Taste cell-expressed α-glucosidase enzymes contribute to gustatory responses to disaccharides.

The primary sweet sensor in mammalian taste cells for sugars and noncaloric sweeteners is the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by Tas1r2 and Tas1r3 genes). However, in the absence of T1R2+T1R3 (e.g., in Tas1r3 KO mice), animals still respond to sugars, arguing for the presence of T1R-independent detection mechanism(s). Our previous findings that several glucose transporters (GLUTs), sodium glucose cotransporter 1 (SGLT1), and the ATP-gated K(+) (KATP) metabolic sensor are preferentially expressed in the same taste cells with T1R3 provides a potential explanation for the T1R-independent detection of sugars: sweet-responsive taste cells that respond to sugars and sweeteners may contain a T1R-dependent (T1R2+T1R3) sweet-sensing pathway for detecting sugars and noncaloric sweeteners, as well as a T1R-independent (GLUTs, SGLT1, KATP) pathway for detecting monosaccharides. However, the T1R-independent pathway would not explain responses to disaccharide and oligomeric sugars, such as sucrose, maltose, and maltotriose, which are not substrates for GLUTs or SGLT1. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we found that taste cells express multiple α-glycosidases (e.g., amylase and neutral α glucosidase C) and so-called intestinal "brush border" disaccharide-hydrolyzing enzymes (e.g., maltase-glucoamylase and sucrase-isomaltase). Treating the tongue with inhibitors of disaccharidases specifically decreased gustatory nerve responses to disaccharides, but not to monosaccharides or noncaloric sweeteners, indicating that lingual disaccharidases are functional. These taste cell-expressed enzymes may locally break down dietary disaccharides and starch hydrolysis products into monosaccharides that could serve as substrates for the T1R-independent sugar sensing pathways.

Resistance to Mutant Group 2 Influenza Virus Neuraminidases of an Oseltamivir-Zanamivir Hybrid Inhibitor.

Influenza virus neuraminidase (NA) drug resistance is one of the challenges to preparedness against epidemic and pandemic influenza virus infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug resistance of the influenza virus N1 NA is well characterized. Yet drug resistance of the N2 strain is not well understood. Here, we confirm that replacement of N2 E119 or I222 results in multidrug resistance, and when the replacements occur together, the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we showed that E119 replacement results in a loss of hydrogen bonding to oseltamivir and zanamivir, whereas I222 replacement results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we found that MS-257, a zanamivir-oseltamivir hybrid inhibitor, is less susceptible to drug resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug resistance. MS-257 serves as a next-generation anti-influenza virus drug candidate and serves also as a scaffold for further design of NAIs. IMPORTANCE: Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.

A Second, Druggable Binding Site in UDP-Galactopyranose Mutase from Mycobacterium tuberculosis?

UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.

Synthesis and evaluation of influenza A viral neuraminidase candidate inhibitors based on a bicyclo[3.1.0]hexane scaffold.

This manuscript describes a novel class of derivatives based on a bicyclo[3.1.0]hexane scaffold, proposed as mimics of sialic acid in a distorted boat conformation that is on the catalytic pathway of neuraminidases (sialidases). A general synthetic route for these constrained-ring molecules was developed using a photochemical reaction followed by a Johnson-Corey-Chaykovsky cyclopropanation. Functionalization with the goal of occupying the 150-cavity was also exploited. Inhibition assays demonstrated low micromolar inhibition against both group-1 (H5N1) and group-2 (H9N2) influenza neuraminidase subtypes, indicating good affinity for the alpha and beta sialic acid mimics and 150-cavity-targeted derivatives. These results provide a validation of a bicyclo[3.1.0]hexane scaffold as a mimic of a distorted sialic acid bound in the neuraminidase active site during catalysis.

Naturally occurring sulfonium-ion glucosidase inhibitors and their derivatives: a promising class of potential antidiabetic agents.

In humans, four different enzymes mediate the digestion of ingested carbohydrates. First salivary and pancreatic α-amylases, the two endoacting retaining glucosidases, break down the complex starch molecules into smaller linear maltose-oligomers (LM) and branched α-limit dextrins (αLDx). Then two retaining exoglucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), convert those molecules into glucose in the small intestine. The small intestinal brush-border epithelial cells anchor MGAM and SI, and each contains a catalytic N- and C-terminal subunit, ntMGAM, ctMGAM, ntSI, and ctSI, respectively. All four catalytic domains have, to varying extents, α-1,4-exohydrolytic glucosidase activity and belong to the glycoside hydrolase family 31 (GH31). ntSI and ctSI show additional activity toward α-1,6 (isomaltose substrates) and α-1,2 (sucrose) glycosidic linkages, respectively. Because they mediate the final steps of starch digestion, both MGAM and SI are important target enzymes for the treatment of type-2 diabetes. Because of their potent inhibitory activities against the mammalian intestinal α-glucosidases, sulfonium-ion glucosidase inhibitors isolated from the antidiabetic herbal extracts of various Salacia species have received considerable attention recently. Thus far, researchers have isolated eight sulfonium-ion glucosidase inhibitors from Salacia species: salaprinol, salacinol, ponkoranol, kotalanol, and four of their corresponding de-O-sulfonated compounds, the structures of which comprise a 1,4-anhydro-4-thio-d-arabinitol and a polyhydroxylated acyclic side chain. Some of these compounds more strongly inhibit human intestinal α-glucosidases than the currently available antidiabetic drugs, acarbose and miglitol, and could serve as lead candidates in the treatment of type-2 diabetes. In this Account, we summarize progress in the field since 2010 with this class of inhibitors, with particular focus on their selective inhibitory activities against the intestinal glucosidases. Through structure-activity relationship (SAR) studies, we have modified the natural compounds to derive more potent, nanomolar inhibitors of human MGAM and SI. This structural optimization also yielded the most potent inhibitors known to date for each subunit. Furthermore, we observed that some of our synthetic inhibitors selectively blocked the activity of some mucosal α-glucosidases. Those results led to our current working hypothesis that selective inhibitors can dampen the action of a fast digesting subunit or subunits which places the burden of digestion on slower digesting subunits. That strategy can control the rate of starch digestion and glucose release to the body. Decreasing the initial glucose spike after a carbohydrate-rich meal and extending postprandial blood glucose delivery to the body can be desirable for diabetics and patients with other metabolic syndrome-associated diseases.

Combined molecular dynamics, STD-NMR, and CORCEMA protocol yields structural model for a UDP-galactopyranose mutase-inhibitor complex.

UDP-galactopyranose mutase (UGM) is an enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall, and is essential for the growth and survival of the organism. A micromolar inhibitor developed by tetrafluorination of the UGM substrate has been previously studied by saturation transfer difference (STD) NMR spectroscopy. To elucidate the bioactive conformation of the inhibitor bound to UGM, we employ molecular dynamics (MD) simulations to construct a structural model. The MD model is subsequently validated by a good fit between experimental and theoretical STD effects, the latter calculated by a complete relaxation and conformational exchange matrix (CORCEMA) analysis. This structural model is used to explain the relative binding affinities of the inhibitor and the parent substrate.

Synthesis and biological evaluation of nonionic substrate mimics of UDP-Galp as candidate inhibitors of UDP galactopyranose mutase (UGM).

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.

Specificity of a UDP-GalNAc pyranose-furanose mutase: a potential therapeutic target for Campylobacter jejuni infections.

Pyranose-furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP-Gal). We report here the detailed structural characterization of the first bifunctional pyranose-furanose mutase, which recognizes both UDP-Gal and UDP-GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X-ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA-ST calculations, we have characterized the binding of the enzyme to both UDP-Galp and UDP-GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc-containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP-Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.

Serendipitous discovery of a potent influenza virus a neuraminidase inhibitor.

We have previously reported a potent neuraminidase inhibitor that comprises a carbocyclic analogue of zanamivir in which the hydrophilic glycerol side chain is replaced by the hydrophobic 3-pentyloxy group of oseltamivir. This hybrid inhibitor showed excellent inhibitory properties in the neuraminidase inhibition assay (Ki =0.46 nM; Ki (zanamivir) =0.16 nM) and in the viral replication inhibition assay in cell culture at 10(-8) M. As part of this lead optimization, we now report a novel spirolactam that shows comparable inhibitory activity in the cell culture assay to that of our lead compound at 10(-7) M. The compound was discovered serendipitously during the attempted synthesis of the isothiourea derivative of the original candidate. The X-ray crystal structure of the spirolactam in complex with the N8 subtype neuraminidase offers insight into the mode of inhibition.

Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase†2.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

Metabolic inhibition of sialyl-Lewis X biosynthesis by 5-thiofucose remodels the cell surface and impairs selectin-mediated cell adhesion.

Sialyl-Lewis X (sLe(X)) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe(X) biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe(X) in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe(X) presentation on HepG2 cells with an EC(50) in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe(X) levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe(X) formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.

Modulation of starch digestion for slow glucose release through "toggling" of activities of mucosal α-glucosidases.

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.

Exploitation of the catalytic site and 150 cavity for design of influenza A neuraminidase inhibitors.

We report here the exploitation of the 150 cavity in the active site of influenza A viral neuraminidases for the design of novel C-6 triazole-containing Tamiflu derivatives. A general and convenient synthetic route was developed by utilizing a highly substituted cyclic Baylis-Hillman acetate as an active precursor for azide substitution via suprafacial allylic azide [3,3]-sigmatropic rearrangement. Virus replication inhibitory assays in vitro of these triazole derivatives containing either an amino or guanidino function indicated that the guanidinium compound showed the higher efficacy against a strain with N2 subtype at a concentration of 2 × 10(-5) M but did not inhibit replication of a strain with N1 subtype even at a concentration of 10(-4) M. In order to probe the nature of the enzyme-inhibitor interactions, molecular dynamics simulations were performed on complexes of these compounds with different neuraminidase enzymes. The results indicated that the candidate inhibitors occupy both the 150 cavity and catalytic site but with alternating occupancy.

Synthesis and immunological characterization of modified hyaluronic acid hexasaccharide conjugates.

The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the N-acetyl moiety of the N-acetyl-D-glucosamine residue is replaced with an N-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites.

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus.

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS-TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.

5-thiomannosides block the biosynthesis of dolichol-linked oligosaccharides and mimic class I congenital disorders of glycosylation.

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum.

Sequence-dependent structural dynamics of primate adenosine-to-inosine editing substrates.

Humans have the highest level of adenosine-to-inosine (A-to-I) editing amongst primates, yet the reasons for this difference remain unclear. Sequence analysis of the Alu Sg elements (A-to-I RNA substrates) corresponding to the Nup50 gene in human, chimp, and rhesus reveals subtle sequence variations surrounding the edit sites. We have developed three constructs that represent human (HuAp5), chimp (ChAp5), and rhesus (RhAp5) Nup50 Alu Sg A-to-I editing substrates. Here, 2-aminopurine (2-Ap) was substituted for edited adenosine (A5) so as to monitor the fluorescence intensity with respect to temperature. UV and steady-state fluorescence (SSF) T(M) plots indicate that local and global unfolding are coincident, with the human construct displaying a T(M) of approximately 70°C, compared to 60°C for chimp and 54°C for rhesus. However, time-resolved fluorescence (TRF) resolves three different fluorescence lifetimes that we assign to folded, intermediate(s), and unfolded states. The TRF data fit well to a two-intermediate model, whereby both intermediates (M, J) are in equilibrium with each other, and the folded/unfolded states. Our model suggests that, at 37°C, human state J and the folded state will be the most heavily populated in comparison to the other primate constructs. In order for adenosine deaminase acting on RNA (ADAR) to efficiently dock, a stable duplex must be present that corresponds to the human construct, globally. Next, the enzyme must "flip out" the base of interest to facilitate the A-to-I conversion; a nucleotide in an intermediate-like position would enhance this conformational change. Our experiments demonstrate that subtle variations in RNA sequence might contribute to the high A-to-I editing levels found in humans.

Probing hyperconjugation experimentally with the conformational deuterium isotope effect.

Hyperconjugation underlies many chemical phenomena of fundamental and practical importance. Owing to a great deal of interest in the anomeric effect, anomeric-like hyperconjugative effects have been thoroughly investigated in oxygen-containing systems. However, such interactions in the second- and third-row chalcogens are less well-understood and have generated some controversy. Here, we show that the conformational deuterium isotope effect, in combination with Saunders' isotopic perturbation method, permits sensitive and direct experimental probing of the conformational equilibria in dioxane, dithiane, and diselenane analogues by variable-temperature, dynamic NMR spectroscopy. We find that the magnitude of the conformational deuterium isotope effect is 252.1, 28.3, and 7.1 J/mol (±10%) for the oxygen, sulfur, and selenium analogues, respectively. These results reveal the periodic trend for hyperconjugation in the chalcogens, which reflect a decreasing n(x)→σ(C-H(D)) interaction throughout the period, as supported by IR spectroscopy and in agreement with DFT calculations and a natural bond order analysis.

Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.

N-glycosylation controls trafficking, zymogen activation and substrate processing of proprotein convertases PC1/3 and subtilisin kexin isozyme-1.

The limited proteolysis of proteins by the proprotein convertases (PCs) is a common means of producing bioactive proteins or peptides. The PCs are associated with numerous human pathologies and their activity can be reduced through the use of specific inhibitors. Here, we demonstrate an alternative approach to inhibiting PCs by altering their N-glycosylation. Through site-directed mutagenesis, we show that the convertase PC1/3 contains two N-glycans, only one of which is critical for its prosegment cleavage. The exact structure of PC1/3 N-glycans does not significantly affect its zymogen activation within endocrine cells, but glycosylation of Asn(146) is critical. Processing of the PC1/3's substrate proopiomelanocortin (POMC) was used in a cell-based assay to screen a collection of 45 compounds structurally related to known glycosidase inhibitors. Two 5-thiomannose-containing disaccharide derivatives were discovered to block PC1/3 and POMC processing into the analgesic peptide ß-endorphin. These compounds also reduced the zymogen activation of the convertase subtilisin kexin isozyme-1 (SKI-1), blocked the processing of its substrate the sterol regulatory element-binding protein SREBP-2 and altered its glycosylation. Thus, modification of PC glycosylation may also be a means of blocking their activity, an effect which, in the case of SKI-1, may be of possible therapeutic use since SREBP-2 regulates sterol levels including cholesterol biosynthesis and its metabolism.