Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc₃Man₉GlcNAc₂ ...precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endoacting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile ...via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
JCO
The purpose of this update was to determine differences in patient-reported chronic toxicity and disease outcomes with intensity-modulated radiation therapy (IMRT) compared with conventional ...pelvic radiation. Patients with cervical and endometrial cancers who received postoperative pelvic radiation were randomly assigned to conventional radiation therapy (CRT) or IMRT. Toxicity and quality of life were assessed using Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events, Expanded Prostate Cancer Index Composite (EPIC) bowel and urinary domains, and Functional Assessment of Cancer Therapy-General. Between 2012 and 2015, 279 eligible patients were enrolled to the study with a median follow-up of 37.8 months. There were no differences in overall survival (
= .53), disease-free survival (
= .21), or locoregional failure (
= .81). One year after RT, patients in the CRT arm experienced more high-level diarrhea frequency (5.8% IMRT
15.1% CRT,
= .042) and a greater number had to take antidiarrheal medication two or more times a day (1.2% IMRT
8.6% CRT,
= .036). At 3 years, women in the CRT arm reported a decline in urinary function, whereas the IMRT arm continued to improve (mean change in EPIC urinary score = 0.5, standard deviation = 13.0, IMRT
-6.0, standard deviation = 14.3, CRT,
= .005). In conclusion, IMRT reduces patient-reported chronic GI and urinary toxicity with no difference in treatment efficacy at 3 years.
Objectives The results of treatment for subclavian vein effort thrombosis were assessed in a series of competitive athletes. Methods A retrospective review was conducted of high-performance athletes ...who underwent multidisciplinary management for venous thoracic outlet syndrome in a specialized referral center. The overall time required to return to athletic activity was assessed with respect to the timing and methods of diagnosis, initial treatment, operative management, and postoperative care. Results Between January 1997 and January 2007, 32 competitive athletes (29 male and 3 female) were treated for venous thoracic outlet syndrome, of which 31% were in high school, 47% were in college, and 22% were professional. The median age was 20.3 years (range, 16-26 years). Venous duplex ultrasound examination in 21 patients had a diagnostic sensitivity of 71%, and the mean interval between symptoms and definitive venographic diagnosis was 20.2 ± 5.6 days (range, 1-120 days). Catheter-directed subclavian vein thrombolysis was performed in 26 (81%), with balloon angioplasty in 12 and stent placement in one. Paraclavicular thoracic outlet decompression was performed with circumferential external venolysis alone (56%) or direct axillary-subclavian vein reconstruction (44%), using saphenous vein panel graft bypass (n = 8), reversed saphenous vein graft bypass (n = 3), and saphenous vein patch angioplasty (n = 3). In 19 patients (59%), simultaneous creation of a temporary (12 weeks) adjunctive radiocephalic arteriovenous fistula was done. The mean hospital stay was 5.2 ± 0.4 days (range, 2-11 days). Seven patients required secondary procedures. Anticoagulation was maintained for 12 weeks. All 32 patients resumed unrestricted use of the upper extremity, with a median interval of 3.5 months between operation and the return to participation in competitive athletics (range, 2-10 months). The overall duration of management from symptoms to full athletic activity was significantly correlated with the time interval from venographic diagnosis to operation ( r = 0.820, P < .001) and was longer in patients with persistent symptoms ( P < .05) or rethrombosis before referral ( P < .01). Conclusions Successful outcomes were achieved for the management of effort thrombosis in a series of 32 competitive athletes using a multidisciplinary approach based on (1) early diagnostic venography, thrombolysis, and tertiary referral; (2) paraclavicular thoracic outlet decompression with external venolysis and frequent use of subclavian vein reconstruction; and (3) temporary postoperative anticoagulation, with or without an adjunctive arteriovenous fistula. Optimal outcomes for venous thoracic outlet syndrome depend on early recognition by treating physicians and prompt referral for comprehensive surgical management.
α‐Mannosidases and α‐mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α‐mannosides, and the potential of mannosidase ...inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α‐mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an OS2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on‐enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through OS2 and B2,5≠ conformations, information that should inspire the development of new antifungal agents.
Family GH76 endo‐α‐mannanases participate in construction and breakdown of fungal cell wall mannoprotein. A combined synthetic, structural, and theoretical study discloses the first inhibitors of this family of enzymes and quantifies how the enzyme distorts an azasugar inhibitor into a transition‐state‐mimicking boat conformation.
Protein production and purification Gräslund, Susanne; Nordlund, Pär; Weigelt, Johan ...
Nature methods,
02/2008, Letnik:
5, Številka:
2
Journal Article
Recenzirano
Odprti dostop
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to ...try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Glycoside hydrolase family 99 (GH99) was created to categorize sequence‐related glycosidases possessing endo‐α‐mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N‐glycan ...precursors (Glc1–3Man9GlcNAc2), releasing mono‐, di‐ and triglucosylated‐mannose (Glc1–3‐1,3‐Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α‐mannans, releasing α‐1,3‐mannobiose by hydrolysing αMan‐1,3‐αMan→1,2‐αMan‐1,2‐αMan sequences within branches off the main α‐1,6‐mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo‐α‐1,2‐mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol−1 preference for mannose‐configured substrates in the −2 subsite (relative to glucose), which inspired the development of a new inhibitor, α‐mannopyranosyl‐1,3‐isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X‐ray structures of ManIFG or a substrate in complex with wild‐type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D‐mannose‐ rather than D‐glucose‐configured substrates.
Remote control: A remote stereocentre defines a new activity for glycosidases within glycoside hydrolase family GH99. Biochemical and structural analysis of substrates and inhibitors reveal the preference for a D‐mannose‐configuration in the −2 subsite that distinguishes endo‐α‐1,2‐mannanase activity from the −2 subsite D‐glucose‐configuration of endo‐α‐mannosidases in the same family.
NRG/RTOG 1203 compared 3-D conformal radiotherapy (3D CRT) to intensity-modulated radiotherapy (IMRT) in patients with endometrial or cervical cancer requiring post-operative radiotherapy after ...hysterectomy. The purpose of this study was to report the first quality-adjusted survival analysis comparing the two treatments.
NRG/RTOG 1203 randomized patients having undergone hysterectomy to either 3DCRT or IMRT. Stratification factors included RT dose, chemotherapy, and disease site. The EQ-5D, both index and visual analog scale (VAS), were obtained at baseline, 5 weeks after the start of RT, 4–6 weeks post RT and 1 and 3-years post RT. EQ-5D index and VAS scores along with quality-adjusted survival (QAS) were compared between treatment arms using the t-test at a two-sided significance level of 0.05.
NRG/RTOG 1203 enrolled 289 patients of which 236 consented to participate in the patient reported outcome (PRO) assessments. QAS was higher in women treated with IMRT, 1374 vs 1333 days (p = 0.5) compared to patients treated with 3DCRT, but this difference was not statistically different. Patients treated with IMRT had less of a decline in VAS score 5 weeks post RT, −5.04, compared to patients treated with 3DCRT, −7.48, although not statistically significant (p = 0.38).
This is the first report of the use of the EQ-5D comparing two radiotherapy techniques in the treatment of gynecologic malignancies after surgery. While there were no significant differences in QAS and VAS scores between patients who received IMRT vs. 3DCRT, RTOG 1203 was not powered to show statistical differences in these secondary endpoints.
•The first randomized clinical trial comparing two types of radiotherapy in gynecologic malignancies.•Utilities were measured a priori to calculate quality-adjusted survival between the two treatment arms.•Although not powered for these endpoints, non-statistically significant improvements were seen with the use of IMRT.
Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary ...polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are alpha-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca(2+)-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca(2+) plays a key catalytic role in helping distort the mannoside away from its ground-state (4)C(1) chair conformation toward the transition state.
Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic ...resolution structures along the reaction coordinate of an inverting α‐mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α‐D‐mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.
PDF
