High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.     

Incidence and Risk Factors for Neonatal Tetanus in Admissions to Kilifi County Hospital,Kenya

Background

Neonatal Tetanus (NT) is a preventable cause of mortality and neurological sequelae that occurs at higher incidence in resource-poor countries, presumably because of low maternal immunisation rates and unhygienic cord care practices. We aimed to determine changes in the incidence of NT, characterize and investigate the associated risk factors and mortality in a prospective cohort study including all admissions over a 15-year period at a County hospital on the Kenyan coast, a region with relatively high historical NT rates within Kenya.

Methods

We assessed all neonatal admissions to Kilifi County Hospital in Kenya (1999–2013) and identified cases of NT (standard clinical case definition) admitted during this time. Poisson regression was used to examine change in incidence of NT using accurate denominator data from an area of active demographic surveillance. Logistic regression was used to investigate the risk factors for NT and factors associated with mortality in NT amongst neonatal admissions. A subset of sera from mothers (n = 61) and neonates (n = 47) were tested for anti-tetanus antibodies.

Results

There were 191 NT admissions, of whom 187 (98%) were home deliveries. Incidence of NT declined significantly (Incidence Rate Ratio: 0.85 (95% Confidence interval 0.81–0.89), P<0.001) but the case fatality (62%) did not change over the study period (P = 0.536). Younger infant age at admission (P = 0.001) was the only independent predictor of mortality. Compared to neonatal hospital admittee controls, the proportion of home births was higher among the cases. Sera tested for antitetanus antibodies showed most mothers (50/61, 82%) had undetectable levels of antitetanus antibodies, and most (8/9, 89%) mothers with detectable antibodies had a neonate without protective levels.

Conclusions

Incidence of NT in Kilifi County has significantly reduced, with reductions following immunisation campaigns. Our results suggest immunisation efforts are effective if sustained and efforts should continue to expand coverage.     

The Response of Cyclic Electron Flow around Photosystem I to Changes in Photorespiration and Nitrate Assimilation

Photosynthesis captures light energy to produce ATP and NADPH. These molecules are consumed in the conversion of CO₂ to sugar, photorespiration, and NO₃⁻ assimilation. The production and consumption of ATP and NADPH must be balanced to prevent photoinhibition or photodamage. This balancing may occur via cyclic electron flow around photosystem I (CEF), which increases ATP/NADPH production during photosynthetic electron transport; however, it is not clear under what conditions CEF changes with ATP/NADPH demand. Measurements of chlorophyll fluorescence and dark interval relaxation kinetics were used to determine the contribution of CEF in balancing ATP/NADPH in hydroponically grown Arabidopsis (Arabidopsis thaliana) supplied different forms of nitrogen (nitrate versus ammonium) under changes in atmospheric CO₂ and oxygen. Measurements of CEF were made under low and high light and compared with ATP/NADPH demand estimated from CO₂ gas exchange. Under low light, contributions of CEF did not shift despite an up to 17% change in modeled ATP/NADPH demand. Under high light, CEF increased under photorespiratory conditions (high oxygen and low CO₂), consistent with a primary role in energy balancing. However, nitrogen form had little impact on rates of CEF under high or low light. We conclude that, according to modeled ATP/NADPH demand, CEF responded to energy demand under high light but not low light. These findings suggest that other mechanisms, such as the malate valve and the Mehler reaction, were able to maintain energy balance when electron flow was low but that CEF was required under higher flow.Photosynthesis must balance both the amount of energy harvested by the light reactions and how it is stored to match metabolic demands. Light energy is harvested by the photosynthetic antenna complexes and stored by the electron and proton transfer complexes as ATP and NADPH. It is used primarily to meet the energy demands for assimilating carbon (from CO₂) and nitrogen (from NO₃⁻ and NH₄⁺; Keeling et al., 1976; Edwards and Walker, 1983; Miller et al., 2007). These processes require different ratios of ATP and NADPH, requiring a finely balanced output of energy in these forms. For example, if ATP were to be consumed at a greater rate than NADPH, electron transport would rapidly become limiting by the lack of NADP⁺, decreasing rates of proton translocation and ATP regeneration. Alternatively, if NADPH were consumed faster than ATP, proton translocation through ATP synthase would be reduced due to limiting ADP and the difference in pH between lumen and stroma would increase, restricting plastoquinol oxidation at the cytochrome b₆f complex and initiating nonphotochemical quenching (Kanazawa and Kramer, 2002). The stoichiometric balancing of ATP and NADPH must occur rapidly, because pool sizes of ATP and NADPH are relatively small and fluxes through primary metabolism are large (Noctor and Foyer, 2000; Avenson et al., 2005; Cruz et al., 2005; Amthor, 2010).The balancing of ATP and NADPH supply is further complicated by the rigid nature of linear electron flow (LEF). In LEF, electrons are transferred from water to NADP⁺, oxidizing water to oxygen and reducing NADP⁺ to NADPH. This electron transfer is coupled to proton translocation and generates a proton motive force, which powers the regeneration of ATP. The stoichiometry of ATP/NADPH produced by these reactions is thought to be 1.29 based on the ratio of proton pumping and the requirement for ATP synthase in the thylakoid (Sacksteder et al., 2000; Seelert et al., 2000). However, under ambient CO₂, oxygen, and temperature, the ATP/NADPH required by CO₂ fixation, photorespiration, and NO₃⁻ assimilation is approximately 1.6 (Edwards and Walker, 1983). The ATP/NADPH demand from central metabolism changes significantly from 1.6 if the ratio of CO₂ or oxygen changes, driving different rates of photosynthesis and photorespiration (see “Theory”). Such changes in energy demand require a flexible mechanism to balance ATP/NADPH that responds to environmental conditions.The difference between ATP/NADPH supply from LEF and demand from primary metabolism could be balanced via cyclic electron flow around PSI (CEF; Avenson et al., 2005; Shikanai, 2007; Joliot and Johnson, 2011; Kramer and Evans, 2011). During CEF, electrons from either NADPH or ferredoxin are cycled around PSI into the plastoquinone pool and regenerate ATP without reducing NADP⁺ (Golbeck et al., 2006). Therefore, CEF has been suggested to be important for optimal photosynthesis and plant growth, but its physiological role in energy balancing is not clear (Munekage et al., 2002, 2004; Livingston et al., 2010). For example, there was no shift in CEF in Arabidopsis (Arabidopsis thaliana) measured under low light (less than 300 μmol m⁻² s⁻¹) and different oxygen partial pressures, which would significantly change the ATP/NADPH demand of primary metabolism (Avenson et al., 2005). Similar results were seen under low light in leaves of barley (Hordeum vulgare) and Hedera helix (Genty et al., 1990). While CEF did not shift with energy demand in steady-state photosynthesis under low light, it did increase with photorespiration as expected at high light (Miyake et al., 2004, 2005). These observations could be explained if CEF becomes more important for energy balancing under high irradiances when other mechanisms become saturated.To determine under which conditions CEF responded to ATP/NADPH demand, we used biochemical models of leaf CO₂ fixation to model ATP and NADPH demand under a variety of conditions (see “Theory”). We then used in vivo spectroscopy to measure the relative response of CEF to modeled ATP/NADPH demand from CO₂ fixation and NO₃⁻ assimilation in hydroponically grown Arabidopsis. Our findings indicate that CEF responded to modeled ATP/NADPH demand under high light but not under low light or nitrate availability.     

The Coordination of C4 Photosynthesis and the CO2-Concentrating Mechanism in Maize and Miscanthus × giganteus in Response to Transient Changes in Light Quality

Unequal absorption of photons between photosystems I and II, and between bundle-sheath and mesophyll cells, are likely to affect the efficiency of the CO₂-concentrating mechanism in C₄ plants. Under steady-state conditions, it is expected that the biochemical distribution of energy (ATP and NADPH) and photosynthetic metabolite concentrations will adjust to maintain the efficiency of C₄ photosynthesis through the coordination of the C₃ (Calvin-Benson-Bassham) and C₄ (CO₂ pump) cycles. However, under transient conditions, changes in light quality will likely alter the coordination of the C₃ and C₄ cycles, influencing rates of CO₂ assimilation and decreasing the efficiency of the CO₂-concentrating mechanism. To test these hypotheses, we measured leaf gas exchange, leaf discrimination, chlorophyll fluorescence, electrochromatic shift, photosynthetic metabolite pools, and chloroplast movement in maize (Zea mays) and Miscanthus × giganteus following transitional changes in light quality. In both species, the rate of net CO₂ assimilation responded quickly to changes in light treatments, with lower rates of net CO₂ assimilation under blue light compared with red, green, and blue light, red light, and green light. Under steady state, the efficiency of CO₂-concentrating mechanisms was similar; however, transient changes affected the coordination of C₃ and C₄ cycles in M. giganteus but to a lesser extent in maize. The species differences in the ability to coordinate the activities of C₃ and C₄ cycles appear to be related to differences in the response of cyclic electron flux around photosystem I and potentially chloroplast rearrangement in response to changes in light quality.The CO₂-concentrating mechanism in C₄ plants reduces the carbon lost through the photorespiratory pathway by limiting the oxygenation of ribulose-1,5-bisphosphate (RuBP) by the enzyme Rubisco (Brown and Smith, 1972; Sage, 1999). Through the compartmentalization of the C₄ cycle in the mesophyll cells and the C₃ cycle in the bundle-sheath cells (Hatch and Slack, 1966), C₄ plants suppress RuBP oxygenation by generating a high CO₂ partial pressure around Rubisco (Furbank and Hatch, 1987). To maintain high photosynthetic rates and efficient light energy utilization, the metabolic flux through the C₃ and C₄ cycles must be coordinated. However, coordination of the C₃ and C₄ cycles is likely disrupted due to rapid changes in environmental conditions, particularly changes in light availability (Evans et al., 2007; Tazoe et al., 2008).Spatial and temporal variations in light environments, including both light quantity and quality, are expected to alter the coordination of the C₃ and C₄ cycles. For example, it has been suggested that the coordination of C₃ and C₄ cycles is altered by changes in light intensity (Henderson et al., 1992; Cousins et al., 2006; Tazoe et al., 2006, 2008; Kromdijk et al., 2008, 2010; Pengelly et al., 2010). However, more recent publications indicate that some of the proposed light sensitivity of the CO₂-concentrating mechanisms in C₄ plants can be attributed to oversimplifications of leaf models of carbon isotope discrimination (Δ13C), in particular, errors in estimates of bundle-sheath CO₂ partial pressure and omissions of respiratory fractionation (Ubierna et al., 2011, 2013). Alternatively, there is little information on the effects of light quality on the coordination of C₃ and C₄ cycle activities and the subsequent impact on net rate of CO₂ assimilation (A_net).In C₃ plants, A_net is reduced under blue light compared with red or green light (Evans and Vogelmann, 2003; Loreto et al., 2009). This was attributed to differences in absorbance and wavelength-dependent differences in light penetration into leaves, where red and green light penetrate farther into leaves compared with blue light (Vogelmann and Evans, 2002; Evans and Vogelmann, 2003). Differences in light quality penetration into a leaf are likely to have profound impacts on C₄ photosynthesis, because the C₄ photosynthetic pathway requires the metabolic coordination of the mesophyll C₄ cycle and the bundle-sheath C₃ cycle. Indeed, Evans et al. (2007) observed a 50% reduction in the rate of CO₂ assimilation in Flaveria bidentis under blue light relative to white light at a light intensity of 350 µmol quanta m⁻² s⁻¹. This was attributed to poor penetration of blue light into the bundle-sheath cells and subsequent insufficient production of ATP in the bundle-sheath cells to match the rates of mesophyll cell CO₂ pumping (Evans et al., 2007). Recently, Sun et al. (2012) observed similar low rates of steady-state CO₂ assimilation under blue light relative to red, green, and blue light (RGB), red light, and green light at a constant light intensity of 900 µmol quanta m⁻² s⁻¹.Because the light penetration into a leaf depends on light quality, with blue light penetrating the least, this potentially results in changes in the energy available for carboxylation reactions in the bundle-sheath (C₃ cycle) and mesophyll (C₄ cycle) cells. Changes in the balance of energy driving the C₃ and C₄ cycles can alter the efficiency of the CO₂-concentrating mechanisms, often represented by leakiness (ϕ), the fraction of CO₂ that is pumped into the bundle-sheath cells that subsequently leaks back out (Evans et al., 2007). Unfortunately, ϕ cannot be measured directly, but it can be estimated through the combined measured and modeled values of Δ13C (Farquhar, 1983). Using measurements of Δ13C, it has been demonstrated that under steady-state conditions, changes in light quality do not affect ϕ (Sun et al., 2012); however, it remains unknown if ϕ is also constant during the transitions between different light qualities. In fact, sudden changes of light quality could temporally alter the coordination of the C₃ and C₄ cycles.To understand the effects of light quality on C₄ photosynthesis and the coordination of the activities of C₃ and C₄ cycles, we measured transitional changes in leaf gas exchange and Δ13C under RGB and broad-spectrum red, green, and blue light in the NADP-malic enzyme C₄ plants maize (Zea mays) and Miscanthus × giganteus. Leaf gas exchange and Δ13C measurements were used to estimate ϕ using the complete model of C₄ leaf Δ13C (Farquhar, 1983; Farquhar and Cernusak, 2012). Additionally, we measured photosynthetic metabolite pools, Rubisco activation state, chloroplast movement, and rates of linear versus cyclic electron flow during rapid transitions from red to blue light and blue to red light. We hypothesized that the limited penetration of blue light into the leaf would result in insufficient production of ATP in the bundle-sheath cells to match the rate of mesophyll cell CO₂ pumping. We predicted that rapid changes in light quality would affect the coordination of the C₃ and C₄ cycles and cause an increase in ϕ, but this would equilibrate as leaf metabolism reached a new steady-state condition.