Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells

We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.     

Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

Biosynthesis and intracellular transport of the mouse macrophage Fc receptor

The membrane insertion, processing, and intracellular transport of the mouse macrophage Fc receptor for IgG1/IgG2b was studied using specific mono- and polyclonal anti-receptor antibodies. By immunoprecipitation from Triton X-114 lysates of radiolabeled J774 cells, we determined that the mature 60-kDa receptor is a transmembrane glycoprotein which is synthesized in the rough endoplasmic reticulum as a 53-kDa precursor. Digestion of the precursor with endo-beta-N-acetylglucosaminidase F demonstrated that the receptor consisted of a 37-kDa polypeptide to which four asparagine-linked oligosaccharides were attached. Proteinase K treatment of isolated microsomes indicated that the receptor also has a putative 15-kDa cytoplasmic domain apparently recognized by at least one anti-Fc receptor monoclonal antibody. An additional 15-kDa domain was found to be inaccessible to proteolysis from either side of the membrane. Pulse-chase experiments using [35S]methionine-, [3H]mannose-, and [3H]galactose-labeled cells showed that processing of the receptor's N-linked oligosaccharides occurred rapidly (t1/2 = 15 min) and resulted in the conversion of at least three of the chains to complex endo-beta-N-acetylglucosaminidase H-resistant forms. O-Linked oligosaccharides were not detected. Fc receptor was detected on the plasma membrane 30 min after its synthesis. Transport of newly synthesized receptors to the plasma membrane was slowed but not blocked by incubation of J774 cells at 20 degrees C or by the carboxylic ionophore monensin, although monensin completely inhibited the galactosylation of the receptor.     

Role of glycosylation for beta 2-adrenoceptor function in A431 cells

A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.     

The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides

The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.     

Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Biosynthesis and acquisition of biological activity of the fibronectin receptor

The biosynthesis of the 140-kilodalton fibronectin receptor complex by cultured 3T3 mouse cells was characterized and compared with that of chick embryo fibroblasts. Three murine glycoprotein components of 140-150 kilodaltons (band 1), 125 kilodaltons (band 2), and 105 kilodaltons (band 3) could be immunoprecipitated from metabolically labeled 3T3 cells using polyclonal antibodies. In pulse-chase experiments, bands 1 and 3 of the mouse receptor were labeled to maximal levels immediately after completion of the labeling pulse. However, band 2 was not detected at short chase times, and it reached maximal labeling only after approximately 12 h of chase. The appearance of band 2 occurred at the same rate as the disappearance of band 3. Only bands 1 and 2 could be affinity purified by binding to immobilized fibronectin cell-binding fragment, indicating that they represent mature functional receptor components. When 3T3 cells were incubated with radioactive sugars, band 1 of the receptor labeled well with both [3H]mannose and [3H]glucosamine. However, band 2 labeled well with [3H]glucosamine but contained low amounts of mannose, and band 3 labeled well with [3H]mannose but contained low amounts of glucosamine. Digestion of both bands 2 and 3 with endoglycosidase F yielded similar-sized products of approximately 88,000 daltons, suggesting that post-translational asparagine-linked oligosaccharide processing can account for most of the size difference between these bands. These data suggest that in the mouse fibronectin receptor, band 3 is a biologically inactive precursor of band 2 that does not appear on the cell surface. In contrast, pulse-chase experiments using chicken embryo fibroblasts indicated that the three components of the chicken 140k complex were distinct moieties. Our results demonstrate distinct types of processing for fibronectin receptor complexes from different species. In mammalian cells, this receptor undergoes a surprisingly long (20 h) maturation process involving asparagine-linked oligosaccharides before reaching its final, biologically active form.     

Action of low-density lipoprotein and compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, on the synthesis of dolichol-linked oligosaccharides and low-density-lipoprotein receptor in human skin fibroblasts.

To explore whether there is an inter-relationship between the rate of low-density (LD) lipoprotein binding to its receptor and the formation of dolichol-linked oligosaccharides, experiments were performed with human fibroblasts where the synthesis of lipoprotein receptor and dolichyl saccharides was under control of LD lipoprotein and compactin. Pretreatment of the cells with nonlabelled LD lipoprotein resulted in a suppression of both the binding of 125I-labelled LD lipoprotein to the receptor and the synthesis of dolichyl saccharides from [14C]acetate and [3H]mannose, but not from [3H]mevalonolactone. Compactin, in contrast, inhibited only the formation of dolichol-linked oligosaccharides. Mevalonolactone (1 microM) abolished the inhibitory effect of LD lipoprotein on dolichyl saccharide formation, but was not able to restore the receptor-binding capacity, thus suggesting that the synthesis of lipoprotein receptor is not coupled to the formation of dolichyl saccharides.     

Calmodulin antagonists stimulate LDL receptor synthesis in human skin fibroblasts

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.     

Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.).

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).     

High-density lipoprotein-associated 17beta-estradiol fatty acyl ester uptake by Fu5AH hepatoma cells: implications of the roles of scavenger receptor class B, type I and the low-density lipoprotein receptor

17beta-estradiol (E2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E2 cellular uptake and to determine the intracellular distribution of E2 and its fatty acyl esters (E2-FAE) after uptake. [3H]E2 or [3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [3H]E2-FAE or [3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[3H]cholesterol, cellular uptake of HDL-[3H]E2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E2 uptake. HDL-E2-FAE was hydrolyzed into free E2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E2-FAE into free E2 ensues.     

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.     

Fatty acylation of yeast glycoproteins proceeds independently of N-linked glycosylation

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild-type and asparagine-linked glycosylation-deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non-permissive temperature (37 degrees C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS-PAGE. In contrast, the wild-type yeast cells contained [3H]palmitate-labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS-PAGE. In addition, labeling of the wild-type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non-permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N-glycosylation in yeast cells.     

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound ''high-mannose'' oligosaccharides.

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.     

Uptake and metabolism of lactosylceramide on low density lipoproteins in cultured proximal tubular cells from normal and familial hypercholesterolemic homozygotes

The metabolism of low density lipoproteins (LDL), and LDL modified by reductive methylation (M-LDL) of lysine residues, was studied in proximal tubular (PT) cells both from normal human kidney and from urine of patients with homozygous (LDL receptor-negative) familial hypercholesterolemia (FH). LDL and M-LDL was labeled either in the protein moiety with 125I or in the lactosylceramide moiety with 3H. The binding and degradation of 125I-LDL in normal cells was saturable and displaced by unlabeled LDL but not by M-LDL. The uptake of [3H]lactosylceramide (LacCer) low density lipoprotein in normal renal cells was saturable, and time and temperature-dependent. Exogenously derived [3H]LacCer on LDL was rapidly taken up and catabolized to monoglycosylceramide, or it was utilized for the endogenous synthesis of globotriaosylceramide (trihexosylceramide) and globotetraosylceramide (tetraglycosylceramide). [3H]LacCer M-LDL was taken up less avidly and metabolized less extensively than [3H]LacCer-LDL in normal cells. In homozygous FH renal cells the binding of 125I-LDL was not saturable and not displaced by unlabeled LDL. 125I-LDL degradation did not occur in FH cells. The homozygous FH PT cells took up a 2-fold greater amount of exogenously derived [3H]LacCer on LDL than normal cells. Yet, most of the [3H]LacCer taken up by FH PT cells accumulated as LacCer, and only small amounts were metabolized to monoglycosylceramide, globotriaosylceramide (trihexosylceramide), or globotetraosylceramide (tetraglycosylceramide). When normal and FH PT cells were preincubated with LDL (0-100 micrograms/ml medium), there was a 5-fold increase in cellular LacCer levels in FH cells at saturating levels of LDL, whereas there was about a 50% decrease in LacCer levels in normal cells. While the high affinity binding of LDL was not essential for the delivery of LacCer to cells, the data support the conclusion that LDL binding to the LDL receptor facilitates further LacCer processing and metabolism in normal renal cells. We speculate that [3H] LacCer is taken up by FH homozygous cells via a LDL receptor-independent mechanism and accumulates in the cells without significant metabolism. LacCer taken up by this mechanism contributes to the storage of LacCer in FH PT cells.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

4-Deoxy-4-fluoro-D-mannose inhibits the glycosylation of the G protein of vesicular stomatitis virus

The effect of 4-deoxy-4-fluoro-D-mannose (4F-Man), a synthetic analog of D-mannose, on the synthesis of the glycoprotein (G) of vesicular stomatitis virus was examined. Nearly confluent monolayers of cultured BHK21 cells infected with vesicular stomatitis virus were incubated for 2 h with 4F-Man (0-10 mM) or for 1 h with tunicamycin (2 micrograms/ml) and then pulse-labeled with [35S]methionine or [3H]glucosamine. After a 90-min chase period, the cells were lysed and the viral proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The 35S-labeled G protein from cells exposed to greater than or equal to 1 mM 4F-Man migrated more rapidly than G protein isolated from control cells and with the same electrophoretic mobility as the glycoprotein produced by cells treated with tunicamycin. When infected cells were labeled with [3H]glucosamine, little or no radioactivity was associated with G protein synthesized in the presence of greater than or equal to 1 mM 4F-Man. The conclusion that 4F-Man blocks the glycosylation of the G protein was supported by experiments which demonstrated that the fluorosugar inhibits the synthesis of lipid-linked oligosaccharides.