Biomedical subjects
Publications and source records attributed to Raymond A Dwek.
We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.
Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.
Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.
Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain. We present the analysis of both the N- and O-glycans on the individual peptides from this complex. Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains. The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked beta1-4 and beta1-3 to GlcNAc, fucose-linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, and alpha2-3 and alpha2-6-linked sialic acids. These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity. We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannose-binding lectin, by disrupting the SC-H chain noncovalent interactions.
Prostate-specific antigen (PSA) is a glycoprotein secreted by prostate epithelial cells. PSA is currently used as a marker of prostate carcinoma because high levels of PSA are indicative of a tumor situation. However, PSA tests still suffer from a lack of specificity to distinguish between benign prostate hyperplasia and prostate cancer. To determine whether PSA glycosylation could provide a means of differentiating between PSA from normal and tumor origins, N-glycan characterization of PSA from seminal fluid and prostate cancer cells (LNCaP cell line) by sequencing analysis and mass spectrometry was carried out. Glycans from normal PSA (that correspond to low and high pI PSA fractions) were sialylated biantennary complex structures, half of them being disialylated in the low pI PSA fraction and mostly monosialylated in the high pI PSA. PSA from LNCaP cells was purified to homogeneity, and its glycan analysis showed a significantly different pattern, especially in the outer ends of the biantennary complex structures. In contrast to normal PSA glycans, which were sialylated, LNCaP PSA oligosaccharides were all neutral and contained a higher fucose content. In 10-15% of the structures fucose was linked alpha1-2 to galactose, forming the H2 epitope absent in normal PSA. GalNAc was increased in LNCaP glycans to 65%, whereas in normal PSA it was only present in 25% of the structures. These carbohydrate differences allow a distinction to be made between PSA from normal and tumor origins and suggest a valuable biochemical tool for diagnosis and follow-up purposes.
The N-alkylated imino sugars have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase. A therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to reduce biosynthetic capability in the cell to match the reduced lysosomal catalytic activity seen in lysosomal storage disorders. The use of N-alkylated imino sugars to establish this therapeutic strategy is described in cell culture and gene knockout mouse disease models. One imino sugar, N-butyl-DNJ (NB-DNJ) has been in clinical trials for type 1 Gaucher disease and has shown to be an effective therapy for this disorder.
The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.
During mammalian spermatogenesis, male germ cells undergo a dramatic transformation, which includes a change of shape, nuclear condensation, and development of specialised structures, such as an acrosome, and a flagellum with a mitochondrial sheath. We have found a previously undescribed pharmacological approach to intervene in these events. After oral administration of the alkylated imino sugar N-butyldeoxynojirimycin (NB-DNJ) to mice, epididymal spermatozoa displayed a spectrum of abnormal head shapes, and acrosomal antigens were mostly absent or displayed irregular patterns. In addition, the mitochondria of these cells often had an aberrant morphology, and were arranged in relatively short and wide mitochondrial sheaths. The motility of the affected spermatozoa was severely impaired. After 3 weeks of administration of NB-DNJ, male mice became sterile, and regained their fertility during the fourth week off drug. The NB-DNJ-induced infertility was not associated with a reduction in the serum testosterone level. Biochemically, the capacity of imino sugars to impair spermatogenesis was associated with their potential to attenuate the biosynthesis of glucosylceramide-based sphingolipids. Our study reveals that male fertility is affected by partial glycosphingolipid depletion, or, alternatively, by a distinct as yet unidentified property that is shared by alkylated imino sugars that inhibit glucosylceramide biosynthesis. These compounds therefore may be new leads in the development of a male contraceptive, especially because NB-DNJ has already been through extensive evaluation in various mammals, including man.
Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.
Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.
Glycosylation analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to the major histocompatibility complex class I heavy chains. Free heavy chains were isolated from the beta(2)-microglobulin-negative cell line Daudi and from the B-lymphoblastoid cell line Raji. Heavy chains were also isolated from Raji cells in multimolecular complexes (peptide loading complexes) containing the transporter associated with antigen processing, tapasin and ERp57 with and without the lectin-like folding chaperone, calreticulin. Calreticulin is a soluble protein that recognizes primarily the terminal glucose of Glc(1)Man(7-9)GlcNAc(2) glycans. This paper shows that monoglucosylated glycoforms of heavy chain, which exist transiently in the endoplasmic reticulum in the initial stages of the glycosylation processing pathway, are present in the peptide loading complex. The data are consistent with a model in which the release of peptide-loaded major histocompatibility complex class I molecules from calreticulin, induced by deglucosylation of the heavy chain N-linked glycan, signals the dissociation of the complex. This is consistent with the hypothesis that the class I loading process is an adaptation of the quality control mechanism involving calreticulin and ERp57.
Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, closely related to, and used as a surrogate model for the hepatitis C virus. Its envelope contains the E1 and E2 glycoproteins, disulfide linked into homo- and heterodimers. In this study, we investigate the role of disulfide bond formation in the folding, assembly, and stability of BVDV glycoproteins. We provide molecular evidence that intact disulfide bonds are critical for the acquirement of a stable conformation of E2 monomers. Forcing the E2 glycoproteins to adopt a reduced conformation either co- or post-translationally before assembly into dimers, determines their misfolding and degradation by proteasome. In contrast, dimerization of E2 glycoproteins results in a conformation resistant to reducing agents and degradation. Furthermore, inhibition of the ER-alpha-mannosidase activity leads to impairment of misfolded E2 degradation, demonstrating the involvement of this enzyme in targeting viral proteins towards proteasomal degradation.
We have reductively alkylated deoxynojirimycin imino sugars using sodium cyanoborohydride to provide an efficient means of generating a series of N-alkylated compounds containing 4-18 carbon side chains. The yields were greater than 90% using a variety of aldehydes of different chain lengths, and after purification were >95% pure using (1)H-NMR. Radiolabelled compounds were prepared using sodium cyanoborotriti-ide to selectively label the first carbon atom in the alkyl chain and used in protein-binding and cell- and tissue-uptake experiments. Protein binding was chain-length-dependent with compounds of intermediate chain length (C(9)-C(12)), demonstrating an equal distribution between the aqueous and protein-bound phase. The extent of cell uptake also increased proportionally with increased chain length in a time-dependent manner. When administered to mice, the longer alkyl-chain compounds showed reduced absorption from the intestine and a marked deposition of compound in the liver and brain, suggesting that the more hydrophobic compounds were poorly cleared by the major tissues. In tissue-culture cells compounds with 8 or fewer carbon atoms were non-toxic and had CC(50) (the concentration at which the number of cells or cell proliferation is reduced by 50%) values greater than 1 mM. Compounds with chain lengths above C(8) showed a chain-length-dependent increase in cytotoxicity. N-alkylated deoxynojirimycins (C(4)-C(18)) were evaluated for their inhibitory effects on ceramide-specific glucosyltransferase and glycoprotein-processing alpha-glucosidase. Increasing the alkyl chain length had little effect on alpha-glucosidase activity, but inhibition of ceramide-specific glucosyltransferase increased 10-fold when C(4) and C(9)-C(18) compounds were compared. Overall these data provide further definition of the molecular features of alkylated imino sugars that influence tissue selectivity and efficacy for cellular enzyme inhibition.
The crystal structure of IgG1 b12 represents the first visualization of an intact human IgG with a full-length hinge that has all domains ordered and visible. In comparison to intact murine antibodies and hinge-deletant human antibodies, b12 reveals extreme asymmetry, indicative of the extraordinary interdomain flexibility within an antibody. In addition, the structure provides an illustration of the human IgG1 hinge in its entirety and of asymmetry in the composition of the carbohydrate attached to each C(H)2 domain of the Fc. The two separate hinges assume different conformations in order to accommodate the vastly different placements of the two Fab domains relative to the Fc domain. Interestingly, only one of two possible intra-hinge disulfides is formed.
Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.
A sensitive, rapid, quantitative strategy has been developed for O-glycan analysis. A structural database has been constructed that currently contains analytical parameters for more than 50 glycans, enabling identification of O-glycans at the subpicomole level. The database contains the structure, molecular weight, and both normal and reversed-phase HPLC elution positions for each glycan. These observed parameters reflect the mass, three-dimensional shape, and hydrophobicity of the glycans and, therefore, provide information relating to linkage and arm specificity as well as monosaccharide composition. Initially the database was constructed by analyzing glycans released by mild hydrazinolysis from bovine serum fetuin, synthetic glycopeptides, human glycophorin A, and serum IgA1. The structures of the fluorescently labeled sugars were determined from a combination of HPLC data, mass spectrometric composition and mass fragmentation data, and exoglycosidase digestions. This approach was then applied to human neutrophil gelatinase B and secretory IgA, where 18 and 25 O-glycans were identified, respectively, and the parameters of these glycans were added to the database. This approach provides a basis for the analysis of subpicomole quantities of O-glycans from normal levels of natural glycoproteins.
The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.
Recently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress.