Armando J. Parodi

The role of dolichol monophosphate in sugar transfer.

Abstract The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate- N -acetylgalactosamine gave negative results. The optimal conditions for transfer from dolichol monophosphate glucose to endogenous acceptor was determined. Studies were carried out on the glucosylation of ceramide by brain extracts and of collagen by skin enzymes in order to find out if dolichol monophosphate glucose is an intermediate in these reactions. The results, while not definite, were not in favor of this assumption.

Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells

Proteins entering the secretory pathway may be glycosylated upon transfer of an oligosaccharide (Glc3Man9GlcNAc2) from a dolichol-P-P derivative to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). Oligosaccharides are then deglucosylated by glucosidases I and II (GII). Also in the ER, glycoproteins acquire their final tertiary structures, and species that fail to fold properly are retained and eventually degraded in the proteasome. It has been proposed that in mammalian cells the monoglucosylated oligosaccharides generated either by partial deglucosylation of the transferred compound or by reglucosylation of glucose-free oligosaccharides by the UDP-Glc:glycoprotein glucosyltransferase (GT) are recognized by ER resident lectins (calnexin and/or calreticulin). GT is a sensor of glycoprotein conformation as it only glucosylates misfolded species. The lectin-monoglucosylated oligosaccharide interaction would retain glycoproteins in the ER until correctly folded, and also facilitate their acquisition of proper tertiary structures by preventing aggregation. GII would liberate glycoproteins from the calnexin/calreticulin anchor, but species not properly folded would be reglucosylated by GT, and so continue to be retained by the lectins. Only when the protein becomes properly folded would it cease to be retained by the lectins. This review presents evidence suggesting that a similar quality control mechanism of glycoprotein folding is operative in Schizosaccharomyces pombe and that the mechanism in Saccharomyces cerevisiae probably differs substantially from that occurring in mammalian and Sch. pombe cells.

Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues.

The size of a lysosomal cysteine proteinase from epimastigotes of Trypanosoma cruzi decreased from 60 to 54 kDa upon treatment with endo beta-N-acetylglucosaminidase H. A lower-molecular weight component (30-35 kDa), which usually accompanies the 60-kDa protein also increased its electrophoretic mobility, and seems to consist of a mixture of degradation products of the enzyme, since both the larger and the smaller components had the same N-terminal sequence as the 60-kDa protein. The amino acid composition of the protein moiety and the composition of the oligosaccharide chains have been determined. The oligosaccharide chains are of the high-mannose type, and contain 6, 7, 8 or 9 mannose residues, as shown both by in vivo labeling with [U-14C]glucose, and by labeling the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of the purified enzyme with tritiated sodium borohydride. The oligosaccharide chains did not contain phosphate residues. Further studies with [U-14C]-labeled total glycoproteins of T. cruzi and enzyme assays, suggest that T. cruzi and other trypanosomatids do not target their lysosomal enzymes to the organelle through the mannose-6-phosphate marker pathway.

Glycoprotein assembly in Leishmaniamexicana

Abstract The oligosaccharide transferred from a dolichol-P-P derivative to proteins in the assembly of N-linked glycoproteins in Leishmania mexicana appeared to be Man6GlcNAc2. It was found that this compound underwent transient glucosylation once bound to protein but that Man6GlcNAc2 was the oligosaccharide present in mature glycoproteins. No complex type saccharides were detected. The structure of the oligosaccharide appeared to be similar to that of the core of compounds transferred from dolichol-P-P derivatives in protein glycosylation in Trypanosoma cruzi or animal cells.

Further studies on a glycolipid formed from dolichyl-d-glucosyl monophosphate

Abstract Incubation of liver microsomes with dolichyl- d -glucosyl- 14 C monophosphate led to the labelling of an endogenous acceptor. This compound seems to be also a dolichol derivative. It contains a high-molecular weight oligosaccharide bound to dolichol through a phosphate or pyrophosphate bond. Various treatments of the labelled oligosaccharide afforded further information on its structure: Reduction with sodium borohydride, followed by acid hydrolysis gave only radioactive d -glucose indicating that the labelled d -glucose is not incorporated at the reducing end of the oligosaccharide. The percentage of radioactivity, liberated as formic acid after periodate oxidation, indicates that more than one molecule of d -glucose is incorporated and that at least one of them is inside the oligosaccharide chain. Alkaline treatment of the otherwise neutral oligosaccharide gave two positively charged derivatives which could be neutralized by N -acetylation, indicating the presence of two hexosamine residues. The oligosaccharides isolated from different tissues by the same method as that used for rat liver, were similar as judged by their migration in paper chromatography and by the pattern of products liberated by acetolysis.

Subcellular distribution of dolichol phosphate.

A crude preparation of liver microsomes was shown to catalyze the transfer of glucose from UDP-Glc to Dol-P** to form Dol-P-Glc [ 1 ] . ~ub~quently it was observed that microsomes also transferred the glucose from Dol-P-Glc to an endogenous acceptor. This latter compound is probably another dolichol derivative containing about 20 monosaccharide units bound to dolichol through a phosphate or pyrophosphate bridge [2,3]. The abbreviation Glc-acceptor will be used for this dolichol derivative until the details of its structure are known with certainty, Liver microsomes also catalyze the transfer of mannose to Dol-P from GDP-mannose and of N-acetyl ~ucos~ine from UDP-GlcNAc to form what seems to be dolichol monophosphate mannose and dolichol monophosphate N-aeetyl glucosamine [4, S] . It is possible that the dolichol derivatives are intermediates involved in the synthesis of the sugar portions of the various glycoproteins and glycolipids occurring in smooth microsomes, the Golgi system and other cellular membranes. It is therefore necessary to establish the subcellular loc~ization of these derivatives.

Glucose transfer from dolichol monophosphate glucose The lipid moiety of the endogenous microsomal acceptor

Abstract Further studies on the structure of the microsomal endogenous substance which accepts labeled glucose from dolichol monophosphate glucose are reported. In order to obtain sufficient amounts of substance for measuring bound dolichol monophosphate, a concentrate was prepared by treating liver with mixtures of chloroform-methanol-water which lead to purification of the radioactive compound. Estimation of dolichol monophosphate, by its accelerating action on dolichol monophosphate glucose formation, showed that it is liberated by mild acid treatment of the concentrate. The dolichol monophosphate-yielding substance behaved like the labeled glucosylated acceptor when tested by its solubility properties in some solvent mixtures, thin-layer and DEAE-cellulose chromatographies and acid or alkaline treatments. Therefore the results provide further evidence that the lipid moiety of the endogenous acceptor of microsomes is dolichol phosphate.

In vitro synthesis of particulate glycogen from uridine diphosphate glucose

Abstract High molecular weight glycogen has been prepared in vitro with liver glycogen synthetase (uridine diphosphate glucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11) and branching enzyme (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase, EC 2.4.1.18) and uridine diphosphate glucose as glucose donor. The product obtained did not differ significantly from the native glycogen as judged by iodine spectrum, sedimentation coefficient in sucrose gradients, and by the effect of treatment with acid or alkali. Glycogen obtained from uridine diphosphate glucose differed from that prepared with glucose 1-phosphate as glucosyl donor.

Some properties of rat liver amylase

Abstract Some properties of rat liver amylase were studied. It was confirmed that this enzyme is located mainly in the microsomal fraction and that it is activated by detergents. The effects of digitonin, Triton X–100 and sodium deoxycholate on the amylase were compared. It was observed that amylase requires a higher concentration of Triton X–100 for maximal activation than lysosomal acid phosphatase. When lysosomes and microsomes were submitted to a detergent treatment capable of activating completely acid phosphatase and α-amylase, the former was solubilized, while the second remained particulate. The activation of α-amylase was found to be reversible in the first stages. The microsomal α-amylase is not bound to glycogen in fed rats. It was observed that the free α-amylase did not change when the liver was induced to accumulate different amounts of glycogen by administration of sugar, but that latent amylase was doubled when large amounts of glycogen accumulated. The K m of liver amylase for glycogen is 2.5 mg/ml as compared with 0.4 mg/ml for serum amylase. The significance of these observations is discussed.

Properties of synthetic and native liver glycogen

Abstract The properties of high molecular weight glycogen extracted from rat liver and of that prepared in vitro with muscle phosphorylase and liver branching enzyme have been compared. The stability at different pH values was measured spectrophotometrically for liver, corn, and synthetic glycogen. The former is more labile, but the shape of the pH-stability curve is very similar for all of them. Borate, copper, and iron accelerate the decomposition of the three types of glycogen. Sonication produces breakdown but affects in the same way synthetic and liver glycogen. After shortening the outer chains with β-amylase, native liver glycogen becomes slightly more stable to acid treatment and decomposes giving smaller molecules than the untreated glycogen. Glycogen extracted from livers of toad and pigeon was similar in molecular weight distribution and acid lability to that of rat liver. Rat muscle glycogen had a molecular weight of about 8 million.