man9-oligosaccharide-lipid has been studied during estrogen-induced chick oviduct differentiation. The process was studied as two distinct reactions: transfer of Glc from UDP-Glc to Dol-P, forming Glc-P-Dol; and transfer of Glc from Glc-P-Dol to Man9-OL (oligosaccharide-lipid), forming a series of glucosylated oligosaccharide-lipids. Kinetic analysis of [14C]Glc transfer from UDP-[14C]Glc to endogenous Dol-P shows that Dol-P is limiting in membrane preparations and that, concomitant with estrogen-induced differentiation, there is an increase in Dol-P available for Glc transfers. There is also greater glucosyl transferase activity present in membranes from mature hens and estrogenized chicks than in membranes from immature chicks. In order to study the second phase of glucosylation, transfer to the oligosaccharide, it was necessary to develop an assay in which membranes could be reacted with exogenously added substrates, [14C]Glc-P-Dol and [3H]Man9-OL. This reaction is dependent on detergent (0.02% NP-40 was used) and is stimulated by EDTA. The apparent Km for Glc-P-Dol was about 1.5 microM. A series of double-labeled oligosaccharides having sizes consistent with Glc1-, Glc2-, and Glc3-Man9-OL were formed. Chemical and enzymatic analysis of [14C]Glc oligosaccharides formed by incubation with the exogenous substrates, or by incubation with UDP-[14C]Glc and endogenous acceptors, indicated that the glucosylated oligosaccharides were similar. Assays of membranes from estrogenized chicks, mature hens, and hormone-withdrawn chicks showed increased glucosyl transferase activity upon hormone treatment. Similar assays in the absence of exogenous Man9-OL indicated that hormone treatment was also accompanied by increased levels of endogenous oligosaccharide-lipid acceptors.

The biosynthesis of crustacean chitin. Isolation and characterization of polyprenol-linked intermediates from brine shrimp microsomes.

The biosynthesis of crustacean chitin appears to involve the participation of a lipid-linked intermediate. A microsomal preparation from larval stages of the brine shrimp Artemia salina was found to catalyze the glycosylation of exogenous [3H]dolichol phosphate, yielding a product which was insoluble in chloroform:methanol (2:1) but soluble in chloroform:methanol:water (10:10:3). Artemia microsomes catalyze the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to a lipid acceptor. After extraction of labeled lipids with either chloroform:methanol (2:1) or chloroform:methanol:water (10:10:3), labeled compounds could be purified by ion-exchange chromatography on DEAE-Sephacel. Mild acid hydrolysis of 3H-N-acetylglucosamine labeled material soluble in chloroform:methanol:water (10:10:3) yielded a series of oligosaccharides ranging from 2 to about 8 glycosyl units in size. The larger components were shown to be sensitive to chitinase digestion but resistant to treatment with alpha-mannosidase. Such 3H-N-acetylglucosamine containing compounds, prepared by both in vivo and in vitro procedures, appear to be chitin oligosaccharides. Brine shrimp microsomes also catalyze the transfer of mannose from GDP-mannose to a lipid acceptor. Mild acid hydrolysis of mannosyl lipids soluble in chloroform:methanol:water (10:10:3) yielded oligosaccharides which were sensitive to alpha-mannosidase digestion and resistant to treatment with endochitinase. The results suggest 3H-N-acetylglucosamine-labeled oligosaccharide-lipids are distinct from the mannose-labeled fraction and may participate in the formation of an endogenous primer for chitin synthesis after their transfer to a protein acceptor.

Studies on the synthesis and processing of the asparagine-linked carbohydrate units of glycoproteins.

It has become apparent in recent years from the work of a number of laboratories that the N-glycosylation of both membrane and secretory glycoproteins is effected by the transfer en bloc to nascent polypeptides of a glucose-containing oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier; this is followed by a series of modifying reactions to yield the mature polymannose and complex asparagine-linked carbohydrate units. The enzymic steps involved in the assembly of the precursor oligosaccharide, its transfer to protein and its subsequent processing represent potential sites for the regulation of glycoprotein synthesis. Studies performed in our laboratory have dealt primarily with thyroid slices and particulate enzymes with special regard to the role of glucose in these events. Thyroglobulin, the major secretory glycoprotein of this tissue, has well defined complex and polymannose saccharide units, and indeed the most complete form of the latter (Man9GlcNAc2) has the same structure as the lipid-linked oligosaccharide without the glucose. Our studies indicate that effective N-glycosylation requires a complete glucose chain (Glc3) and that the glucose sequence is assembled from dolichol-P-glucose in a stepwise manner through the concerted action of at least two transferases in a fashion complementary to the subsequent excision of this sugar by glucosidases. Pulse-chase studies indicate that, after the transfer to protein, the removal of all three glucose residues as well as of the first mannose takes place in the endoplasmic reticulum and three additional mannoses are excised in the Golgi complex, because in the presence of an inhibitor of intracellular transport, carbonyl cyanide m-chlorophenylhydrazone (CCCP), there is a pronounced accumulation of protein-linked Man8GlcNAc2. Studies with metabolic inhibitors (CCCP, antimycin, N2) indicate that, under conditions of energy depletion, glucosylation of oligosaccharide-lipid is selectively impaired, resulting in an accumulation, as measured chemically or metabolically, of high-mannose-containing (Man9GlcNAc2 and Man8GlcNAc2) lipid-linked saccharides. Further evidence that the glucosylation reaction is very sensitive to the metabolic state is suggested by the observation that tissues not rapidly frozen after removal from the animal show a similar depletion of the glucose-containing oligosaccharide lipids. Another important aspect for the regulation of N-glycosylation of proteins is the availability of dolichyl phosphate for the formation of the lipid-linked mono- and oligosaccharides. Our studies with puromycin suggest that there is a limited supply of the lipid carrier, because in the presence of this inhibitor there is no accumulation of any of the oligosaccharide-lipid species.

Topological and regulatory aspects of dolichyl phosphate mediated glycosylation of proteins.

From the time of their synthesis in the rough endoplasmic reticulum until they are secreted, packaged in lysosomes, or appear as membrane components at the cell surface, the polypeptide chains of N- and O-linked glycoproteins remain associated with intracellular membranes that are components of the secretory pathway. The various co-translational and post-translational modifications of the carbohydrate moieties of glycoproteins have been shown to occur within morphologically and functionally distinct regions of this complex membrane system. However, the sugar nucleotides, which serve as precursors to the oligosaccharide moieties of these glycoproteins, are synthesized almost exclusively in the cytoplasm. These findings raise a number of questions about the mechanisms involved in the transmembrane assembly of membrane and secretory glycoproteins. In this paper these questions are reviewed and recent studies directed towards providing answers to them are summarized. In addition, information related to the possible role of dolichyl phosphate in regulating the glycosylation of proteins is presented.

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins.

The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.