SummaryGlycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.PaperFlick

SUMMARY Malaria-specific antibody responses are short-lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1+CXCR5+CD4+ Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population PD-1+CXCR5+CXCR3− Tfh cells are superior to Th1-polarized PD-1+CXCR5+CXCR3+ Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children, and suggest that vaccine strategies that promote CXCR3− Tfh cell responses may improve malaria vaccine efficacy.

Despite years of exposure to intense P. falciparum transmission, there is no evidence of acquired, sterile immunity to P. falciparum infection in this population, even as clinical immunity to blood-stage malaria is clearly acquired. Understanding why repeated P. falciparum infections do not induce sterile protection may lead to insights for developing vaccines that target the liver stage in malaria-endemic populations.

We have identified a third member of the junctional adhesion molecule (JAM) family. At the protein level JAM3 displays 36 and 32% identity to JAM2 and JAM1, respectively. The coding region is distributed over 9 exons and maps to chromosome 11q25. The gene shows widespread tissue expression with higher levels apparent in the kidney, brain, and placenta. At the cellular level we show expression of JAM3 transcript within endothelial cells. Our major finding is that JAM3 and JAM2 are binding partners. Thus, JAM3 ectodomain binds firmly to JAM2-Fc. This heterotypic interaction is maintained when JAM3-Fc is used to capture Chinese hamster ovary cells expressing full-length JAM2. In static adhesion assays we show that JAM3 is unable to bind to leukocyte cell lines. This is consistent with the lack of JAM2 expression. However, using JAM2-Fc pulldown experiments in combination with polyclonal anti-JAM3 serum, we demonstrate that JAM3 is the previously uncharacterized 43-kDa counter-receptor that mediates JAM2 adhesion to T cells. Most significantly we demonstrate up-regulation of JAM3 protein on peripheral blood lymphocytes following activation. Finally we show the utility of JAM3 ectodomain as an inhibitor of JAM2 adhesion.

Agonist-stimulated desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to ␤-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human ␤ 2 AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant ␤ 2 ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E 1 , and forskolin at maximum concentrations stimulated phosphorylation of the ␤ 2 AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC 50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC 50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC 50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still Ϸ200-fold greater than for the PKA site. The EC 50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the ␤ 2 AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agoniststimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine Ն formoterol ϭ fenoterol Ͼ terbutaline ϭ zinterol ϭ albuterol Ͼ salmeterol Ͼ Ͼ dobutamine Ն ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous ␤ 2 AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.The ␤ 2 -adrenergic receptor (␤ 2 AR) plays significant roles in relaying signals from the autonomic sympathetic nervous system to the cardiovascular and pulmonary systems in particular. There are...

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a hightemperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNAsequencing technologies, enables high-throughput mapping of enzyme sequence space.protein engineering | droplet-based microfluidics | high-throughput DNA sequencing E nzymes are powerful biological catalysts capable of remarkably accelerating the rates of chemical transformations (1). The molecular bases of these rate accelerations are often complex, using multiple steps, multiple catalytic mechanisms, and relying on numerous molecular interactions, in addition to those provided by the main catalytic groups. This complexity imposes a significant barrier to understanding how an enzyme's sequence impacts its function and, thus, on our ability to rationally design biocatalysts with new or enhanced functions (2-4).Comprehensive mappings of sequence-function relationships can be used to dissect the molecular basis of protein function in an unbiased manner (5). Growth selections or in vitro binding screens can be combined with next-generation DNA sequencing to generate detailed mappings between a protein's sequence and its biochemical properties, such as binding affinity, enzymatic activity, and stability (6-9). This deep mutational scanning approach has been used to study the structure of the protein fitness landscape, discover new functional sites, improve molecular energy functions, and identify beneficial combinations of mutations for protein engineering. However, these methods rely on functional assays coupled to cell growth or protein binding, severely limiting the types of proteins that can be analyzed. For example, most enzymes of biological or industrial relevance cannot be analyzed using existing methods because they do not catalyze a reaction that can be directly coupled to cell growth. Experimental advances are needed to broaden the applicability of deep mutational scanning to the diverse palette of functions performed by enzymes.In this paper, we present a general method for mapping protein sequence-func...

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquitoes to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the host or compromising host survival is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5-to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of subjects with febrile malaria in the transmission season, reflecting longer circulation within each replicative cycle, of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication, but rather increased splenic clearance of longer-circulating infected erythrocytes. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

NCT01322581.