Synthesis Of N-linked Glycoproteins Research Articles

Generalizing a Ligation Site at the N-Glycosylation Sequon for Chemical Synthesis of N-Linked Glycopeptides and Glycoproteins.

Chemical synthesis can generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites. The precision and flexibility of the chemical synthetic approach provide a solution to the heterogeneity problem of glycopeptides/glycoproteins obtained through biological approaches. In this study, we reported that the conserved N-glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation to achieve N-linked glycoprotein synthesis. We developed an N + 2 strategy to prepare the corresponding glycopeptide salicylaldehyde esters for Ser/Thr ligation and demonstrated that Ser/Thr ligation at the sequon was not affected by the steric hindrance brought about by the large-sized glycan structures. The effectiveness of this strategy was showcased by the total synthesis of the glycosylated receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

N-RasG12D-Mediated Dysregulation of IRE1alpha-Xbp1s Signaling Promotes Pre-Leukemic Hematopoietic Stem Cell Expansion

Protein homeostasis is tightly regulated to ensure cell integrity. Upon stimulation by nutrition deprivation, increased reactive oxygen species (ROS), or high protein synthesis demand, increased ER stress triggers unfolded protein response (UPR) pathways to induce either adaptation, including reduced protein synthesis and increased expression of chaperones to promote protein refolding, or apoptosis after severe or prolonged damage. The fate of the cell under ER stress therefore depends on the balance between adaptive and pro-apoptotic signalings. Within the hematopoietic populations, HSCs have the lowest protein synthesis rate. In addition, HSCs are more sensitive to ER-stress-induced apoptosis compared to progenitors. Recent studies have shown that constitutive activation of ER-stress leads to depletion of HSCs. Promotion of adaptation to ER-stress by overexpressing co-chaperones such as ERDJ4, increase the HSC repopulation capacity. Taken together, these studies suggest that protein homeostasis plays an important role in hematopoietic stem cell (HSC) regulation.Genetic lesions that activate oncogenes or inactivate tumor suppressor genes transform HSCs and progenitors first to pre-leukemic stem cells (pre-LSCs) which often exhibit increased proliferation and therefore increased protein synthesis demand. Employing an Mx-Cre+; LSL-NrasG12D/+ conditional knock-in mouse model, our previous studies have shown that N-RasG12Dincreases both self-renewal and proliferation of HSCs. To determine whether UPR pathways contribute to N-RasG12D-mediated pre-leukemic stem cell expansion, we first assessed the response of Nras mutant HSCs to ER stress either by exposure to tunicamycin(TM), which blocks synthesis of N-linked glycoproteins, or thapsigargin(TG), which disrupts Ca2+ homeostasis in the ER. Consistent with recent reports, both TM and TG treatment induced apoptosis in murine SLAM HSCs (CD150+CD48-Lin-sca1+ckit+). Nras mutant HSCs, however, exhibited a significant reduction of apoptosis as measured by Annexin V staining and caspase activation, and their colonogenic potential was unaffected. These effects were mitigated by inhibition of MEK/ERK signaling but not STAT5, and were specific to hyperactive Nras mutant HSCs, since FLT3-ITD leukemic mutation had no effect on HSC survival under ER stress.To identify the mechanism underlying the N-RasG12D-mediated HSC protection, we examined the major signaling branches of UPR: IRE1alpha/XBP1s, ATF6 and PERK/eIF2alpha. We found that N-RasG12D activated the adaptive branch of the UPR, Inositol-requiring enzyme 1 (IRE-1)/XBP1s signaling, as increased level of phospho-IRE1alpha and XBP1s mRNA were observed. Furthermore, inhibition of this pathway by either a small molecular inhibitor specific to the RNase activity of IRE1alpha, or overexpression of a dominant negative mutant of XBP1s, reduced the adaptation to ER-stress and competitive advantage in transplant recipients of Nras mutantHSCs. Moreover, heterozygous knockout of IRE1alpha blocked N-RasG12D-mediated splenomegaly and myeloid expansion in Mx1-cre+; Ire1alphafl/+; LSL-NrasG12D/+ mice. These results indicate that N-RasG12D-mediated activation of IRE1alpha/XBP1s signaling protects HSCs from ER stress and promotes pre-LSC clonal expansion.To identify the downstream targets of IRE1alpha/XBP1s, we examined the potential transcription targets of XBP1s and found that the co-chaperone dnajb8 is significantly upregulated in Nras mutant HSCs. Overexpression of dnajb8 also significantly enhanced the long term reconstitution capacity of HSCs in transplant recipients, suggesting that N-RasG12D-mediated activation of IRE1alpha/XBP1s promotes protein refolding. Consistent with this, in vivo level of protein aggregation was much lower in Nras mutant HSCs as compared to wild type HSCs. Interestingly, unlike Runx1 knockout, which confers resistance to ER stress by inhibiting ribosome biogenesis and protein synthesis, we observed increased protein synthesis in NrasG12D HSCs. Taken together, we conclude that N-RasG12D dysregulates IRE1alpha-Xbp1s signaling to promote adaptation to ER-stress, re-balancing protein homeostasis, and maintaining the integrity of the HSC pool to promote pre-leukemic hematopoietic stem cell expansion. DisclosuresNo relevant conflicts of interest to declare.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

The iodosulfonamidation of peracetylated glycals revisited: access to 1,2-di-nitrogenated sugars

Iodosulfonamidation of peracetylated glycals was investigated using either a combination of N-iodosuccinimide/iodine or iodine chloride as a source of iodonium ion. 1,2- trans- and 1,2- cis-2-deoxy-2-iodo-1-sulfonamido hexoses were, respectively, obtained depending on the reagent system used. Both series of isomers were successfully converted to 1,2-di-nitrogenated compounds, for example, 1-azido-1,2-dideoxy-2-sulfonamido sugars, which are useful intermediates for the synthesis of N-linked glycoproteins or glycoconjugates.

Novel mannosidase inhibitors probe glycoprotein degradation pathways in cells

Multiple isoforms of mammalian alpha-mannosidases are active in the pathways of N-linked glycoprotein synthesis and catabolism. They differ in specificity, function and location within the cell and can be selectively inhibited by imino sugar monosaccharide mimics. Previously, a series of structurally related novel 7-membered iminocyclitols were synthesised and found to be inhibitors of alpha-mannosidase using in vitro assays. The present study aimed to delineate alpha-mannosidases hydrolytic pathways in azepane inhibitor treated cells by the analysis of free oligosaccharides (FOS) as markers of endoplasmic reticulum (ER), Golgi, lysosomal and cytosolic alpha-mannosidase activities. Two compounds were identified as potent and selective cytosolic alpha-mannosidase inhibitors. Two related compounds were shown to be potent inhibitors of lysosomal alpha-mannosidase with different potencies towards alpha1,6 mannosidase. The specificities of these novel 7-membered imino sugars are related to differences in their structure and D: -mannose-like stereochemistry. Specific ER-mannosidase inhibition by kifunensine also reveals significant non-proteasomal degradation following FOS analysis and appears to be cell line dependent. The availability of more selective inhibitors allows the pathways of N-linked oligosaccharide metabolism to be dissected.

High extracellular glucose impairs cardiac E-C coupling in a glycosylation-dependent manner.

Hyperglycemia is a major manifestation of all forms of diabetes mellitus and is associated with increased risk of cardiovascular disease. It is well established that cardiac excitation-contraction (E-C) coupling is adversely affected in diabetic animals such that ventricular myocyte action potential duration is prolonged and intracellular Ca2+ clearing and mechanical relaxation are slowed. We now report that ventricular myocytes incubated in a culture medium containing high extracellular glucose (25.5 mM) also exhibit these same changes in E-C coupling. These effects are not manifested for approximately 24 h after exposure. Furthermore, in the presence of normal glucose (5.5 mM), relaxation is also prolonged by fructose (20 mM), yet is unaffected by equimolar concentrations of nonmetabolizable sugars such as L-glucose and mannitol, implying that the high glucose effects require glucose entry into the cell and metabolic processing. The prolonged relaxation can also be produced by 5 mM glucosamine (an intermediate of glycosylation) and is blocked by 0.5 microgram/ml tunicamycin (an inhibitor of N-linked glycoprotein synthesis). Culturing myocytes with an inhibitor of glycation (10 mM aminoguanidine) does not prevent the high extracellular glucose concentration effects. Thus our data indicate that high extracellular glucose impairs cellular mechanisms contributing to myocardial relaxation and that this impairment may involve glycosylation of nascent proteins.

Inhibition of Glycosylation by Amphomycin and Sugar Nucleotide Analogs PP36 and PP55 Indicates That Haloferax volcanii β-Glucosylates Both Glycoproteins and Glycolipids through Lipid-Linked Sugar Intermediates: Evidence for Three Novel Glycoproteins and a Novel Sulfated Dihexosyl-Archaeol Glycolpid

Archaebacteria have been recently placed in evolution as a separate kingdom of organisms between procaryotes and eucaryotes. Although these organisms contain both glycolipids and glycoproteins, they possess no Golgi. No biosynthetic work has been published on the complex carbohydrates of these newly reassigned organisms. This report describes preliminary results from one member of this kingdom, Haloferax volcanii, which suggest that all glycosylation proceeds through lipid intermediates. Evidence for novel glycolipid structure was also found during this study. H. volcanii plasma membranes contain all of the enzyme activities for synthesis of N-linked glycoproteins and archaeol-based glycolipids. For glucose transfer, all reactions apparently proceed through glucose-phosphopolyisoprenol using UDP-glucose as primary donor. Incorporation of D-[3H]glucose from UDP-D-[3H]glucose into glycoproteins and glycolipids of H. volcanii was stimulated by addition of C55-polyisoprenol phosphate, but not by C85-105 dolichol phosphate, and was inhibited by amphomycin and two recently described sugar nucleotide analogs, PP36 (5′-[N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxy carbonyl)glycyl]amino]-5′-deoxyuridine) and PP55 (5′-O-[[(2-decanoylamino-3-phenylpropyloxycarbonyl)amino]sulfonyl]uridine). All three inhibitors are reported to block transfer of sugar from UDP-sugars to phosphopolysioprenols in eucaryotes. However, in H. volcanii these inhibitors apparently block transfer of glucose from polyprenyl intermediates to final glycoproteins and glycolipid products. The sulfodihexosyl archaeol glycolipid fraction was partially characterized by mass spectrometry and was found to contain a previously unreported structure with sulfate on the reducing-end sugar. Four major glycoproteins 190, 105, 56, and 52 kDa and an archaeol-based glycolipid fraction were labeled by amphomycin-sensitive pathways. Photoaffinity labeling of H. volcanii homogenate with 5-azido-[32P]UDP-Glc tagged only one 45-kDa polypeptide which is a probable glucosyl-phosphoryl-polyisoprenol synthase. The fact that only one polypeptide band was photoaffinity-labeled indicated that no other transferase utilized UDP-glucose directly in H. volcanii. The salt requirement of the UDP-glucose-dependent pathways suggests that cytoplasmic enzymes function in a high salt environment in H. volcanii. The archaebacterial plasma membrane thus expresses many functions for glycosylation of both glycoproteins and glycolipids, normally found in the endoplasmic reticulum and Golgi of eucaryotes.

Biosynthetic and Structural Studies on Thy-1 in a Rat Neuronal Tumor Cell Line

Because of the limited information available about the synthesis of N-linked glycoproteins in nerve cells, in regard to both processing steps and enzyme characterization, the biosynthetic processing of Thy-1 of the rat neuronal tumor cell line BN-1010-1 has been investigated using several inhibitors of biosynthesis and transport. (i) Tunicamycin completely inhibited mannose incorporation into Thy-1. Unglycosylated Thy-1 was not transported to the cell surface and was probably degraded rapidly following synthesis. (ii) Brefeldin A completely inhibited the transport of all [3H]mannose-labeled proteins releasable by phosphatidylinositol-specific phospholipase C, including Thy-1, to the surface of BN-1010-1 cells. Removal of the inhibitor led to rapid reversal of the inhibition. Pulse-chase experiments demonstrated that ≍50% of Thy-1 was degraded after 4 h in the presence of brefeldin A. (iii) Castanospermine treatment slowed the appearance of Thy-1 on the cell surface. The surface Thy-1 contained mainly normal Man5, Man6, and Man7 oligosaccharides, suggesting that Golgi endo-α-D-mannosidase effected the removal of glucose. (iv) Treatment with deoxymannojirimycin resulted in the synthesis of Thy-1 that contained Man8 and Man9 oligosaccharides compared to Man5, Man6, and Man7 in the control. Neither the rate of appearance nor the level of surface expression was affected by the drug. (v) Swainsonine did not affect either the rate of appearance or the level of surface expression of Thy-1. The HPLC elution profile of neutral oligosaccharides resulting from Endo-H digestion of Thy-1 synthesized in the presence of swainsonine was indistinguishable from controls. The lack of an effect of swainsonine is explained by the unexpected absence of a complex type oligosaccharide in Thy-1 of BN-1010-1 cells, as shown in experiments with a variety of lectins as well as digestions with Endo-H or glycopeptidase F, or digestions with both enzymes in sequence. The fact that, after [3H]fucose-labeling, Endo-H digestion produced Thy-1 still labeled with fucose indicates that hybrid oligosaccharide is present in Thy-1.

Effects of growth factors on cell cycle arrest in dolichyl phosphate-depleted cultures.

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth-limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N-linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133-140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382-389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin-like growth factor-1 (IGF-1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface.

Glycosylation site-binding protein is not required for N-linked glycoprotein synthesis.

In prior studies we identified a 57-kDa protein in the lumen of the endoplasmic reticulum that, in addition to having both protein disulfide isomerase and thyroid hormone-binding protein activities, bound a photoaffinity probe containing the N-glycosylation-site sequence Asn-Xaa-Ser/Thr. It was hypothesized that this multifunctional protein, called glycosylation site-binding protein (GSBP), participated in the process of N-glycosylation of proteins. To test this hypothesis we have employed various conditions to deplete the lumen of GSBP and then assess the level of N-glycosylation catalyzed by oligosaccharyltransferase (OTase). Although most conditions leading to depletion resulted in partial loss of OTase activity, this loss was independent of the extent of GSBP depletion. Indeed, virtually complete loss (greater than 99%) of GSBP with partial retention of OTase activity was frequently observed. Moreover, repletion of the microsomal lumen with GSBP did not restore OTase activity to control levels. Thus, no correlation between GSBP content and OTase activity before or after reconstitution was found. These results suggest that this multifunctional 57-kDa protein is not an essential component of the enzymatic reaction in which oligosaccharide chains are transferred from dolichyl-P-P-GlcNAc2Man9Glc3 to nascent polypeptides or to synthetic tripeptide acceptors.

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [ 3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.

Selective activation of the N-glycosylation apparatus in uteri by estrogen.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional level.

Estrogen induces N-linked glycoprotein expression by immature mouse uterine epithelial cells.

Characterization of complex glycoconjugates and the effects of estrogen on their expression in immature mouse uterine epithelial cells are reported. The secreted fraction contained nonanionic, O-linked lactosaminoglycan (LAG)-bearing proteins of Mr 30,000-40,000 as well as anionic, O-linked, LAG-bearing glycoproteins with very high apparent molecular weight (greater than 670K). Heparan sulfate (HS) proteoglycans and HS linked to little or no protein were found in the secreted fraction as well. A very similar array of glycoconjugates was found in the nonhydrophobic fraction of cell-associated macromolecules. In addition, the hydrophobic cell-associated fraction contained nonanionic, LAG-bearing glycoproteins of approximately 250K, anionic LAG-bearing glycoproteins distributing over a wide range of molecular weights, and HS proteoglycans with median molecular weights of approximately 250K. In contrast to the glycoproteins produced by their mature counterparts, virtually all glycoproteins produced by immature cells were O-linked. Estrogen treatment of immature mice caused uterine epithelial cells to secrete anionic, high molecular weight (greater than 670K) N-linked glycoproteins as a major product. These estrogen-responsive glycoproteins did not appear to contain LAGs. Estrogen treatment also markedly decreased the proportion of all hydrophobic glycoconjugates in the cell-associated fraction. Collectively, these observations indicate that one aspect of the estrogen-induced maturation of uterine epithelial cells is the stimulation of N-linked glycoprotein synthesis and secretion. Furthermore, stimulation of N-linked glycoprotein synthesis by itself is insufficient to support N-linked LAG glycoprotein production.

Dolichyl phosphate: rapid increase and predominant form of brain dolichol compounds during early brain development.

Of brain dolichol compounds, dolichyl phosphate is most critical since it serves as the carrier of saccharide moieties to be utilized for N-linked protein glycosylation. The objectives of the current study were to determine (1) whether the proportion of dolichyl phosphate to dolichol in brain is relatively large and/or changes with development, and (2) whether the subcellular distribution of these isoprenoid compounds differs and/or changes with development. A recently described, improved technique for isolation and quantitation of dolichyl phosphate was employed. The data show that in the first 4 weeks of postnatal life in the rat dolichyl phosphate is, in fact, the predominant form of total dolichol compounds in brain. Subsequently, concentrations of dolichol exceeded those of dolichyl phosphate in adult brain, particularly with aging. Dolichyl phosphate was found to be localized primarily in microsomes; dolichol was found in only small amounts in microsomes but was enriched in the fraction that contained lysosomes. The observations indicate that during early brain development, an active time period for differentiating events, many of which are mediated by glycoproteins, dolichyl phosphate is present in high concentration at the subcellular site of N-linked glycoprotein synthesis, i.e., endoplasmic reticulum.

Dolichol metabolism in the rat

Dolichyl phosphate (Dol-P) is a long chain polyisoprenoid which transfers saccharide residues in N-linked glycoprotein synthesis. It is synthesized by a quantitatively small branch of the cholesterol pathway at a rate which, at least in liver, appears to be independent of the rate of sterol synthesis. This article reviews recent work on the fate of dolichol compounds and the regulation of Dol-P synthesis.

Regulation of Glycoprotein Synthesis During Embryonic Development of the Sea Urchin

We are interested in the regulation of glycoprotein synthesis in the sea urchin embryo and the role that these glycoproteins play in gastrulation. Earlier reports from this laboratory on the effect of tunicamycin on embryonic development in sea urchins have provided strong evidence that a marked increase in the synthesis of N-linked glycoproteins occurs at gastrula? tion. If N-linked glycoprotein synthesis is blocked, gastrulation does not occur (Schneider et al, 1978; Heifetz and Lennarz, 1979). It has been established that in a number

Do sugar residues contribute to the antigenic determinants responsible for protection and/or abolition of protection in Leishmania-infected BALB/c mice?

Intravenous inoculation of irradiated virulent promastigotes of Leishmania mexicana, or intradermal inoculation of avirulent temperature-sensitive clones of the same parasite, can protect BALB/c mice from progressive disease when challenged with parental organisms. However, if animals are prechallenged s.c. with irradiated parental parasites before (or shortly after, i.e., within 10 days) any other immunization regime is used, the s.c. challenge effectively suppresses development of protective immunity. The role of N-linked sugars on promastigotes in providing the determinants responsible for protection and/or suppression of protection in these assays has been examined. Growth of parasite in 2 micrograms/ml tunicamycin has no effect on incorporation of [3H]leucine but decreases incorporation of [3H]mannose by some 40 to 50%. Such tunicamycin-treated avirulent clones or irradiated virulent organisms are now unable to induce a protective response. However, tunicamycin-treated parental parasites given s.c. could still suppress the protection seen when irradiated parasites were given i.v. as immunogen. Tunicamycin-treated avirulent organisms given s.c. subcutaneously unlike the untreated avirulent clones, now also caused suppression of protection. These data suggest that the determinants responsible for development of protective immunity to L. mexicana in BALB/c mice are dependent on N-linked glycoproteins for their expression, unlike the determinants responsible for suppression of that protection. By using swainsonine as an alternative inhibitor of N-linked glycoprotein synthesis, the data additionally suggest that no complex processing of N-linked sugars takes place to reveal the immunogenic determinants in this system.

A comparison of glycoprotein biosynthesis in basal cell carcinoma and normal epidermis.

Summary Protein and N-linked glycoprotein biosynthesis in normal epidermis and basal cell carcinomas were measured by the incorporation of 3[H] leucine and 3[H] mannose. Compared with normal epidermis, basal cell carcinomas had a reduced overall protein synthesis accompanied by an increased N-linked glycoprotein synthesis. Polyacrylamide gel electrophoresis showed that in the tumours the glycoprotein synthesis was restricted to molecular weight ranges with peaks at 95K (74K) and 38K, whereas in normal epidermis they were found at 95K, 62K, 52K and 43K.

Regulation of levels of dolichol and dolichyl phosphate during germination and early development of soybeans

In a previous study (Ravi, K., Rip, J.W. and Carroll, K.K. (1983) Biochem. J. 213, 513–518) soybeans were found to contain considerable amounts of dolichol but only traces of dolichyl phosphate (Dol-P). Further experiments have suggested that dolichol intermediates are involved in glycolipid and glycoprotein biosynthesis during germination, and have shown that the dolichol content of soybeans decreases over the first few days after germination, while the Dol- p content increases. These observations suggested the presence of an enzyme capable of phosphorylating preexisting endogenous dolichol (accompanying paper, Ravi, K., Rip, J.W. and Carroll, K.K. (1986) Biochim. Biophys. Acta 875, 618–625). Assays using soybean microsomes indicated that dolichol kinase activity increased over the first 3–4 days of germination and then became less active. After the third day, the dolichol content of seedlings increased gradually, whereas the concentration of Dol- p decreased until it was not detectable. The activity of Dol- P-phosphatase, a second enzyme involved in regulating Dol- p concentrations, increased over the first 5 days after germination. The phosphatase may have a biosynthetic role in supplying dolichol from de novo synthesis for (re)phosphorylation by dolichol kinase. All portions of 15-day-old seedlings: roots, stems, leaves and cotyledons contained dolichol, with the highest concentration in cotyledons. There appeared to be no difference in distribution of homologues among different parts of the plant or at different stages of development. Dol- p formed during germination may be utilized for synthesis of N-linked glycoproteins required during germination.

Regulation of synthesis of N-linked glycoproteins and their lipid intermediates in human T lymphoblastoid cells

1. 1. The synthesis of N-linked glycoproteins and their lipid intermediates was investigated in cell-free preparations of human T lymphoblastoid cells during two phases of cell growth. 2. 2. The incorporation of 14C-labeled Man into glycoproteins and dolichol-linked oligosaccharides was greater during the logarithmic growth phase than the stationary phase. 3. 3. The incorporation of 14C-labeled GlcNAc into dolichol derivatives was increased in the logarithmic phase. 4. 4. However, the synthesis of Dol-P-Man was not significantly different. 5. 5. These data suggest that the differences are due, at least partially, to the increased synthesis of Dol-P-P-GlcNAc.