Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.

The catalytical role of the hydroxy amino acid in the "marker sequence" Asn-Xaa-Thr(Ser) for the N-glycosylation step of glycoprotein formation was investigated by using a series of hexapeptides derived from Tyr-Asn-Gly-Xaa-Ser-Val by substituting threonine, serine, cysteine, valine and O-methylthreonine respectively for Xaa. The results, which were obtained with calf liver microsomal fractions as enzyme source and dolichyl diphosphate di-N-acetyl [14C] chitobiose as glycosyl donor showed that the threonine-, serine- and cysteine-containing derivatives could be glycosylated, although at very different rates, whereas the valine and O-methylthreonine analogues did not work as glycosyl acceptors. Replacement of threonine by serine resulted in a 4-fold decrease in Vmax, and about a 10-fold increase in Km for glycosyl transfer. Replacement of serine by cysteine again decreased acceptor activity 2-3-fold. The various results, taken together, indicate an absolute requirement for a hydrogen-bond-donor function in the side chain of the hydroxy amino acid of the "marker sequence" and furthermore, point to a considerable influence of the structure of this amino acid on binding as well as on the glycosyl transfer itself. In order to explain the observed differences in the glycosyl-transfer rates, a model is proposed with a hydrogen-bond interaction between the amide of asparagine as the hydrogen-bond donor and the oxygen of the hydroxy group of the hydroxy amino acid as the hydrogen-bond acceptor. The participation of the hydroxy group in the catalytic mechanism of glycosyl transfer in the kind of proton-relay system is discussed.

Many secretory and membrane proteins are glycoproteins carrying asparagine-linked (N-linked) oligosaccharides. There are two types of N-linked glycans, referred to as high-mannose and complex type, respectively. Biosynthesis of N-linked glycans of the complex type proceeds via a high-mannose intermediate. After the initial transfer of a high-mannose oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2 from a lipid carrier to the nascent polypeptide chain, trimming reactions take place. Trimming glucosidases remove the glucose residues quantitatively and mannosidases IA/B and II can remove all but three mannose residues. After trimming, terminal sugars such as N-acetylglucosamine, galactose, sialic acid and fucose may be added and result in the conversion to a glycan of the complex type. Because suitable inhibitors were lacking, it was difficult to assess the importance of the trimming reactions for proper intracellular traffic, modification reactions other than the addition of terminal sugars, or as regulatory steps in glycoprotein processing. Here we describe the action of 1-deoxymannojirimycin (1,5-dideoxy-1,5-imino-D-mannitol, dMM; Fig. 1) on the biosynthesis of IgM and IgD. dMM is the mannose analogue of 1-deoxynojirimycin (dNM; Fig. 1), itself a glucosidase inhibitor. We present evidence that dMM is a mannosidase inhibitor. In vivo dMM inhibits the equivalent of the mannosidase IA/B activities and blocks conversion of high-mannose to complex oligosaccharides. It is the first such inhibitor to be reported. Interference with the biosynthetic pathway of N-linked glycans could prove to be a powerful way to manipulate carbohydrate structure in vivo.

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

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This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

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Trimming glucosidase 1 and I1 have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and I1 and the nonspecific cc-glucosidase(s) were inhibited by 1 -deoxynojirimycin with median inhibitory concentration of 3 pM, 20 pM, 12 pM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of I-deoxynojirimycin and formed the basis for the design of the affinity ligand.Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecullar mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was % 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 -'C in the presence of detergent (t,,, z 20 days) and showed high specificity for the hydrolysis of the terminal ( E 1,2)-linked glucose residue in the natural substrate Glc,-Man,-(GlcNAc),.During N-glycoprotein formation oligosaccharides of the composition Glc,-Man,-(GlcNAc), are preassembled on a lipid carrier (dolichyl diphosphate, Doil-PP) and subsequently transferred 'en bloc' onto target asprragine residues of the nascent polypeptide chain. The protein-bound oligosaccharides are then modified by a sequence of reactions, generating N-glycans with either high mannoge orland complex type structures. The trimming sequence is initiated by the stepwise removal ofthe three glucose units, which is catalysed by at least two specific glucosidases (glucosidase I and 11). Cleavage of the glucose residues is followed by an einzymatic hydrolysis of several ( a 1,2)-linked mannose residues, resulting in high mannosc structures, which may be preserved as such or further modified to complex type oligosaccharides, the latter process requiring the concerted action of presumably other rx-mannosidases and various glycosyltransferhses (for review, see [I]).Since glucose residues are generally not found in mature N-glycoproteins, it is suggested that their transient occurrence may have an important function in the regulation of oligosaccharide trimming and processing. First evidence that the presence of the Glc, unit may give a signal for the transfer of the lipid-linked precursor oligosaccharide to protein, came from studies ofTurco et al. [2], who demonstrated that, at least under conditions in vitvo, the Glc,-containing structure is transferred more rapidly to protein acceptors than the intermediate devoid of glucose. A similar regulatory function was recently put forward by Spiro et al. [3], who discussed that the extent of N-glycosylation might depend...