The molecular basis underpinning the potency and specificity of MAIT cell antigens.

Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.

Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Aortic Arch Debranching and Thoracic Endovascular Aortic Repair (TEVAR) for Type B Aortic Dissection.

BACKGROUND: Since its introduction, thoracic endovascular aortic repair (TEVAR) has revolutionized the treatment of type B aortic dissections (TBADs). However, the proximal aspect of the aortic pathology treated may infringe on the origin of the left subclavian artery or even more proximally. Hence, to ensure durable outcomes, the origin of these vessels needs to be covered, but an extra-anatomical bypass is required to perfuse vital branches, known as aortic arch debranching. This series aims to describe and delineate the disparities of aortic arch debranching during TEVAR for TBAD. METHODS: A retrospective review and analysis of a multicenter international database was conducted to identify patients with TBAD treated with TEVAR between 2005 and 2021. Data analyzed included patient demographics, disease characteristics, operative characteristics, and postoperative outcomes with follow-up on mortality and reintervention. All statistical analyses were carried out using IBM SPSS 26. Patient survival was calculated using a Kaplan-Meier survival analysis, and a P value of less than 0.05 was considered statistically significant. RESULTS: A total of 58 patients were included in the analysis, of which 27 (46.6%) presented with complicated disease and 31 were uncomplicated, of which 10 (17.2%) were classed as high risk and 21 (36.2%) low risk. Zone 2 was the most common proximal landing zone for the stent graft. Left subclavian artery bypass was performed selectively (26%), with 1 stroke occurring, likely due to embolic reasons. A further 6 underwent more proximal aortic debranching before TEVAR (10%) and was a significant risk factor for mortality and the number of stents deployed. The overall rates of reintervention and mortality were 17.2% (n = 10) and 29.3% (n = 17). CONCLUSIONS: Aortic arch debranching and TEVAR for TBAD is associated with significant mortality. Future developments to treat aortic arch pathology could incorporate branched graft devices, eliminating the need for debranching, improving stroke rates, and reducing future reinterventions.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor.

Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I-related protein 1 MR1.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC50 < 1 µM), moderate (1 µM < IC50 < 100 µM), and weak (IC50 > 100 µM). We demonstrated a clear correlation between ligand-binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed fluorescence polarization-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the A'-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

Myths and methodologies: Cardiopulmonary exercise testing for surgical risk stratification in patients with an abdominal aortic aneurysm; balancing risk over benefit.

The extent to which patients with an abdominal aortic aneurysm (AAA) should exercise remains unclear, given theoretical concerns over the perceived risk of blood pressure-induced rupture, which is often catastrophic. This is especially pertinent during cardiopulmonary exercise testing, when patients are required to perform incremental exercise to symptom-limited exhaustion for the determination of cardiorespiratory fitness. This multimodal metric is being used increasingly as a complementary diagnostic tool to inform risk stratification and subsequent management of patients undergoing AAA surgery. In this review, we bring together a multidisciplinary group of physiologists, exercise scientists, anaesthetists, radiologists and surgeons to challenge the enduring 'myth' that AAA patients should be fearful of and avoid rigorous exercise. On the contrary, by appraising fundamental vascular mechanobiological forces associated with exercise, in conjunction with 'methodological' recommendations for risk mitigation specific to this patient population, we highlight that the benefits conferred by cardiopulmonary exercise testing and exercise training across the continuum of intensity far outweigh the short-term risks posed by potential AAA rupture.

Uncomplicated Type B Aortic Dissection: Challenges in Diagnosis and Categorization.

BACKGROUND: Acute type B aortic dissection (TBAD) is a rare disease that is likely under-diagnosed in the UK. As a progressive, dynamic clinical entity, many patients initially diagnosed with uncomplicated TBAD deteriorate, developing end-organ malperfusion and aortic rupture (complicated TBAD). An evaluation of the binary approach to the diagnosis and categorisation of TBAD is needed. METHODS: A narrative review of the risk factors predisposing patients to progression from unTBAD to coTBAD was undertaken. RESULTS: Key high-risk features predispose the development of complicated TBAD, such as maximal aortic diameter > 40 mm and partial false lumen thrombosis. CONCLUSION: An appreciation of the factors that predispose to complicated TBAD would aid clinical decision-making surrounding TBAD.

Ligand-dependent downregulation of MR1 cell surface expression.

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

An Optimized Ensemble Deep Learning Model for Predicting Plant miRNA-IncRNA Based on Artificial Gorilla Troops Algorithm.

MicroRNAs (miRNA) are small, non-coding regulatory molecules whose effective alteration might result in abnormal gene manifestation in the downstream pathway of their target. miRNA gene variants can impact miRNA transcription, maturation, or target selectivity, impairing their usefulness in plant growth and stress responses. Simple Sequence Repeat (SSR) based on miRNA is a newly introduced functional marker that has recently been used in plant breeding. MicroRNA and long non-coding RNA (lncRNA) are two examples of non-coding RNA (ncRNA) that play a vital role in controlling the biological processes of animals and plants. According to recent studies, the major objective for decoding their functional activities is predicting the relationship between lncRNA and miRNA. Traditional feature-based classification systems' prediction accuracy and reliability are frequently harmed because of the small data size, human factors' limits, and huge quantity of noise. This paper proposes an optimized deep learning model built with Independently Recurrent Neural Networks (IndRNNs) and Convolutional Neural Networks (CNNs) to predict the interaction in plants between lncRNA and miRNA. The deep learning ensemble model automatically investigates the function characteristics of genetic sequences. The proposed model's main advantage is the enhanced accuracy in plant miRNA-IncRNA prediction due to optimal hyperparameter tuning, which is performed by the artificial Gorilla Troops Algorithm and the proposed intelligent preying algorithm. IndRNN is adapted to derive the representation of learned sequence dependencies and sequence features by overcoming the inaccuracies of natural factors in traditional feature architecture. Working with large-scale data, the suggested model outperforms the current deep learning model and shallow machine learning, notably for extended sequences, according to the findings of the experiments, where we obtained an accuracy of 97.7% in the proposed method.

Cardiothoracic surgery leadership and learning are indispensable to each other.

Cardiothoracic surgery is facing a multitude of challenges in leadership and training on the global scale, these being a complex and aging patient population, shortage of cardiac surgeons, diminishing student interest and trainee enthusiasm, increasingly challenging training obstacles and work-life imbalances, suboptimal job prospects, reports of discrimination and bullying and lack of diversity as well as gap between innovation and technology, clinical application, and training of future surgeons. The survival of cardiac surgery hinges on the leadership attracting and retaining young surgeons into the specialty. Mentoring, leading through example, recognizing the work-life imbalances, adapting to diverse and modern training models and embracing diversity with respect to gender and race, will ultimately be required to create and cultivate a nurturing environment of training and preparing future leaders. The vision for training future generations of cardiothoracic surgeons must rely heavily on strengthening the unity of the heart team. In doing so we can provide the best possible care for our patients and a most fulfilling career for the future generation of cardiac surgeons.

Effect of Transcatheter Aortic Valve Implantation vs Surgical Aortic Valve Replacement on All-Cause Mortality in Patients With Aortic Stenosis: A Randomized Clinical Trial.

Importance: Transcatheter aortic valve implantation (TAVI) is a less invasive alternative to surgical aortic valve replacement and is the treatment of choice for patients at high operative risk. The role of TAVI in patients at lower risk is unclear. Objective: To determine whether TAVI is noninferior to surgery in patients at moderately increased operative risk. Design, Setting, and Participants: In this randomized clinical trial conducted at 34 UK centers, 913 patients aged 70 years or older with severe, symptomatic aortic stenosis and moderately increased operative risk due to age or comorbidity were enrolled between April 2014 and April 2018 and followed up through April 2019. Interventions: TAVI using any valve with a CE mark (indicating conformity of the valve with all legal and safety requirements for sale throughout the European Economic Area) and any access route (n = 458) or surgical aortic valve replacement (surgery; n = 455). Main Outcomes and Measures: The primary outcome was all-cause mortality at 1 year. The primary hypothesis was that TAVI was noninferior to surgery, with a noninferiority margin of 5% for the upper limit of the 1-sided 97.5% CI for the absolute between-group difference in mortality. There were 36 secondary outcomes (30 reported herein), including duration of hospital stay, major bleeding events, vascular complications, conduction disturbance requiring pacemaker implantation, and aortic regurgitation. Results: Among 913 patients randomized (median age, 81 years [IQR, 78 to 84 years]; 424 [46%] were female; median Society of Thoracic Surgeons mortality risk score, 2.6% [IQR, 2.0% to 3.4%]), 912 (99.9%) completed follow-up and were included in the noninferiority analysis. At 1 year, there were 21 deaths (4.6%) in the TAVI group and 30 deaths (6.6%) in the surgery group, with an adjusted absolute risk difference of -2.0% (1-sided 97.5% CI, -∞ to 1.2%; P < .001 for noninferiority). Of 30 prespecified secondary outcomes reported herein, 24 showed no significant difference at 1 year. TAVI was associated with significantly shorter postprocedural hospitalization (median of 3 days [IQR, 2 to 5 days] vs 8 days [IQR, 6 to 13 days] in the surgery group). At 1 year, there were significantly fewer major bleeding events after TAVI compared with surgery (7.2% vs 20.2%, respectively; adjusted hazard ratio [HR], 0.33 [95% CI, 0.24 to 0.45]) but significantly more vascular complications (10.3% vs 2.4%; adjusted HR, 4.42 [95% CI, 2.54 to 7.71]), conduction disturbances requiring pacemaker implantation (14.2% vs 7.3%; adjusted HR, 2.05 [95% CI, 1.43 to 2.94]), and mild (38.3% vs 11.7%) or moderate (2.3% vs 0.6%) aortic regurgitation (adjusted odds ratio for mild, moderate, or severe [no instance of severe reported] aortic regurgitation combined vs none, 4.89 [95% CI, 3.08 to 7.75]). Conclusions and Relevance: Among patients aged 70 years or older with severe, symptomatic aortic stenosis and moderately increased operative risk, TAVI was noninferior to surgery with respect to all-cause mortality at 1 year. Trial Registration: isrctn.com Identifier: ISRCTN57819173.

Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2+ αß T cell receptor (TCR). In addition, a more diverse TRAV1-2- MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2- TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2+ TCR (termed D462-E4) recognizes an MR1-antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 ß-chain of the D462-E4 TCR binds over the F'-pocket of MR1, whereby the complementarity-determining region (CDR) 3ß loop surrounded and projected into the F'-pocket. Nevertheless, the CDR3ß loop anchored proximal to the MR1 A'-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2+ and TRAV36+ TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.

Mechanical circulatory support-Challenges, strategies, and preparations.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is usually mild, but patients can present with pneumonia, acute respiratory distress syndrome (ARDS), and circulatory shock. Although the symptoms of the disease are predominantly respiratory, the involvement of the cardiovascular system is common. Patients with heart failure (HF) are particularly vulnerable when suffering from COVID-19. AIM OF THE REVIEW: To examine the challenges faced by healthcare organizations, and mechanical circulatory support management strategies available to patients with heart failure, during the COVID-19 pandemic. RESULTS: Extracorporeal membrane oxygenation (ECMO) can be lifesaving in patients with severe forms of ARDS, or refractory cardio-circulatory compromise. The Impella RP can provide right ventricular circulatory support for patients who develop right side ventricular failure or decompensation caused by COVID-19 complications, including pulmonary embolus. HT are reserved for only those patients with a high short-term mortality. LVAD as a bridge to transplant may be a viable strategy to get at-risk patients home quickly. Elective LVAD implantations have been reduced and only patients classified as INTERMACS profile 1 and 2 are being considered for LVAD implantation. Delayed recognition of LVAD-related complications, misdiagnosis of COVID-19, and impaired social and psychological well-being for patients and families may ensue. Remote patient care with virtual or telephone contacts is becoming the norm. CONCLUSIONS: HF incidence, prevalence, and undertreatment will grow as a result of new COVID-19-related heart disease. ECMO should be reserved for highly selected cases of COVID-19 with a reasonable probability of recovery. Special considerations are needed for patients with advanced HF, including those supported by durable LVADs.

Transcatheter aortic valve implantation versus surgical aortic valve replacement during the COVID-19 pandemic-Current practice and concerns.

COVID-19 has had a dramatic impact on the provision of healthcare. COVID-19 can manifest with cardiac and thrombotic presentations. Additionally, patients with cardiovascular comorbidities are at an increased risk of adverse outcomes related to COVID-19 infection. This in turn has led to a significant reduction in the provision of cardiac surgery with alternative management options utilized to address patients with significant disease. In terms of aortic valve disease, transcatheter aortic valve implantation (TAVI) provides advantages over surgical aortic valve replacement in with a lower burden on healthcare resources. COVID-19 also resulted in changes in management strategies and as such TAVI is now being considered in younger- and low-risk patients. However, long term data with regard to TAVI is still unknown, and the use in patient groups that have been excluded in the large pivotal studies that established TAVI as an alternative to surgery has raised specific concerns in the use of TAVI as the preferred treatment choice. With the long term ramification unknown, it is essential that decisions are made with caution.

Aortic valve replacement with sutureless Perceval S valve: A case report of aortic root homograft failure in the setting of Streptococcus constellatus endocarditis.

BACKGROUND: Surgery for failed homograft aortic root replacement with extensive calcification in the setting of endocarditis alone is very challenging. CASE SUMMARY: We report the case of redo aortic valve replacement and mitral valve replacement, in a 39 years old presenting with a rare Streptococcus constellatus endocarditis of a previously implanted homograft root and native mitral valve, where conventional valve replacement proved nonfeasible. S. constellatus had caused severe tissue destruction and the extensive calcification in the homograft prevented conventional valve replacement with sutures. In this case, a sutureless valve provided a useful alternative surgical strategy. DISCUSSION: We consider heavily calcified failed homografts to be a good indication for sutureless (rapid deployment) valves.

Impact of COVID-19 on cardiac surgical training: Our experience in the United Kingdom.

The current evolving global pandemic caused by coronavirus disease-2019 (COVID-19) has dramatically impacted global health care systems, resulting in governments taking unprecedented measures to contain the spread of the infection, with adaptations by health care organizations. Research into understanding the pathophysiology behind this virus, to ascertain best medical management and treatment, has been accelerated to keep up with the rapidly evolving situation. There has been redeployment of medical and nursing staff to the frontlines and redistribution of health care resources. In addition, the cancellation of elective surgery and centralization of services to treat high-risk surgical cases will all, undeniably, have an impact on current surgical training with possible future implications. We aim to explore the impact COVID-19 is having on cardiac surgical training in the UK and what future implications this may have.

Coronary artery bypass vs percutaneous coronary intervention in under 50s.

BACKGROUND: Young patients with coronary artery disease are undergoing percutaneous coronary intervention (PCI) primarily, with a view to deferring coronary artery bypass grafting (CABG). We investigated the validity of this approach, by comparing outcomes in patients ≤50 years undergoing CABG or PCI. METHODS: One hundred consecutive patients undergoing PCI and 100 undergoing CABG in 2004 were retrospectively studied to allow for 5 and 12 years follow-up. The two groups were compared for the primary endpoints of major adverse cardiac or cerebrovascular event (MACCE). RESULTS: Diabetes, peripheral vascular disease, and left ventricular ejection fraction <50% were higher in the CABG group. At 5 years, rates of myocardial infarction (MI) (9% vs 1%, P = .02), repeat revascularization (31% vs 7%, P < .01), and MACCE (34 vs 12, P < .01) were greater in the PCI vs the CABG group. Similarly, at 12 years, rates of MI (27.4% vs 19.4%, P = .19), repeat revascularization (41.1% vs 20.4%, P < .01), and MACCE (51 vs 40, P = .07) were greater in the PCI group. There were no differences in major outcomes in patients with 1 or 2VD, at 5 or 12 years. Rates of MI, revascularization, and MACCE were higher in patients with 3VD undergoing PCI (n = 21; MI, 47.6%; revascularization, 66.7%; and MACCE, 19 events) vs CABG (n = 78; MI, 19.2%; revascularization, 20.5%; and MACCE, 31 events); P < .01, for all end points. CONCLUSIONS: MACCE was lower in young patients undergoing CABG vs PCI at both 5 and 12 years follow-up, primarily as a consequence of patients with 3VD undergoing PCI having more MI and repeat revascularization. CABG should remain the preferred method of revascularization in young patients with 3VD.

Structural and Biophysical Characterization of Human EXTL3: Domain Organization, Glycosylation, and Solution Structure.

Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of ß sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.

Mucosal-associated invariant T cell receptor recognition of small molecules presented by MR1.

The major histocompatibility complex (MHC) class-I related molecule MR1 is a monomorphic and evolutionary conserved antigen (Ag)-presenting molecule that shares the overall architecture of MHC-I and CD1 proteins. However, in contrast to MHC-I and the CD1 family that present peptides and lipids, respectively, MR1 specifically presents small organic molecules. During microbial infection of mammalian cells, MR1 captures and presents vitamin B precursors, derived from the microbial biosynthesis of riboflavin, on the surface of antigen-presenting cells. These MR1-Ag complexes are recognized by the mucosal-associated invariant T cell receptor (MAIT TCR), which subsequently leads to MAIT cell activation. Recently, MR1 was shown to trap chemical scaffolds including drug and drug-like molecules. Here, we review this metabolite Ag-presenting molecule and further define the key molecular interactions underlying the recognition and reactivity of MAIT TCRs to MR1 in an Ag-dependent manner.